UNIVERSIDADE ESTADUAL DO CEARÁ FACULDADE DE …€¦ · ENVOLVIMENTO DO ESTRESSE OXIDATIVO E DO...
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UNIVERSIDADE ESTADUAL DO CEARÁ FACULDADE DE VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS DOUTORADO EM CIÊNCIAS VETERINÁRIAS BELARMINO EUGÊNIO LOPES NETO ENVOLVIMENTO DO ESTRESSE OXIDATIVO E DO INFILTRADO LINFOCÍTICO NO MICROAMBIENTE TUMORAL MAMÁRIO EM CADELAS FORTALEZA - CEARÁ 2017
Transcript of UNIVERSIDADE ESTADUAL DO CEARÁ FACULDADE DE …€¦ · ENVOLVIMENTO DO ESTRESSE OXIDATIVO E DO...
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UNIVERSIDADE ESTADUAL DO CEARÁ
FACULDADE DE VETERINÁRIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS
DOUTORADO EM CIÊNCIAS VETERINÁRIAS
BELARMINO EUGÊNIO LOPES NETO
ENVOLVIMENTO DO ESTRESSE OXIDATIVO E DO INFILTRADO
LINFOCÍTICO NO MICROAMBIENTE TUMORAL MAMÁRIO EM
CADELAS
FORTALEZA - CEARÁ
2017
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BELARMINO EUGÊNIO LOPES NETO
ENVOLVIMENTO DO ESTRESSE OXIDATIVO E DO INFILTRADO
LINFOCÍTICO NO MICROAMBIENTE TUMORAL MAMÁRIO EM CADELAS
FORTALEZA - CEARÁ
2017
Tese apresentada ao Programa de Pós-Graduação em
Ciências Veterinárias da Faculdade de Veterinária da
Universidade Estadual do Ceará, como requisito
parcial para a obtenção do título de Doutor em
Ciências Veterinárias.
Área de Concentração: Reprodução e Sanidade
Animal.
Linha de Pesquisa: Reprodução e sanidade de
carnívoros, onívoros, herbívoros e aves.
Orientadora: Prof.ª Dra. Diana Célia Sousa Nunes
Pinheiro
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BELARMINO EUGÊNIO LOPES NETO
ENVOLVIMENTO DO ESTRESSE OXIDATIVO E DO INFILTRADO
LINFOCÍTICO NO MICROAMBIENTE TUMORAL MAMÁRIO EM CADELAS
Aprovado em: 19 / 12 / 2017
BANCA EXAMINADORA
Tese apresentada ao Programa de Pós-Graduação em
Ciências Veterinárias da Faculdade de Veterinária da
Universidade Estadual do Ceará, como requisito
parcial para a obtenção do título de Doutor em
Ciências Veterinárias.
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Dedico este trabalho aos meus
pais, Francisco e Celina, a minha
esposa Adália e ao meu filho
Bento.
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AGRADECIMENTOS
Agradeço e bendigo a Deus, pai supremo e fidedigno, por ter me concedido o dom da
vida e da sabedoria, me dando forças e providenciando os meios para concretizar mais
uma etapa da minha vida.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pelo apoio
financeiro concedido, especial
mente por ter concedido a bolsa de Doutorado Sanduiche.
Ao PPGCV, aos docentes pesquisadores e seu quadro de pessoal de apoio, pelos
ensinamentos, critérios científicos e oportunidade de crescimento intelectual.
À Profa. Dra. Diana Célia Sousa Nunes Pinheiro, por sempre ter acreditado no meu
potencial, por sua inestimável orientação, colaboração, compreensão, incentivo e acima
de tudo amizade e paciência, meus sinceros agradecimentos.
À Profa. Dra. Fátima Gärtner pela co-orientação e colaboração do desenvolvimento do
projeto e estágio doutoral, permitindo a realização de etapas fundamentais na execução
deste trabalho.
Aos colegas do Laboratório de Imunologia e Bioquímica de Animais (LIBA), sem
mencionar nomes, pois todos tiveram fundamental importância para a realização desse
trabalho.
Aos novos colegas no Instituto de Ciências Biomédicas Abela Salazar (ICBAS),
Universidade do Porto, que conheci durante o doutorado sanduiche. Em especial, Aline
Alvarenga, que compartilhou e me acompanhou nesse período de desafios e conquistas.
A todos os funcionários da Faculdade de Veterinária (FAVET), especialmente aos
professores do Programa de Pós-Graduação em Ciências Veterinárias (PPGCV) da
UECE, pelos conhecimentos e experiências compartilhadas.
Aos meus pais, Francisco Barroso Pinto e Maria Celina Lopes Barroso, que são base da
minha vida, por todo amor, carinho e dedicação, e por estarem sempre ao meu lado, me
apoiando em qualquer situação.
A minha esposa, Adália Freitas de Oliveira Lopes, que teve a coragem e ousadia de
acompanhar esse cientista maluco. Ela que em nenhum momento hesitou nessa jornada,
pelo contrário, me incentivou e ainda vai me dar o melhor presento do mundo, nosso filho
Bento.
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Por fim, mas não menos importante, aos animais que são criaturas de Deus e merecem
todo o respeito a ajuda de nós, seres um pouco mais pensantes. Que um dia possamos
respeitá-los e não mais explorarmos em todas as dimensões.
“Nossa tarefa deveria ser nos libertarmos ... aumentando o nosso círculo de compaixão
para envolver todas as criaturas viventes, toda a natureza e sua beleza." - Albert Einstein
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RESUMO
O câncer de mama é a principal neoplasia diagnosticada em mulheres e cadelas. No
entanto, a etiologia do tumor mamário canino (TMC) ainda não está esclarecida. Este
trabalho teve como objetivos investigar o nível sérico de oxidantes e antioxidantes não
enzimáticos, estudar o infiltrado celular (IC) e infiltrado linfocítico tumoral (TIL), além
da expressão de biomarcadores no microambiente tumoral, bem como a associação de
características clínico-patológicas e tempo de sobrevivência das cadelas. Para tanto, as
cadelas foram divididas no grupo com TMC benigno (Be), TMC maligno (Ma) e animais
controles (Co). O protocolo foi aprovado pelo Comitê de Ética para o Uso de Animais
(CEUA-UECE), número 12247080-2. Os níveis séricos de proteína total, globulina,
bilirrubina e malondialdeído (MDA) foram maiores (P < 0,05) nos grupos Ma e Be
comparado ao grupo Co. Todas as características malignas apresentaram níveis elevados
de MDA e estavam associados com necrose e tipo de tumor (P < 0,05). Não foi observada
associação entre o MDA e o tempo de sobrevida geral. A idade média dos animais foi de
9,3 anos, os tumores estavam localizados nas regiões inguinais e predomínio de grau I-II
do TMC. IC estava distribuído nas regiões peri e intratumoral, tendo linfócitos como
principal população encontrada dentro dos tumores. A correlação do IC com o grau do
TMC demostrou que os linfócitos (ρ = 0,28) e as células plasmáticas (ρ = 0,22)
apresentaram uma ligeira correlação positiva, por outro lado, neutrófilos (ρ = -0,1) e
macrófagos (ρ = -0,38) apresentaram correlação negativa. Cadelas com TIL moderado (<
800 linfócitos) TMC apresentaram maior taxa de sobrevivência (P = 0,01) em relação aos
animais com infiltrado linfocítico intenso (≥ 800 linfócitos). Foram observados linfócitos
FoxP3 em menor intensidade enquanto que CD4 e CD8 estava em maior quantidade e
concentrada no TIL. HSP60 foi observado nas células inflamatórias e tumorais. No
estroma tumoral pode-se observar que 65,2% dos tumores apresentavam baixo TIL e
tumores com alto TIL diferiu significativamente (P < 0,001) em relação ao baixo TIL. O
TIL estromal foi associado (P < 0,009) a expressão de PD-L1 (39%) no TMC. Nas
metástases dos linfonodos (n = 5), PD-L1 estava presente no epitélio maligno e estava
associado (P < 0,034) ao envolvimento de linfonodos regionais. As curvas de
sobrevivência demonstraram maior sobrevida nos animais com baixo TIL (P < 0,01)
comparado ao com auto TIL. As variáveis clínico-patológicas correlacionadas com
sobrevida global por análise univariada foram grau de tumor (P < 0,009), envolvimento
dos linfonodos (P < 0,004), TIL (P < 0,016) e PD-L1+/auto TIL vs. PD -L1-/baixo TIL
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(P <0,01). A análise multivariada revelou que o grupo de tumores com grau II-III era um
fator independente e pior prognósticos para a sobrevida global. Conclui-se que os TMC
apresentam intensa peroxidação lipídica, presença de CD4, CD8 e FoxP3 nas regiões peri
e intratumoral e a expressão de PD-L1 está associada com TIL estromal. Estes marcadores
podem estar relacionados ao prognóstico dos tumores mamários caninos e podem ser
utilizados como alvos para terapias anticâncer.
Palavras-chave: Câncer. Tumor mamário canino. Estresse oxidativo. Linfócitos. PD-L1
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ABSTRACT
Breast cancer is the major neoplastic diagnosticated in both dogs and human. However,
canine mammary tumor (CMT) etiology still unknown. This study investigates the serum
levels of oxidant and antioxidant non-enzymatic, cell infiltrate (CI) and tumor-infiltrating
lymphocytes (TILs), and tumor microenvironment biomarker expressions, as well as the
association of clinic-pathological features and survival time of animals. Female dogs were
divided into benign (Be) CMT, malignant (Ma) CMT, and healthy control dogs (Co). This
study was approved by the Committee for Ethics in Research using Animals (CEUA-
UECE), protocol 12247080-2. The levels of total proteins, globulin, bilirubin and MDA
was higher (P < 0.05) in Ma and Be CMT than Co group. All malignancy features present
high MDA levels, and the parameters as necrosis and type of tumor had difference (P <
0.05) between categories. No association was observed between MDA and overall
survival (OS). Data showed a mean age of 9.3 years old, tumors were located in inguinal
mammary glands, and CMTs shows I-II grade. CI was distributed both in peri and
intratumoral regions, and lymphocytes cells were the major populations into tumors. In
relationship to CI with CMT grade it was observed that lymphocytes (ρ = 0.28) and
plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative
correlation of neutrophil (ρ = -0.1) and macrophage (ρ = -0.38). CMT presents moderate
lymphocytic infiltrate (< 800 lymphocytes), shows higher (P = 0.01) survival rates as
compared to intense lymphocytic infiltrate (≥ 800 lymphocytes). FoxP3 showed lower
intensity while CD4 and CD8 lymphocytes expression was higher and concentrated
within TIL. HSP60 was observed in the inflammatory and tumor cells. TILs evaluation
within tumor stroma revealed that 65.2% had TILs-Low, and TILs-High differed (P <
0.001) from TILs-Low. PD-L1 and stromal TILs were associated (P < 0.009).
Immunostaining of PD-L1 (39%) in TILs has shown strong labeled in lymphocytes within
tumor stroma. PD-L1 in malignant epithelium was present in all lymph node metastasis
(n = 5), and was associated with regional lymph nodes involvement (P < 0.034). Survival
curves demonstrated difference between TILs-Low and TILs-High (P < 0.01). The clinic-
pathologic variables significantly correlated with OS by univariate analysis were grade
of tumor (P < 0.009), lymph node involvement (P < 0.004), stromal TILs (P < 0.016), and
PD-L1+/TILs-High vs. PD-L1–/TILs-Low (P < 0.01). Multivariate analysis revealed that
group of tumors with grade II-III was independent and negative prognostic factors for
OS. We concluded that MDA support the lipid peroxidation in MGT-bearing dogs. CD4,
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CD8 and FoxP3 labeling were distributed in peri and intratumoral regions. PD-L1
expression was associated with stromal TILs. These markers may represent important
survival prognostic biomarkers for canine mammary tumors and can be used as potential
target for anticancer therapies.
Key-words: Cancer. Canine mammary tumor. Oxidative stress. Lymphocytes. PD-L1
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LISTA DE FIGURAS
Figura 1 - Alteração maligna de um ducto mamário demostrando as
alterações nos componentes da MEC. 23
CAPÍTULO 1
Figura 1 - Relationship between two different levels of MDA and
overall survival time (P = .710). 40
Figura 2 - MDA levels in dogs with different MGT types. Co: control
group; Ad: Adenoma; BMT: Benign Mixed Tumor; Ca:
carcinoma; CoC: complex carcinoma; CMT: carcinoma in
mixed tumor; CSC: carcinosarcoma. a,b,c Differet letters are
significant (P = .05). Bars indicate standard deviation.
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CAPÍTULO 2
Figura 1 - Composition of inflammatory infiltrate in CMT.
Lymphocytes (L), Neutrophils (N), Macrophages (M) and
Plasma Cells (P). Cell count performed on eight fields
(400X). Results were expressed in mean ± standard deviation.
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Figura 2 - Presence of inflammatory infiltrate in mammary carcinoma
in mixed tumor (MCMT) of bitches and CD4+, CD8+, FoxP3+
and HSP60+ expressions in cellular infiltrate. Where, (A)
Lymphocytes associated with a bone matrix, (B)
Macrophages between bone marrow and cartilaginous
matrix, (C) CD4+ T lymphocytes moderately immunostained,
(D) CD8+ T lymphocytes moderately immunostained, (E)
FoxP3+ T regulatory lymphocytes mildly immunostained and
(F) HSP60+ strongly immunostained in tumor and
inflammatory cells.
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Figure 3 - Survival rates of animals with MCMT categorized in two
intervals of the lymphocytic infiltrate (< 800 and ≥ 800
lymphocytes).
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CAPÍTULO 3
Figura 1 - TILs assessment and PD-L1 expression within CMT and
stromal TILs. A. Carcinoma in benign mixed tumor with
few stromal TILs (H&E, 200x); B. Solid carcinoma with
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high stromal TILs (H&E, 200x); C. Complex carcinoma
with few infiltrate cells expressing PD-L1 (200x); D.
Tubular carcinoma with high infiltrate cells expressing
PD-L1 (200x); E. Complex carcinoma with negative PD-
L1 expression (100x); F. Tubular carcinoma with positive
PD-L1 expression (100x); Malignant epithelium with
strong PD-L1 expression (400x); Lymph node metastasis
with PD-L1 expression (200x). I. Lymph node metastasis
with AE1/AE3 positive expression (100x)
Figura 2 - Relationship between PD-L1 expression and stromal TILs.
A. Figures within this bar graph depict percentage of cases
with PD-L1 expression and stromal TILs intensity. B.
Analysis of TILs status and stromal TILs(%). C. Analysis
of PD-L1 status and stromal TILs(%). Significant
differences at P < 0.05 are highlighted by asterisk.
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Figura 3 - Overall survival for stromal TILs (A), PD-L1 expression
(B), and PD-L1* TILs. 83
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LISTA DE TABELAS
Tabela 1 - Critérios para estadiamento clínico dos TMC. 20
Tabela 2 - Classificação das lesões e neoplasias da glândula mamária
canina (MISDORP et al., 1998; GOLDSCHMIDT et al.,
2011).
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Tabela 3 - Critérios para graduação do TMC (ELSTON; ELLIS,
1998). 22
Tabela 4 - Principais componentes da matriz extracelular. 24
Tabela 5 - Principais componentes solúveis da matriz extracelular. 25
CAPÍTULO 1
Tabela 1 - Clinic-pathological data of MGT-bearing dogs. 39
Tabela 2 - Serum levels of oxidative and antioxidant non-enzymatic
parameters in MGT-bearing dogs. 40
Tabela 3 - Association of serum MDA levels with clinic-pathological
parameters in malignant MGT-bearing dogs. 41
CAPÍTULO 2
Tabela 1 - Distribution, location and intensity of cellular infiltrate in
tumor environment. Data were expressed as percentage. 59
CAPÍTULO 3
Tabela 1 - Clinicopathologic data of CMT. 75
Tabela 2 - Association between PD-L1expression, stromal TILs and
Clinicopathologic data of CMT.
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Tabela 3 - Univariate and multivariate cox proportional hazard model
for overall survival 80
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LISTA DE ABREVIATURAS E SIGLAS
% Percentagem
μL Microlitro
Ad Adenomas
BC Breast cancer
Be Benign
BMT Benign mixed tumors
Ca Carcinomas
CA Simple carcinoma
CAPES Improvement of Higher Education Personnel
CC Complex carcinoma
CEUA-
UECE
Committee for Ethics in Research using Animals
CI Cell infiltrate
CIs Confidence intervals
CMCMT Canine mammary carcinoma in mixed tumor
CMT Canine mammary tumor
Co Control dogs
CoC Complex carcinomas
CSC Carcinosarcoma
CTLA-4 Antígeno-4 associado ao linfócito-T citotóxico
DAB Diaminobenzidine
DAB 3-diaminobenzidine tetrahydrochloride
FAVET Faculdade de Veterinária
g/dL Grama/decilitro
H&E Hematoxylin-eosin
HER2 Human epidermal growth factor receptor 2
HPF High-power field
HR Hazard ratios
HSP Heat shock proteins
i3S Instituto de Investigação e Inovação em Saúde
ICBAS-UP Instituto de Ciências Biomédicas Abel Salazar, da Universiade do
Porto
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IPATIMUP Instituto de Patologia e Imunologia Molecular da Universidade do
Porto
L Lymphocytes
LDH Lactate dehydrogenase
M Metástase à distância
Ma Malignant
MCMT Mammary carcinoma in mixed tumor
MDA Malondialdeído
MEC Matrix extracelular
mg/dL Miligrama/decilitro
MGT Mammary gland tumors
MMT Mammary mixed tumor
N Envolvimento do linfonodo com metástase regional
n° Numbers
NK Célula natural killer
nmo/mL Nanomol/mililitro
OS Overall survival
P Plasma Cells
PBS Phosphate buffered saline
PD-1 receptor de morte programada-1
PD-L1 Ligante do receptor de morte programada-1
PD-L1 Programmed death 1 ligand 1
ROS Reactive oxygen species
T Tamanho do tumor
TBARS Thiobarbituric acid-reactive substances
TBS Tris buffered saline
TCA Trichloroacetic acid
TILs Tumor-infiltrating lymphocytes
Treg T lymphocytes
U/L Unidade/litro
UECE Universidade Estadual do Ceará
UHV Unidade Hospitalar Veterinária
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SUMÁRIO
1 INTRODUÇÃO…………………………..…………………………….… 17
2 REVISÃO DE LITERATURA………………………..……………….... 19
2.1 Etiologia dos Tumores da Glândula Mamário em cadelas……………….... 19
2.2 Diagnóstico dos tumores da glândula mamário canina……………….….... 20
2.2.1 Estadiamento clínico……………………..…………………..…….…….. 20
2.2.2 Classificação e graduação do TMC…………………………….……..… 21
2.3 Microambiente tumoral……………………………………………………. 23
2.3.1 Matriz extracelular 23
2.3.2 Células inflamatórias no câncer………………………………….……… 25
2.4 Estresse oxidativo no câncer……….…………………………………........ 27
2.5 Mecanismos de escape do câncer ao sistema imune………………………. 27
3 JUSTIFICATIVA…………...……………………………………………. 29
4 HIPÓTESE CIENTÍFICA……………...……………………………….. 30
5 OBJETIVOS………………..……………………………………………. 32
5.1 GERAL…….………………………………………………………………. 32
5.2 ESPECÍFICOS…………………………………………………………… 32
6 CAPITULO 1……………………………………………………………... 32
7 CAPÍTULO 2………...…………………………………………………… 50
8 CAPITULO 3………………...…………………………………………… 70
9 CONCLUSÕES………………………………………………………....... 93
10 PERSPECTIVAS………………………………………………………… 94
REFERÊNCIAS………………………………………………………….. 95
APÊNDICE ……………………..………………………………………... 111
APÊNDICE A – CERTIFICADO DO COMITÊ DE ÉTICA PARA O
USO DE ANIMAIS /UECE………………………………………………. 112
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1 INTRODUÇÃO
Os cuidados e prevenção da saúde dos cães tem se tornado cada vez mais um
hábito comum entre os seus proprietários. Dessa forma, esses animais aumentam a sua
expectativa de vida, podendo chegar à senilidade. Em consequência, doenças associadas
ao envelhecimento, como o câncer, passaram a integrar a lista das principais morbidades
nestes pacientes (DAVIS; OSTRANDER, 2014). O tumor de mama é a neoplasia mais
comum em cadelas. Tanto as neoplasias benignas como malignas são frequentemente
diagnosticadas nesta espécie, sendo mais de 50% dos casos podem ser neoplasia maligna
(FILHO et al., 2010; RASOTTO et al., 2017).
A etiologia do tumor mamário canino (TMC) ainda não está bem esclarecida,
sendo atribuída a multifatores, como raça do animal, idade, tamanho do tumor, ausência
de receptores hormonais, uso de estrógenos sintéticos (MATOS; SANTOS, 2015; IM et
al., 2014). O câncer é um processo complexo que se desenvolve em várias etapas
controladas por perturbações genéticas como a ativação de oncogenes, silenciamento de
genes supressores tumorais e o descontrole de eventos epigenéticos que ocorrem dentro
da célula. Além disso, a influências do microambiente tumoral juntamente com os fatores
pró e anti-inflamatórios participam ativamente na manutenção e progressão da neoplasia
(HANAHAN; WEINBERG, 2011).
A inflamação é um processo importante na recuperação de tecidos danificados.
No entanto, a inflamação crônica pode auxiliar na iniciação e progressão do câncer a
partir de citocinas e quimiocinas liberadas pelas células do microambiente tumoral,
incluindo os leucócitos (ELINAV et al., 2013). O papel dos leucócitos no microambiente
ainda não é totalmente compreendido, e vários esforços estão sendo realizados para
entender melhor o papel dessas células no desenvolvimento do tumor. Estudos
demonstraram que a presença de linfócitos em diferentes tipos de TMC pode estar
relacionado tanto a um melhor ou pior prognóstico para os cães (ESTRELA-LIMA et al.,
2010; CARVALHO et al., 2016; LOPES-NETO et al., 2017).
Os tumores de mama são frequentemente infiltrados por populações heterogêneas
das células inflamatórias, cujos linfócitos são as principais células encontradas no
microambiente tumoral (KIM et al., 2013). O infiltrado linfocítico tumoral (TIL) têm
importância considerável como um parâmetro para o prognóstico do TMC, especialmente
os linfócitos T. Estudos tem demonstrado células T CD4+ e T regulatórios podem estar
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associadas com a progressão dos TMC, enquanto um melhor prognóstico para esses
tumores tem apresentado um predomínio de linfócitos T CD8+ (KIM et al., 2012;
Carvalho et al., 2016). Embora a pesquisa de TIL na CMT tenha sido aprimorada, a
ligação entre células imunes e desenvolvimento do câncer ainda não foi bem esclarecida.
Outro mecanismo que está associado a instabilidade promovida pelo câncer na
homeostasia dos animais é o estresse oxidativo (KAWANISHI et al., 2017). As espécies
reativas ao oxigênio (ERO) são agentes oxidantes responsáveis pelo estresse oxidativo,
podendo ser encontradas nas células normais e neoplásicas, e o seu aumento pode
promover sérias consequências, como a proliferação celular, desencadear mutação e
instabilidade genética, além de alterações na sensibilidade celular a agentes anticâncer
(COSTA et al., 2014).
Diante das evidências que o processo inflamatório está relacionado as alterações
provocadas pelo TMC, novos estudos devem ser realizados objetivando investigar o
microambiente tumoral que possam revelar a complexa relação entre a biologia celular e
o sistema imunológico. Para controlar o câncer de mama, uma compreensão profunda do
microambiente do tumor será muito importante.
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2 REVISÃO DE LITERATURA
2.1 Etiologia dos Tumores da Glândula Mamário em cadelas
A neoplasia mamária em cadelas apresenta grande importância na medicina
veterinária e humana, principalmente por servir de modelo para o estudo do câncer de
mama na mulher (LIU et al., 2014). Os tumores da glândula mamários nos caninos
apresentam várias características epidemiológicas, clínicas, biológicas e genéticas
semelhantes aos da espécie humana. Entre estas, podem ser citadas: faixa etária de
aparecimento, morfologia, efeito protetor da ovariohisterectomia, presença de receptores
hormonais no tecido neoplásico, órgãos alvo de metástase, evolução clínica e a
hereditariedade. Também foi demonstrado que neoplasias mamárias apresentam
semelhança na sequência genética entre a espécie canina e humana (DAVIS;
OSTRANDER, 2014; TIMMERMANS-SPRANG et al., 2017).
A glândula mamária canina é um órgão hormônio-dependente cuja atividade cíclica
e sua diferenciação está associada ao controle hormonal orquestrado durante as fases do
ciclo estral. Alguns trabalhos apontam a possibilidade de hormônios esteroides
desempenharem papel importante na etiologia dos tumores mamários. Tanto o estrógeno
quanto a prolactina são necessários ao crescimento dessa enfermidade e a progesterona
apresenta ação carcinogênica quando seus níveis estão aumentados por períodos
prolongados (PENÃ et al., 2013). Receptores para estrógeno, progesterona, andrógenos,
prolactina e para o fator de crescimento epidermal já foram demonstrados nos tumores de
mama em cadelas, podendo haver a coexistência desses receptores em uma mesma
neoplasia. Tem sido demonstrado que a diminuição na expressão dos receptores de
estrógeno e o aumento dos receptores de progesterona está associado com um prognóstico
ruim em carcinomas mamários (KLOPFLEISCH et al., 2011; PENÃ et al., 2014). Outro
indicativo que o fator hormonal contribui para o desenvolvimento das neoplasias
mamárias é a diferente incidência que ela apresenta entre cadelas inteiras e castradas,
sendo um fator de proteção a realização da ovariohisterectomia antes do primeiro estro
(FONSECA; DALECK, 2000).
Outros fatores que podem influenciar no aparecimento do TMC é o aspecto
nutricional do animal, principalmente a obesidade, a idade avançada das cadelas, fatores
ambientais, alterações genéticas que podem reorganizar a estrutura celular, favorecendo
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o desequilíbrio da homeostase das células do tecido glandular mamária (QUEIROGA;
LOPES, 2002; ZIECH et al., 2010).
2.2 Diagnóstico dos tumores da glândula mamário canina
O diagnóstico clínico de CMT geralmente envolve história médica completa,
exame físico com palpação cuidadosa da glândula mamária e o exame laboratorial
complementar (CASSALI et al., 2014). Adicionalmente, radiografia torácicas em três
planos diferentes e avaliação ultrassonográfica podem ser realizados para excluir
metástases pulmonar e abdominal. Os locais mais comuns de metástases à distância são
os pulmões, o fígado e menos frequentemente ossos (CASSALI et al., 2011). Citologia
aspirativa por agulha fina pode auxiliar no diagnóstico da neoplasia além da avaliação
citológica de gânglios linfáticos para rastreio de metástases. Os gânglios linfáticos mais
comumente afetados além dos linfonodos regionais são os linfonodos inguinal,
sublombar, esternal e pré-escapular (LANA et al., 2007). No cão, o TMC pode afetar um
ou mais complexos mamários e se apresenta como nódulos solitários ou múltiplos
(MISDORP, 1999; CASSALI et al., 2013). Estudo observaram que 70% dos cães com
TMC possuem mais de um tumor e que os dois pares caudais de glândula são mais
frequentemente afetados (SORENMO et al., 2011). As características clínicas, como
crescimento rápido, demarcação fraca, ulceração superficial, aumento dos nódulos
linfáticos, emaciação e dispneia podem sugerir comportamento maligno, enquanto o
crescimento lento e com limites definidos indicam neoplasias benignas (MISDORP,
1999). O diagnóstico definitivo requer a histopatologia da massa tumoral, considerado
como a técnica de diagnóstico final para o TMC (SORENMO et al., 2011).
2.2.1 Estadiamento clínico
A classificação do estágio clínico consiste em cinco estágios (I a V) determinados pelo
tamanho do tumor primário (T), envolvimento dos linfonodos regionais (N) e presença
ou ausência de metástases à distância (M) (Tabela 1). O tamanho do tumor é avaliado
medindo o maior diâmetro do maior tumor maligno presente, o linfonodo é considerado
positivo quando há evidências de macrometástases e a metástase a distância é identificada
utilizando as técnicas de imagem (OWEN, 1980; SORENMO et al., 2013).
21
Tabela 1 - Critérios para estadiamento clínico dos TMC.
ESTÁDIO T N M
I T1: < 3cm N0 M0
II T2: 3-5 cm N0 M0
III T3: > 5cm N0 M0
IV T (não interfere) N1(positivo) M0
V T (não interfere) N (não interfere) M1 (positivo)
T: tamanho do tumor; N: envolvimento do linfonodo com metástase regional; M:
metástase à distância. Fonte: Adaptado de Sorenmo et al (2013).
2.2.2 Classificação e graduação do TMC
Os critérios utilizados para classificar neoplasias mamárias são semelhantes aos
utilizados em todos os tipos de neoplasia, como a morfologia e o comportamento
biológico, sendo a classificação ideal a qual combina os dois critérios. Durante o passar
dos anos, muitas classificações foram propostas, sendo a amais utilizada atuçmente está
descrita na tabela 2.
Seguindo as características biológicas, o TMC pode ser benigno ou maligno. Essa
avaliação é feita na análise histopatológica, no qual irá ter como indicadores o
crescimento tumoral, podendo ser expansivo e delimitado no benigno e destrutivo e
invasivo nos tecidos adjacentes nos tecidos malignos (CASSALI et al., 2014). Presença
ou ausência de invasão nos vasos sanguíneos ou linfáticos por êmbolos de células
neoplásicas, presença de necrose, além dos critérios para a graduação do tumor.
O sistema de graduação de Elston e Ellis (1998) é uma ferramenta para avaliar o
nível de diferenciação do câncer de mama. Os três critérios de classificação utilizados são
a formação tubular, pleomorfismo nuclear e contagem mitótica. Cada característica é
dada uma pontuação de um a três pontos. As pontuações para cada critério são
adicionadas e resultam em uma pontuação final, o que corresponde ao grau. O grau I é
um tumor bem diferenciado com uma pontuação final de três a cinco pontos. O grau II é
um tumor moderadamente diferenciado com uma pontuação final de seis ou sete pontos.
O grau III é um tumor pouco diferenciado com uma pontuação final de oito ou nove
pontos (Tabela 3).
22
Tabela 2 - Classificação das lesões e neoplasias da glândula mamária canina.
LESÕES NÃO NEOPLASICAS
Hiperplasia epitelial Lesões de células colunares
Hiperplasia lobular Alteração de célula colunar
Hiperplasia ductal Hiperplasia de célula colunar
Adenose Lesões atípicas de células colunares
NEOPLASIAS BENIGNAS
Adenoma Fibroadenoma
Adenomioepitelioma Papiloma ductal
Adenoma basalóide Tumor misto benigno
NEOPLASIAS MALIGNAS
Carcinomas Carcinomas especiais
Carcinoma in situ ductal ou lobular Carcinoma micropapilar
Carcinoma em tumor misto Carcinoma lobular invasor
Carcinoma papilar Carcinoma lobular pleomórfico
Carcinoma tubular Carcinoma secretor
Carcinoma sólido Carcinoma rico em lipídeos
Carcinoma de células fusiformes
Neoplasia Mioepitelia Carcinoma de células escamosas
Adenomioepitelioma maligno Carcinoma anaplásico
Carcinoma mamário com diferenciação sebácea
Sarcomas Outros sarcomas
Fibrossarcoma Condrossarcoma
Osteossarcoma Lipossarcoma
Carcinossarcoma Hemangiossarcoma
Sarcinoma em tumor misto
Fonte: Adaptado de GOLDSCHMIDT et al., 2011
23
Tabela 3. Critérios para graduação do TMC.
Critérios Escore
Formação de Tubulação
> 75% do tumor 1
10 to 75% do tumor 2
< 10% do tumor 3
Pleomorfismo nuclear
Tamanho nuclear semelhante a uma célula normal (2 a 3 vezes o
tamanho dos glóbulos vermelhos) 1
Moderado aumento de tamanho e
variação nuclear 2
Marcada variação 3
Contagem mitótica CGA
0 a 8 Contagem mitótica / 10 CGA 1
9 a 16 Contagem mitótica / 10
CGA 2
≥ 17 Contagem mitótica / 10 CGA 3
Fonte: Adaptado de Cassali et al., 2013. CGA: Campo de Grande Aumento (400X).
2.3 Microambiente tumoral
O microambiente tumoral está intimamente associado com a carcinogênense,
progressão e metástases dos tumores. Este ambiente tumoral é composto por vasos
sanguíneos circundantes ao tumor, células inflamatórias, células imunes, linfócitos,
fibroblastos, matriz extracelular (MEC) e uma variedade de moléculas biológicas ativas
derivadas de células tumorais. Esses fatores, juntamente com suas vias de sinalização,
interagem uns com os outros no microambiente do tumor. Qualquer alteração nessa rede
formada pode afetar o metabolismo e o comportamento das células tumorais e,
consequentemente, alterar a progressão do tumor de forma direta ou indireta
(HANAHAN; WEINBERG, 2011; HUANG et al., 2017).
2.3.1. Matriz extracelular
A matriz extracelular participa da manutenção de todas as células, sendo
constituída por fibras proteicas e polissacarídeos, numa combinação que confere
resistência aos órgãos, principalmente os tecidos conjuntivos (MOUW et al., 2014). A
24
arquitetura da MEC é organizada por elementos insolúveis (Tabela 4) que conferem tanto
rigidez quanto elasticidade aos tecidos e composta por polímeros solúveis (Tabela 5),
responsáveis por fornecer um ambiente extracelular que regula tanto a proliferação
quanto a diferenciação das células (KIM et al., 2011; KULAR et al., 2014).
Dada a importância crucial da MEC para o desenvolvimento e manutenção da
homeostase dos tecidos, a desregulação dos constituintes da MEC pode contribuir para
várias condições patológicas, tais como a fibrose e o câncer (BONNANS et al., 2014). O
câncer de mama é um tumor sólido composto não por uma mistura aleatória de células e
matriz, mas assemelha-se a estrutura de um órgão, embora seja estruturalmente e
funcionalmente anormal (Figura 1) (KLOPFLEISCH et al., 2011). O TMC contém vários
tipos de células e componentes da MEC que podem desenvolver funções semelhantes às
realizadas nos tecidos normais, como fornecer sustentação, rigidez e substratos para o
crescimento tumoral (EGEBLAD et al., 2010).
Figura 1 - Alteração maligna de um ducto mamário demostrando as alterações nos
componentes da MEC. (A) Ducto mamário normal composto por células do epitélio
luminal, células mioepitéliais e membrana basal íntegra. As moléculas de adesão
(caderinas e integrinas) junto com os componentes da MEC (PGs e colágeno) auxiliam
na manutenção da arquitetura do ducto. (B) Aparecimento das células tumorais no ducto
mamário leva a perda das moléculas de adesão, o rompimento da membrana basal e
consequente invasão do tumor, com mudança na orientação do colágeno transformando
a MEC mais rígida, além da perda de PGs. As moléculas de metaloproteinases da matriz
(MMPs) auxiliam na progressão tumoral aos tecidos adjacentes. Fonte: Elaborado pelo
autor.
25
Tabela 4 - Principais componentes insolúveis da matriz extracelular.
Componente
estromal
Constituinte Função
Colágeno
Homotrímero e
heterotrímero com três
cadeias α polipeptídicas
(MOUW et al., 2014)
Tipo fibrilar: I,
II, III, V e VI
Proteção aos tecidos da ação mecânica de
tensão, corte e pressão (KULAR et al.,
2014)
Tipo não
fibrilar: IV e
VII
Ancoragem das fibras colágenas e da
membrana basal (GORDON; HAHN,
2010)
Elastina Subunidades de tropoelastina que formam
ligações cruzadas com a camada externa das
fibrilas (HALPER; KAJAER, 2014)
Flexibilidade aos tecidos, parede das
grandes artérias e nos ligamentos elásticos
(HALPER; KAJAER, 2014)
Fibronectina Glicoproteína composto por duas cadeias
peptídicas muito longa unidas por pontes
dissulfeto que pode se ligar a integrinas
(SINGH et al., 2010)
Proteína dentro da membrana basal
relacionada a adesão celular e a resposta
cicatricial (MOUW et al., 2014)
Laminina Glicoproteínas triméricas compostas por
cadeias do tipo α, β e γ (IORIO et al., 2014)
Proteínas dentro da membrana basal
envolvida na adesão, diferenciação e
migração celular através da ligação com as
integrinas (IORIO et al., 2014)
Fonte: Elaborado pelo autor.
2.3.2 Células inflamatórias no câncer
A envolvimento do sistema imune no câncer vem sendo amplamente estudado
tanto na medicina humana como na veterinária, e estudos realizados nos cães buscam
novas estratégias no combate ao câncer de mama em cadelas e mulheres, pois o TMC
nessas duas espécies apresentam semelhanças biológicas, particularmente o perfil
hormonal que os tumores expressam. (PEÑA et al., 2014; MATOS; SANTOS, 2015;
TIMMERMANS-SPRANG et al., 2017). O papel da imunidade inata e adaptativa no
controle do TGM tem apresentado resultados paradoxais, visto que linfócitos T
citotóxicos e células natural killer (NK) são responsáveis por controlar e eliminar células
tumorais, enquanto a presença de macrófagos associados a tumores, células B e linfócitos
T regulatórios estão relacionadas a uma tolerância ao tumor (LAW et al., 2017).
Desta forma, o infiltrado inflamatório presente no microambiente tumoral tem sido
estudado como biomarcadores para monitorar a progressão tumoral, especialmente o TIL
(ESTRELA-LIMA et al., 2010; SANTOS; MATOS, 2015). A presença do TIL no tumor
de mama em mulheres demonstrou potencial valor preditiva e prognóstico em subtipos
específicos de câncer de mama, especificamente os que apresentam fator de crescimento
epidérmico humano (HER2) positivo e os triplos negativos (BENSE et al., 2017;
TIMMERMANS-SPRANG et al., 2017).
26
Tabela 5 - Principais componentes solúveis da matriz extracelular
Componente
estromal
Constituinte Tipo Função
GAGs Grande complexo de
moléculas de hidratos de
carbono (GANDHI;
MANCERA, 2008)
Sulfatada (sulfato de condroitina,
sulfato de dermatano, queratan
sulfato, heparina e heparan sulfato)
Inibir ou regular a passagem de
outras moléculas através da
membrana basal, controlar o
acesso de macromoléculas à
superfície celular, afetar o
crescimento, migração, adesão
e a diferenciação celular
(SHAM et al., 2014).
Não sulfatada (ácido hialurônico)
PGs Macromoléculas
caracterizada pela presença
de um ou mais cadeia de
GAGs
(THEOCHARIS et al, 2010)
Diferentes tipos de PGs associados a
localização:
Secretadas na MEC (perlecan, agrin,
agrecan, versican, brevican e
neurocan)
Superfície celular (sindecans e
glipicans)
Intracelular (Serglican)
Organização da MEC, filtração
iônica, modulação dos fatores
de crescimento, multiplicação
e diferenciação celular,
regulação da fibrinogênese e
resistência da pele (AFRATIS
et al., 2012)
CAMs Moléculas de adesão
transmembrana homofílicas
dependentes de Ca2+
(VESTWEBER, 2015)
Caderinas
Subtipos:
E-caderina (epitelial)
N-caderina (neuronal)
P-caderina (placenta)
R-caderina (retina)
Ligada diretamente a catenina
citoplasmático responsável
pelo reconhecimento das
células (ALBERGARI et al.,
2011)
Moléculas de heterodímeros
transmembrana formados
por duas subunidades α e β
associadas (ALBELDA;
BUCK, 1990)
Integrinas Ligação das células à matriz,
auxiliam na ligação das células
às proteínas, fatores de
crescimento, citocinas e
proteases que degradam a
MEC (BARCZYK et al., 2010)
Moléculas de adesão de
células vasculares
dependente de Ca2+
(KANSAS, 1996)
Selectinas
Subtipos:
P-selectina
E- selectina
L-selectina
Moléculas de adesão vascular
aos leucócitos e plaquetas
(LEY et al., 2007)
Proteínas com segmento
extracelular com um ou
mais domínios dobrados
característicos das
imunoglobulinas (WONG et
al., 2012)
Superfamília das imunoglobulinas
(IgSF):
ICAM-1
ICAM-2
VCAM
PECAM-1
N-CAM
Processo de migração dos
leucócitos dentro dos vasos
para o tecido alvo durante a
resposta inflamatória
(LOWSON; WOLF, 2009)
Fonte: Elaborado pelo autor.
27
Em cadelas, foi relatado que a presença marcante do TIL está relacionada a
diversas características de malignidade do TMC (KIM et al., 2013). Além disso, quando
foi identificado as subclassificações de linfócitos T CD4+, T CD8+, FOXP3+ (linfócito
T regulatório) presente no TIL de animais com TMC tinham baixa taxa de sobrevida
associada a uma alta razão entre células CD4+/CD8+, e pior prognóstico associado aos
linfócitos T FoxP3+ (CARVALHO et al., 2014).
2.4 Estresse oxidativo no câncer
O estresse oxidativo desenvolvido no processo inflamatório crônico pode ser o
fator mais importante nas alterações da dinâmica da resposta imune. Durante a
inflamação, leucócitos são recrutados para o local do dano, que podem desencadear
processo que leva a liberação de grande quantidade de ERO. O dano oxidativo pode levar
alterações na estrutura genética das células normais, causando instabilidade genética e
promovendo a carcinogênese (HANAHAN; WEINBERG, 2011; KAWANISHI et al.,
2017).
Em células cancerosas altamente proliferativas, a regulação das ERO é crucia para
sua sobrevivência, pois à presença de mutações oncogênicas que promovem o
metabolismo aberrante e tradução da proteína resultam em aumento das taxas de produção
de moléculas oxidantes o que pode ser deletério para a célula. As células neoplásicas
tentam neutralizar esse acúmulo de ERO por meio da regulação de sistemas antioxidantes,
aparentemente criando um paradoxo de alta produção de ERO na presença de níveis
elevados de antioxidantes (CAIRNS et al., 2011; KAWANISHI et al., 2017). Com isso,
ainda existe dúvidas envolvendo o metabolismo oxidativo das células tumorais para ser
estabelecido e o seu verdadeiro papel patogênico no câncer, podendo tornar-se alvo
terapêutico anticâncer.
2.5 Mecanismos de escape do câncer ao sistema imune
As células tumorais desenvolveram estratégias para escapar da vigilância
imunológica. Sendo assim, a imunoedição é um processo que as neoplasias utilizam para
sobreviver no hospedeiro, em que consiste de uma primeira fase de eliminação das células
tumorais que são imunogênicas, seguido por uma fase de equilíbrio, no qual coexistem
células tumorais e células imunes, por fim resultando no surgimento das variantes
malignas pouco imunogênicas capazes de evadir da resposta imune (GROSS et al., 2013;
GUILLEREY; SMYTH, 2015). Outros mecanismos de evasão do câncer é a
28
imunossupressão das células de defesa por meio da liberação de citocinas (TGF-β e IL-
18) e sua capacidade de atenuar a expressão de recetores que sinalizam a ativação das
células (HASMIM et al., 2015).
Outra via que as células tumorais utilizam para não serem atacadas por células de
defesa é ativando receptores de inibição que reconhecem na superfície celular as
moléculas de MHC classe I (CANTONI et al., 2016). Da mesma forma, células tumorais
podem expressar ou ativar moléculas presentes nas células de defesa que estão associadas
ao ponto de controle imune (immune checkpoint), como o antígeno-4 associado ao
linfócito-T citotóxico (CTLA-4), o receptor de morte programada-1 (PD-1) e seus
ligantes PD-L1/PD-L2. O CTLA-4 e PD-1 atuam limitando a eficácia da resposta
antitumoral induzindo a anergia ou a exaustão das células de defesa ativadas
(BUCHBINDER; DESAI, 2016; JOHNSON et al., 2017).
Foi demostrado que linfócitos circulantes e intratumorais de cães com diferentes
tipos de câncer expressam moléculas de CTLA-4 e PD-1/ PD-L1, como melanoma,
osteossarcoma, sarcoma histiocítico, mastocitoma, inclusive o TGM canina
(MAEKAWA et al., 2016; TAGAWA et al., 2016). Além disso, esses linfócitos podem
apresentar maior quantidade dessas moléculas em cães com câncer comparado aos
animais saudáveis e a terapia com anticorpos anti-PD-L1 pode aumentar a quantidade de
INFγ liberada por linfócitos do infiltrado tumoral (MAEKAWA et al., 2014; TAGAWA
et al., 2016).
29
3 JUSTIFICATIVA
O tumor da glândula mamária em cadelas tem bastante relevância na medicina
veterinária, pois é uma doença de grande morbidade, principalmente em fêmeas não
castradas, além de ter semelhanças fisiopatológicas com as neoplasias mamárias na
mulher. Desta forma, o estudo da patogenia que envolve essas neoplasias é essencial para
o correto emprego de técnicas que possam auxiliar no tratamento e prognósticos de tais
doenças.
O estudo do dano tecidual ligado diretamente à patogenia das neoplasias envolve
agentes oxidantes que participam do desenvolvimento, manutenção e disseminação das
células tumorais. O melhor entendimento da influência do estresse oxidativo nas células
tumorais pode auxiliar na determinação de uma terapia específica para cada tipo de
câncer. Além disso, torna-se necessário relacionar o comportamento biológico de
metástase e angiogênese dos TMC com o desequilíbrio oxirredutivo no meio intra e
extracelular das células tumorais.
30
4 HIPÓTESE CIENTÍFICA
O tumor mamário canino está relacionado diretamente com o processo
inflamatório desenvolvido no microambiente tumoral, além de promover o desequilíbrio
da homeostasia nas cadelas acometidas.
31
5 OBJETIVOS
5.1 GERAL
Estudar mediadores sistêmicos e teciduais induzidos pela progressão do tumor da
glândula mamária canina.
5.2 ESPECÍFICOS
a) Identificar a sobrevida das cadelas a partir de acompanhamento mensal até 12
meses após o procedimento cirúrgico.
b) Avaliar marcadores sistêmicos do estresse oxidativo e antioxidantes em cadelas
com neoplasias da glândula mamária e correlacioná-los com o comportamento
biológico tumoral.
c) Identificar e classificar a distribuição intratumoral do infiltrado inflamatório.
d) Avaliar o infiltrado inflamatório linfocítico intratumoral e correlacionar com a
sobrevida dos animais.
e) Identificar a subpopulção dos linfócitos T utilizando imunohistoquímica para os
marcadores moleculares CD4+, CD8+, FoxP3+.
f) Realizar a identificação da porteína de choque térmico HSP60+.
g) Avaliar a expressão da mólecula PD-L1 no TMC.
32
6 CAPÍTULO 1
Increased serum MDA in different malignant characteristic of canine mammary gland
tumors
(Aumento do MDA sérico em diferentes características malignas dos tumores das
glândulas mamárias caninas)
Periódico: Veterinary Clinical Pathology (Submetido em Out 2017)
ISSN: 0275-6382/QUALIS B1
33
Increased serum MDA in different malignant characteristic of canine mammary
gland tumors
Belarmino Eugênio Lopes-Neto1, Júlio Gil Vale Carvalheira2, Belise Maria Oliveira
Bezerra1, Tiago Cunha Ferreira1, Paulo Ricardo de Oliveira Bersano1, Diana Célia Sousa
Nunes-Pinheiro1
1Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e
Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará
(UECE), Fortaleza, CE, Brazil. 2CIBIO-InBIO, Universidade do Porto, Campus de
Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal.
CORRESPONDENCE: D. Nunes-Pinheiro [[email protected]]. Faculdade de
Veterinária. Universidade Estadual do Ceará. Campus do Itaperi. Av. Dr. Silas Munguba,
n. 1700. CEP 60.714-903. Fortaleza, CE, Brazil.
Background: Mammary gland tumors (MGT) are major neoplasm in female dogs.
Oxidative stress in cancer-bearing dogs has been described. However, the association of
oxidative stress to canine MGT is still unclear.
Objectives: This study investigates the serum levels of oxidant and antioxidant non-
enzymatic, as well as the association of clinic-pathological features and survival time in
MGT-bearing dogs.
Methods: Female dogs were divided into three groups: benign MGT (Be, n = 14),
malignant MGT (Ma, n = 20), and healthy dogs, control (Co, n = 10). Serum levels of
oxidative and antioxidant non-enzymatic parameters in MGT-bearing dogs were
determined. In order to estimate the lipidic peroxidation, it was used malondialdehyde
(MDA, nmol/mL). Clinic-pathological data of MGT-bearing dogs were realized. Data
were expressed in mean ± SD and were analyzed with P < .05.
34
Results: The levels of total proteins (P < .001), globulin (P < .001), and bilirubin (P <
.03) was higher in Ma and Be MGT than Co. MDA was increase (P < .001) in Ma (41.48
± 9.05) and Be (35.76 ± 8.63) in relation to Co (13.62 ± 2.68). All malignancy features
present high MDA levels, and the parameters as necrosis and type of tumor had difference
(P < .05) between categories. No association was observed between MDA and overall
survival time.
Conclusions: The levels of MDA and non-enzymatic antioxidants support the systemic
lipid peroxidation in MGT-bearing dogs. Thus, oxidative stress can be used as a
biomarker and potential target of new therapeutic approaches.
Key Words: Canine, mammary tumor, oxidative stress, malondialdehyde
Introduction
Several conditions cause homeostasis imbalance in animals, including chronic
diseases. Together with instability, the organism undergoes biological changes that can
lead to the formation of reactive ions and molecules, such as reactive oxygen species
(ROS).1 High concentrations of ROS have negative effects on fundamental cellular
processes because they compromise genomic stability and disrupt protein or lipid
structures. Consequently, diseases such as cancer are associated with oxidative stress in
the body, caused by this accumulation of free radicals. Thereby, the capacity to resist and
recover from states of increased oxidative stress can be a central component to avoid
cancer initiation.2
Mammary gland tumors (MGT) are the major common neoplasm in female dogs,
and malignant neoplasms are often diagnosed in this species, which can reach more than
70% of cases.3 The etiology of MGT is still not clear, being attributed to many clinical
and pathological factors, such as animal breed, age, tumor size, not or spayed female
35
dogs, the absence of hormonal receptors, and synthetic estrogens.4,5 Furthermore, genetic
changes are associating with carcinogenesis, and the oxidative stress caused by
inflammation in tumor microenvironment could be participate in both tumor regression
or progression.6,7 The increase of ROS in tumor microenvironment may induce
cytotoxicity of mammary gland cells triggering membrane damage, mutagenesis and
carcinogenesis.8 These oxidative molecules should be scavenged by antioxidant
mechanisms which protect membrane surface and cell components against peroxidation
reactions. However, efforts to prevent ROS request high consumption of antioxidants and
even deplete antioxidants production, promoting oxidative stress.9
Previous research has shown that oxidative stress in dogs is associated with
several types of cancer, including canine MGT.10,11 The disruption of antioxidant and the
increase of oxidant molecules were observed both in tumor tissues and in circulation of
female dogs with MGT.12,13,14 The association of oxidative stress to canine MGT is still
unclear, especially differences based on tumor type or factors associated with prognosis.
In this way, the purpose of this study was to evaluate the serum levels of oxidant and
antioxidant non-enzymatic, as well as the association of clinic-pathological features and
survival time in MGT-bearing dogs.
Materials and Methods
Animals
Dogs were enrolled in the experiment between January and December of 2016 at
the Veterinary Hospital of the State University of Ceará, Brazil. Forty-four female dogs
were included with dog owners consent, whose forty dogs had MGT and ten clinically
healthy intact female dogs from private kennel were used as a control. The animals were
diagnosed by clinical and pathological evaluation. All clinical data were collected and the
36
search for metastasis was accessed through chest radiographs and abdominal ultrasound.
Animals with clinical or laboratorial evidence of any other disease were excluded from
study, and those who had taken previous treatment for the mammary pathology. After
surgery, the follow-up period lasted 365 days, and overall survival was determined in
each case. Tumors were classified by histopathological analysis, according to type15 and
tumor grade,16 and clinical stage was attended following modified criteria.17,18 The
clinical stage classification consists of five stages (I to V) determined by the size of
primary tumor (T), involvement of regional lymph nodes (N) and presence or absence of
distant metastases (M). This study was approved by Committee for Ethics in Research
using Animals (CEUA-UECE), protocol 12247080-2.
Groups and serum samples
A prospective study was performed in MGT-bearing female dogs (n = 44), which
were divided into three groups: benign MGT (Be, n = 14), malignant MGT (Ma, n = 20),
and healthy dogs, control (Co, n = 10). Blood samples were collected before surgery
during preoperative evaluation to obtain serum that was aliquoted and frozen at -80° C
until use.
Biochemical estimations
In order to estimate oxidative stress, serum antioxidants non-enzymatic and
malondialdehyde (MDA) were analyzed. Lactate dehydrogenase (LDH), bilirubin,
triglycerides, uric acid, urea, total proteins, globulin, and albumin were performed using
commercial kits (Bioclin®) in biochemical automation machines (BS-120 Mindray®)
following manufacturer's methodology.
MDA was used as an oxidation biomarker, which is a product of lipid peroxidation
of cell membranes. The technique was performed to measure the MDA serum levels.19
37
Briefly, 100 mL of serum was added to 200 μL of trichloroacetic acid 30% (TCA), 200
μL of TRIS and 200 μL of 0.73% thiobarbituric acid (TBA) were mixed in glass tubes.
The solution was brought to water bath (100°C; 1 h), then it was centrifuged (3000 g, 10
min). The analysis was performed in spectrophotometer (535 nm) and results were
expressed in nmol/mL.
Statistical analysis
Statistical analysis was performed using SPSS software (version 24.0, Chicago,
IL, USA) and expressed as mean ± standard deviation (SD). Normality was assessed using
Shapiro-Wilk test, and ANOVA or Kruskal-Wallis followed by post-hoc test were used
to compare biomarker values in different groups. Survival curves were generated by the
Kaplan–Meier method and survival rates were compared using the log-rank test. The cut-
off points correspond to the mean MDA serum in the group of malignant MGT-bearing
dogs. Statistical significance was set at P < 0.05.
Results
Clinic-pathological data of MGT-bearing dogs are described at Table 1. The mean
age of Be and Ma groups were 9.48 ± 1.35 and 9.89 ± 2.87 years old, respectively. Of
thirty-four female dogs with MGT, the histopathological diagnoses were grouped as
adenomas (Ad; n = 6), benign mixed tumors (BMT; n = 8), carcinomas (4 tubulo-
papillary, 2 solids, 1 anaplasic) (Ca; n = 7), complex carcinomas (CoC; n = 3), carcinomas
in mixed tumors (CMT; n = 7), and carcinosarcoma (CSC; n = 3) tumors. Among the
clinic-pathological characteristics of malignant tumors, there was a predominance of
absence of ulceration (80%), necrosis (75%), and the mean size of tumors was higher than
4 cm. The Ma data showed vascular invasion (25%), involvement of regional lymph
38
nodes (20%), distance metastases (15%), prevalence low-grade of tumor (55%), and
clinical staging II (30%).
Serum levels of biochemical analyses, antioxidant non-enzymatic and oxidative
parameters in MGT-bearing dogs were displayed in Table 2. Both Be and Ma MGT
groups presents higher (P < .001) concentration of total proteins and globulins than
control group. LDH was increase in Ma but not differ between groups. Be and Ma groups
presents bilirubin concentration more elevated (P = .03) in relation to Co, but was not
observed difference between Be and Ma MGT. On the other hand, Be and Ma MGT-
bearing dogs had appeared lower triglycerides levels than Co, but only Be showed
significance (P = .024) in relation to Co. Urea and uric acid did not presents difference
between groups (Table 2).
The serum MDA presented higher levels in Be (P = .002) and Ma (P = .001) than
Co, but similar between groups with MGT (Be and Ma). In Ma group, the MDA detection
was higher (P = .035) in tumor with necrosis compared with non-necrosis. Besides, MDA
levels were significantly lower (P = .009) in Co and CMT than other malignant MGT
(Table 3). The MDA serum concentration was not associated with skin ulceration, tumor
size, and tumor grade. Concerning follow-up study, no statistical association was
observed between different MDA serum levels and overall survival times (Figure 1).
Furthermore, analysis of MDA mean concentration of tumor skin ulceration,
tumor necrosis, tumor size, tumor grade, and others clinic-pathological features reveal a
two-fold increase compared with MDA mean of healthy animals (Table 3).
39
Table 1. Clinic-pathological data of MGT-bearing dogs.
Groups
Be
(n=14)
Ma
(n=20)
Histological Classification Ad BMT Ca CoC and CMT CSC
nº 6 8 7 10 3
Age 9.5 (± 1.64) 9.37 (± 1.06) 10.14 (± 2.97) 9.2 (± 1.62) 10.33 (± 4.04)
Skin ulceration (n°) - - No (5), Yes (2) No (8), Yes (2) No(3), Yes (0)
Tumor necrosis (n°) - - No (3), Yes (4) No (2), Yes (8) No (0), Yes (3)
Vascular invasion (n°) - - No (4), Yes (3) No (9), Yes (1) No (2), Yes (1)
T; cm 1.85 (± 0.99) 1.61 (± 1.03) 4.64 (± 1.91) 5.11 (± 2.07) 3.26 (± 2.56)
N (n°) - - No (5), Yes (2) No (9), Yes (1) No (2), Yes (1)
M (n°) - - No (6), Yes (1) No (9), Yes (1) No (2), Yes (1)
TNM clinical stage (n°) - -
I (1), II(2), III(1),
IV(2), V (1)
I (2), II (4), III (2), IV
(1), V (1)
III (1), IV (1), V (1)
Tumor grade (n°) - - I (4), II (2), III (1) I (7), II (3) II (2), III(1)
T: size of the primary tumor; N: involvement of regional lymph nodes; M: presence of distant metastases;
Ad: Adenoma; BMT: benign mixed tumors; Be: benign tumors; Ma: malignant tumors; Ca: carcinoma, CoC:
complex carcinoma, CMT: carcinoma in mixed tumor, CSC: carcinosarcoma; n°: numbers
40
Table 2. Serum levels of oxidative and antioxidant non-enzymatic parameters in MGT-
bearing dogs.
Groups
Biomarker Co Be Ma P-value
Mean ± SD (Min–Max) Mean ± SD (Min–Max) Mean ± SD (Min–Max)
Total proteins (g/dL) 6.36a ± 0.65 (5.73–7.31) 7.83b ± 0.68 (6.55–8.74) 8.27b ± 0.97 (7.59–11.30) .001
Albumin (g/dL) 3.21 ± 0.27 (2.81–3.62) 2.77 ± 0.74 (1.23–3.62) 2.74 ± 0.65 (1.25–3.67) .170
Globulin (g/dL) 3.14a ± 0.78 (2.36–4.35) 5.22b ± 0.82 (3.54–6.89) 5.23b ± 1.01 (4.01–9.28) .001
Uric acid (mg/dL) 1.59 ± 0.59 (0.18–2.54) 0.73 ± 0.42 (0.24–1.58) 1.10 ± 0.34 (0.14–2.90) .309
Urea (mg/dL) 53.45 ± 16.52 (31.40–78.95) 50.16 ± 21.43 (29.05–97.55) 44.92 ± 20.71 (11.30–98.25) .252
Bilirrubin (mg/dL) 0.16a ±0.06 (0.10–0.28) 0.30b ±0.16 (0.18–0.44) 0.40b ± 0.2 (0.25–0.86) .030
LDH (U/L) 151.25 ± 90.36 (63.00–318.00) 193.02 ± 67.29 (30.50–420.50) 239.25 ± 108.8 (40.50–540.50) .280
Triglycerides (mg/dL) 227.26a ± 62.53 (139.05–289.46) 142.78b ± 69.67 (32.60–267.50) 159.71ab ± 62.37 (43.45–05.60) .024
MDA (nmol/mL) 17.36a ± 7.68 (9.70–32.36) 35.76b ± 8.63 (25.20–52.65) 41.48b ± 9.05 (24.60–60.40 .001
a,b Groups with distinct letters indicate statistical differences. (P < .05).
Figure 1. Relationship between two different levels of MDA and overall survival time (P
= .710).
41
Table 3. Association of serum MDA levels with clinic-pathological parameters in
malignant MGT-bearing dogs.
Clinic-
pathological
parameter
Number of
samples
analyzed
MDA concentration
(nmol/mL)
Mean ± SD (Min–Max)
Fold
increase† P- value
Histologic type
Ca 7 47.4a ± 8.27 (38.85–60.40) 2.73
CoC and CMT 10 35.76b ± 5.45 (24.60–43.70) 2.05 .009
CSC 3 43.24b ± 7.28 (39.03–51.65) 2.49
Tumor grade
I 11 43.26 ± 9.55 (30.95–60.40) 2.49
II 8 36.82 ± 5.24 (24.60–40.53) 2.12 .169
III 1 48.80 2.81
Skin ulceration
No 16 41.3 ± 6.67 (30.95–53.70) 2.37 .733
Yes 4 39.62 ± 8.10 (24.60–60.40) 2.28
Tumor necrosis
No 5 38.7a ± 7.31(24.60–53.70) 2.23 .035
Yes 15 47.73b ± 8.77 (39.03–60.40) 2.75
T (cm)
T1 <3 cm 5 44.19 ± 6.02 (37.75–51.65) 2.55 T2 ≥3 cm and
<5 cm 8 39.57 ± 9.36 (24.60–53.70) 2.28 .635
T3 ≥ 5 cm 7 40.24 ± 9.42 (30.95–60.40) 2.32
N
No 16 42.75 ± 8.19 (31.85–60.40) 2.46 .155
Yes 4 36.8 ± 8.24 (24.60–48.80) 2.12
M
No 17 41.19 ± 8.92 (24.60–60.40) 2.37 .732
Yes 3 38.94 ± 0.12 (38.85–39.03) 2.24
TNM clinical stage
I 3 43.03 ± 6.28 (37.75–51.65) 2.48
II 6 42.27 ± 8.29 (31.85–53.70) 2.43
III 4 44.62 ± 13.85 (34.45–60.40) 2.57 .683
IV 4 36.35 ± 9.13 (24.60–48.80) 2.09
V 3 38.94 ± 1.52 (38.85–39.03) 2.24
*ANOVA test (P < 0.05). Parameter with distinct superscript letters indicate statistical
differences among them (Duncan test, P < .05). † Fold increase represents the augment
of MDA mean concentration relative to MDA mean of control group. T: size of the
primary tumor. N: involvement of regional lymph nodes. M: presence of distant
metastases. Co: control group. Ma: malignant tumors group. Ca: carcinoma, CoC:
complex carcinoma, CMT: carcinoma in mixed tumor, CSC: carcinosarcoma.
42
Discussion
In veterinary medicine, there is an increasing interest in biomarkers as a tool for
diagnosis and monitoring of neoplastic diseases. These tests should be sensitive and
specific with low cost to attend in veterinary clinical oncology.20 Oxidative stress
biomarkers have been pointed as a parameter to access the homeostasis imbalance
produced in dogs with cancer 21,11 To assess the oxidative stress in female dogs with MGT,
serum MDA was used as a biomarker once it is a product of lipid peroxidation in cell
membranes disrupted by ROS activity. Thus, it was report in the present study that MDA
levels were significantly higher in MGT-bearing dogs compared to healthy dogs (Table
2). Moreover, MDA was higher in female dogs with malignant MGT than the group with
benign tumors.
Figure 2. MDA levels in dogs with different MGT types. Co: control group; Ad:
Adenoma; BMT: Benign Mixed Tumor; Ca: carcinoma; CoC: complex carcinoma; CMT:
carcinoma in mixed tumor; CSC: carcinosarcoma. a,b,c Differet letters are significant (P =
.05). Bars indicate standard deviation.
17.36
39.34 38.84
47.41
35.76
43.24
a
bc bc
c
b
c
43
Dogs with different types of cancer have higher serum MDA levels compared with
healthy dogs.10 Likewise, other studies in dogs with mammary tumors reported the
increased lipid peroxidation as evidenced by an increase of thiobarbituric acid-reactive
substances (TBARS) levels.13 TBARS is a byproduct of lipid peroxidation, and similar to
the MDA showed significantly higher levels in MGT tissue than those in the normal
tissue, indicating this trend of systemic and tissue lipid peroxidation associated with
oxidative stress in MGT-bearing dogs. 9,12,13
Researches are linking the high ROS levels produced by cancer cells with the
enhancement of tumor growth and metastasis.7,8 Our study demonstrated the high levels
of MDA in Ma group compared to Be, although group medians were not statistically
significantly different. Besides, all malignant clinic-pathological parameters had an
increase of MDA levels compared with control dogs, suggesting a link between MGT
malignancy and oxidative stress.
Along with increase in lipid peroxidation, high-grade malignant MGT presented
higher serum level of oxidative stress biomarkers such as nitric oxide,11,22 similar to the
present study which showed higher serum MDA levels in female dogs with high-grade
tumor. The serum MDA levels in complex carcinoma and carcinoma in mixed tumor were
lower than other tumors (Figure 2) which may be a relationship between of oxidative
stress growth and tumor malignancy once the anaplasic carcinoma and carcinosarcoma
have worse prognosis to animals.3
Several preventive medicine had purposed that lipid peroxidation levels in breast
ductal cells may represent a promising cancer biomarker to detect, through non-invasive
methods such as nipple fluid aspirate sampling, for example, women at high risk for breast
cancer.23 Although, in cohort study with invasive breast cancer in women did not was
44
associated with oxidative stress and low-grade inflammation.24 The results reported in the
present study did not reveal a relationship between the increase in the serum levels of
MDA and lower overall survival time of Ma group (Figure 1).
Biochemical estimation of lipids can be related to total lipid peroxidation, and in
this work, the triglycerides have shown lower levels in MGT-bearing dogs, unlike the
previous study that did not found serum cholesterol and triglycerides decrease.13 Another
biomarker usually used to reflect the disruption of cells is serum LDH, an enzyme
required for anaerobic glycolysis.25 LDH isoenzymes may be increased in human and dog
breast cancer, and growth of serum levels in canine MGT presented a positive relationship
with tumor staging and disease evolution.26 Although serum LDH concentration between
groups did not show difference, there was a tendency of a higher serum concentration in
MGT-bearing dogs.
Uric acid is a final oxidation product of purine catabolism and may act as a non-
enzymatic antioxidant with the ability to scavenge ROS. Although the uric acid levels did
not present variety between the experimental groups in this work, it was suggested that
antioxidant activity happen only in normal level in the circulation.27 However, the
hyperuricemia may be related to several disorders, such as cardiovascular and renal
diseases, metabolic syndrome, diabetes, and tumor lysis syndrome in cancer patients.28,29
Bilirubin is another non-enzymatic antioxidant from hemoglobin metabolism that
can bind to albumin and prevent oxidation of fatty acids during protein exposure to lipid
peroxidation initiators.30 Data of bilirubin presented higher levels in MGT animals
compared to the healthy group, what could explain the effort of the organism to prevent
oxidative stress producing more antioxidants.
Albumin is an abundant protein in the extracellular fluid and assists on the
transport of substance included free fatty acids in the blood. The non-enzymatic
45
antioxidant albumin is capable of binding ions such copper and iron, inhibiting lipid
peroxidation and formation of the hydroxyl radical and hydrogen peroxide.31 MGT-
bearing dogs presented lower serum albumin concentration, although without a
significant difference. Thus, agreeing with studies that showed hypoalbuminemia in
animals with advanced MGT stage and metastasis, compared to healthy or without
metastasis.32,33,34
Hyperproteinemia and hyperglobulinemia were markedly higher in serum
globulin and serum total proteins levels of Be and Ma groups corroborating with works
in dogs with mammary carcinoma.35,36 Different proteins can be identified in cancer, and
especially gamma globulins participate in defense against cancer and, paradoxically, in
several paraneoplastic syndromes.37 A recent study suggested that higher globulin
concentration, such as acute phase proteins, and immunoglobulins have been linked to an
immune reaction glomerulopathy in malignant MGT-bearing dogs.38
The homeostasis imbalance in animals with cancer demands a strict monitoring,
and oxidative stress is one of the issues which contribute to ongoing disease. Thereby,
oxidants and antioxidants are substantial parameters to consider as biomarkers in canine
MGT. In this way, our results demonstrated the oxidative stress imbalance on serum
MDA levels and non-enzymatic antioxidants biomarkers of MGT-bearing dogs.
Therefore, systemic exacerbation of lipid peroxidation has a markable role in instability
of homeostasis in canine mammary cancer. Besides, our results suggest further studies
are necessary to confirm the participation of oxidative stress in the progression of MGT
phenotype and subsequently new therapeutic approaches.
Acknowledgments
Funding for this study was provided by the Coordination for the Improvement of Higher
Education Personnel (CAPES). FINEP.
46
Disclosure: The authors have indicated that they have no affiliations or financial
involvement with any organization or entity with a financial interest in, or in financial
competition with, the subject matter or materials discussed in this article.
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50
7 CAPÍTULO 2
CD4+, CD8+, FoxP3+ and HSP60+ expressions in cellular infiltrate of canine
mammary carcinoma in mixed tumor
(Expressão de CD4+, CD8+, FoxP3+ e HSP60+ no infiltrado celular em carcinoma
mamário em tumor misto canino)
Periódico: Acta Scientiae Veterinariae (Publicado em Nov 2017)
ISSN: 1679-9216/QUALIS B1
51
CD4+, CD8+, FoxP3+ and HSP60+ expressions in cellular infiltrate of canine mammary
carcinoma in mixed tumor
Belarmino Eugênio Lopes-Neto1, Stephanie Caroline Bezerra Souza1, Lúcio Marinho
Bouty2, Glauco Jonas Lemos Santos1, Emanuele Silva Oliveira3, José Cláudio
Carneiro de Freitas1 & Diana Célia Sousa Nunes-Pinheiro1
1Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e
Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará
(UECE), Fortaleza, CE, Brazil. 2Hospital Veterinário, FAVET, UECE, Fortaleza, CE,
Brazil. 3Laboratório de Genética Molecular, Universidade Federal do Ceará (UFC),
Fortaleza, CE, Brazil. CORRESPONDENCE: D. Nunes-Pinheiro
[[email protected]]. Faculdade de Veterinária. Universidade Estadual do Ceará.
Campus do Itaperi. Av. Dr. Silas Munguba, n. 1700. CEP 60.714-903. Fortaleza, CE,
Brazil.
ABSTRACT
Background: Cancer is a complex process that receive many influences of the tumor
microenvironment. The participation of immune system cells and proteins in tumor
microenvironment is not yet completely understood. Thus, the aim of this study was to
evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine
mammary carcinoma in mixed tumor (CMCMT).
Materials, Methods and Results: Bitches (n = 20) were selected after Canine mammary
tumor (CMT) diagnosis and data were achieved throughout clinical-pathological
information. Clinical staging was evaluated and tumor biopsies were processed by
histology and cellular infiltrate was performed according criteria and grade. Survival
curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by
52
Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8,
anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3.
Clinical-pathological relationship was analyzed using Spearman correlation. This study
was approved by the Committee for Ethics in Research using Animals (CEUA-UECE),
protocol 12247080-2. Our data showed a mean age of 9.3 years old, the size of tumors
presented more than 5 cm (50%), which were located in inguinal mammary glands (70%),
and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both
in peri and intratumoral regions, dispersed multifocally with moderate intensity and
lymphocytes were the major populations found into tumors (n = 826 ± 220). In
relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ =
0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed
negative correlation of neutrophil (ρ = -0.1) and macrophage (ρ = -0.38). CMT presents
moderate lymphocytic infiltrate (< 800 lymphocytes), shows higher (P = 0.01) survival
rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes). FoxP3+ showed
lower intensity while CD4+ and CD8+ expression were concentrated surrounding of
lymphocytic infiltrate tumor region. HSP60+ was observed in the inflammatory and
tumor cells.
Discussion: Our data are according to a greater risk to the development of the breast
tumor in old bitches, not castrated and before or after puberty, as well as the use of
contraceptives based on progesterone and estrogen. In relation to size of tumor, these
findings reinforce that there is a relationship of tumor size with a higher malignancy grade
and with a worse prognosis. The predominant tumor location was in the inguinal breasts
that is attributed to the high activity of the mammary glands to hormonal stimuli. CMT
with low clinical staging are associated with greater overall survival of affected bitches.
In relation to tumor microenvironment, it has been reported that heterogeneous
53
populations of the immune system cells often infiltrate the mammary tumors, whose
lymphocytes are the main cells. It is suggested that tumor lymphocytosis may be
necessary for malignant behavior of the tumor microenvironment. On the other hand,
macrophages and neutrophils play an important role that may favor or inhibit the tumor
cells development in the tumor microenvironment. In our work, CD4, CD8 and FoxP3
labeling were distributed in peri and intratumoral regions, and consequently, these
markers can be used as prognostic for CMT, as well as being a potential target for
anticancer therapies. This is the first work that presents results about the HSP60+
participation in CMT, however this data needs further investigation. HSP60 participates
as a potent activator of the immune system through its peptides and other HSP types were
studied in mammary carcinomas in bitches and presenting results that indicate the
association of these proteins with the carcinogenesis process.
Keywords: canine mammary glands, carcinoma in mixed tumor, T-lymphocytes, Heat
Shock Protein, immunolabeling.
INTRODUCTION
The etiology of canine mammary mixed tumor (CMMT) involves multifactors but
is still not well understood [17]. Cancer is a complex process controlled by the activation
of oncogenes, silencing of tumor suppressor genes and the lack of control of epigenetic
events that occur within the cell. In addition, the influences of the tumor
microenvironment may participate in both, the progression process and tumor regression
[13].
Inflammation is an important process of damaged tissues. The chronic
inflammation may facilitate the tumor progression from cytokines and chemokines
54
released by the microenvironment cells, including leukocytes [8]. The role of leukocytes
in the tumor microenvironment is not yet fully understood, and several efforts have been
made to understand the role of these cells in tumor development. Studies have shown that
the lymphocytes presence in mammary mixed tumor (MMT) may be involved in a better
or worse prognosis for the patient [1,10].
Under stress conditions, the organism undergoes alterations in the cellular
metabolism, leading to the formation of molecules that assist in both the repair and the
new proteins production. The heat shock proteins (HSP) are a large family of molecular
chaperon proteins [5]. Increased HSP in damaged cells may also aid in cell integrity, since
it inhibits the apoptosis [6], and overexpression of the different proteins in this group is
related to a poorer prognosis of the patient, including MMT [22].
Thus, the objective of this study, was to evaluate the infiltrate cellular,
subpopulations of T-lymphocytes (CD4+, CD8+ and FoxP3+) and HSP60 of canine
mammary carcinoma in mixed tumor (CMCMT).
MATERIALS AND METHODS
Animals
For the accomplishment of this work, twenty bitches with varied weight and age,
affected by CMCMT were used. The animals were attended at the Unidade Hospitalar
Veterinária (UHV) of the Universidade Estadual do Ceará (UECE), from January to June
of 2016. All the owners were informed about the study procedures, signing a Free and
Informed Consent Form. This study was approved by the Committee for Ethics in
Research using Animals (CEUA-UECE), protocol 12247080-2.
55
The animals were diagnosed by clinical and radiological evaluation and confirmed
by cytological and histopathological analysis, according criteria [4] and tumor grade [9].
Initially, the tumors were submitted to macroscopic analysis, then tumor fragments were
collected and submitted to classical histology (H & E) and immunohistochemical
analysis. Furthermore, all animals clinical data were also collected for clinical staging
based on the described system [20] and follow up was made during one year (365 days).
Cellular Infiltrate and Immunohistochemical Analysis
Cellular infiltrate analysis was performed for its location, in peri and/or
intratumoral; its distribution as, focal, multifocal or diffuse; its intensity, in absent (0),
mild (1), moderate (2) or intense (3), and quantified in eight random fields, avoiding areas
near necrosis (ECLIPSE E200, 400x magnification)1 [10]. Thus, two intervals of the
lymphocytic infiltrate in CMT were used for data analysis, considering moderate (< 800
lymphocytes) and intense quantity (≥ 800 lymphocytes).
Immunohistochemical analysis were conducted in tumor sections for CD4+,
CD8+, FoxP3+ and HSP60+. For this, 5 µm sections were mounted on silanized glass
slides and subjected to antigen retrieval process (EnVision TMFLEX Target Retrieval
Solution High pH Code DM828)2 or low pH (Code DM829)2 for 20 min at 97ºC using
the Dako pre-treatment (PT) link module2. The endogenous peroxidase activity was
inhibited by peroxidase block2 for 5 min, and slides received the anti-human CD4, anti-
human CD8, anti-human FoxP3 and anti-human HSP60 murine monoclonal antibody3
diluted 1:100 and incubated for 1h, at room temperature. Then, slides were washed three
times in phosphate buffered saline (PBS, pH 7.2), and then incubated with the reagent
polymer (EnVision TMþ Dual LinkSystem/HRP)2 for 30 min at room temperature and
finally diaminobenzidine (DAB)2 for 10 min. The sections were counterstained with
56
Mayer's hematoxylin2 and observed by optical microscopy atributting the scores absent
(0), mild (1), moderate (2) or intense (3). In order to obtain the scores, all slides were
analyzed by two observer and compared to control group.
Statistical Analysis
Data were previously subjected to Grubbs test for outliers exclusion. Then, the
Kolmogorov-Smirnov test and ANOVA for homoscedasticity and homogeneity
evaluation were used. The changes observed in the macroscopic and microscopic
analyzes and the location, distribution and intensity of the cellular infiltrate were
expressed as a percentage. Cellular infiltrate analysis was expressed as mean ± standard
deviation. The correlation between the inflammatory infiltrate and the tumor grade was
analyzed using Spearman correlation test. In addition, survival curve were generated by
Kaplan-Meier estimation method and the two intervals of the lymphocytic infiltrate were
compared by Log-Rank followed Chi-Square χ². Statistical significance was considered
at P < 0.05 and analyses were performed using the software SPSS4. The expressions of
CD4+, CD8+, FoxP3+ and HSP60+ were performed semi-quantitatively, being classified
into scores (0 to 3).
RESULTS
In this study it was observed that 60% of bitches with CMT were Poodle breed,
and the others were non-defined breed, which a higher prevalence of malignancies than
benign ones was observed. The mean of age was 9.3 years, whose bitches were not
castrated, four of them had at least one gestation and two received an injectable
contraceptive. Macroscopically, it was observed that 50% of the tumors had a size bigger
than 5 cm, 30% presented size between 3-5 cm and the remainder with nodules smaller
57
than 3 cm. The predominant tumor location was in the inguinal breasts (70%) and the rest
in the abdominal breasts. In seven animals, there was a greater occurrence of the tumor
mass in the right antimer. Ulceration was evident in 20% of the tumors and in 60% of
cases they had only one tumor formation in the breast, while 40% had two to five
mammary chain formations.
Figure 1. Composition of inflammatory infiltrate in CMT. Lymphocytes (L), Neutrophils
(N), Macrophages (M) and Plasma Cells (P). Cell count performed on eight fields (400X).
Results were expressed in mean ± standard deviation.
All samples evaluated presented histological characteristics of CMT. Foci or
nodules of epithelial cells with high pleomorphism and atypical mitosis were seen in the
middle of a benign mixed tumor. It was observed that the majority of tumors had a tubular
formation index ranging from 10% to 75%, moderate nuclear pleomorphism and mitotic
index of 9 to 16 mitosis in 10 high-power field (HPF). The CMT had a grade I histological
grade in 70% of the cases and 30% were grade II. It was also observed that only 30% of
the cases had areas of tumor necrosis. In the present study, regional metastasis was
identified in one animal, and in none of the cases was identified the presence of distant
metastases.
58
Data of the inflammatory infiltrate were presented in table 1. It was observed that
most of the cells were distributed both in the peri and intratumoral regions (50%, 70%
and 46% for lymphocytes, macrophages, neutrophils, respectivelly), dispersed
multifocally (58%, 63% and 50% for lymphocytes, macrophages and plasma cells) with
moderate intensity (70% and 52% for lymphocytes and macrophages) (Table 1).
Lymphocytes were the major populations found in tumors (n = 826 ± 220), followed by
macrophages, neutrophils and plasma cells (Figures 1, 2A and 2B).
Evaluating the correlation between the type of inflammatory infiltrate with the
CMT grade, it was observed that both lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22)
had a mild positive correlation. The intense lymphocytes infiltration into mamary
carcinomas in bitches was associated with histological alterations of aggressiveness, as
well as presenting higher (ρ = 0.045) lymphocyte infiltration in tumors with a high
histological grade compared to low grade. The correlations between the neutrophil and
macrophage population with the CMT grade were negative (ρ = -0.1 and ρ = -0.38,
respectively).
When analyze the animals overall survival in relation to the two intervals of
lymphocytic infiltrate, it was observed CMT with moderate infiltrate (< 800 lymphocytes)
showed significantly higher (P = 0.008) survival as compared to those with intense
lymphocytic infiltrate (≥ 800 lymphocytes) (Figure 3). The lymphocytic infiltrate
reported in the present study showed animals with intense lymphocytic infiltrate (≥ 800
lymphocytes) in CMT.
59
Table 1. Distribution, location and intensity of cellular infiltrate in tumor environment.
Data were expressed as percentage.
Cellular Infiltrate
Lymphocytes Macrophages Neutrophils Plasma Cells
Lo
cali
zati
on
Peritumoral - 10 14 50
Intratumoral 50 20 40 30
Peri/Intratumoral 50 70 46 20
Dis
trib
uti
on
Focal 22 27 70 50
Multifocal 58 63 30 50
Diffuse 20 10 - -
Inte
nsi
ty
Mild 13 38 56 90
Moderate 70 52 44 10
Intense 17 10 - -
The results of immunostaining for CD4 and CD8 in CMT are presented in figure
2. The CD4 marker was distributed in the tumor infiltrate in both peri and intratumoral
regions. In addition, it was observed that CD4 was more concentrated in the areas
surrounding the malignant tumor region, presenting well-defined cellular marking of
moderate to intense staining (Figure 2C).
60
Figure 2. Presence of inflammatory infiltrate in mammary carcinoma in mixed tumor
(MCMT) of bitches and CD4+, CD8+, FoxP3+ and HSP60+ expressions in cellular
infiltrate. Where, (A) Lymphocytes associated with a bone matrix, (B) Macrophages
between bone marrow and cartilaginous matrix, (C) CD4+ T lymphocytes moderately
immunostained, (D) CD8+ T lymphocytes moderately immunostained, (E) FoxP3+ T
regulatory lymphocytes mildly immunostained and (F) HSP60+ strongly immunostained
in tumor and inflammatory cells.
61
Similar to CD4+ labeling, the CD8+ labeling was distributed in both peri and
intratumoral regions, especially around the areas of carcinoma, from moderate to intense
labeling, but with a higher number of cells compared to CD4+ labeling (Figure 2D).
Figure 3. Survival rates of animals with MCMT categorized in two intervals of the
lymphocytic infiltrate (< 800 and ≥ 800 lymphocytes).
When evaluating the infiltrating leukocytes profile in mamary carcinoma in
bitches, it was demonstrated that animals with CMT without nodal metastasis had a higher
(P < 0.05) amount of T lymphocytes compared to CMT with nodal metastasis, and that
the predominant population in these cases was CD8+ T lymphocytes [10]. On the other
hand, animals with nodal metastasis presented higher (P < 0.05) CD4+ T lymphocytes
and higher (P < 0.05) CD4+/CD8+ ratio. In the present study, it was observed that FoxP3+
labeling were distributed in the intratumoral region, with a mild staining in the
inflammatory infiltrate at lymphocytes (Figure 2E).
HSP60+ immunostaining was observed in the cytoplasm of tumor cells, as well as
in the different cell populations present in the inflammatory infiltrate, with moderate to
intense intensity, independently of the cellular type observed (Figure 2F). This profile
62
was not observed in control samples, with only mild immunostaining in cells of the
mammary alveoli, not being observed in myoepithelial cells or in the cells of intra- and
extra-lobular ducts.
DISCUSSION
In recent years, oncology specialty has been gaining great prominence in
veterinary medicine. This was due to the great advances in research and in the diagnosis
of tumors subtypes, especially those that affect pets. Among the main ones, we can
highlight the mammary tumors, which present a large number of reports in the veterinary
clinic, especially in older bitches. One of the main challenges for veterinary oncologists
is the identification of the mammary tumor and its aggressiveness grade.
In this study it was observed that 60% of bitches with CMT were Poodle breed,
and the others were non-defined breed, of which a higher prevalence of malignancies was
observed. Our data corroborate with works previously described [7,15]. Age, animal
breed and inflammatory infiltrate in the tumor environment are good markers for
assessing tumor malignancy [3,7]. The mean of age was 9.3 years, whose bitches were
not castrated, four of them had at least one gestation and two received an injectable
contraceptive. Based on this, it is possible to emphasize that these factors contribute to a
greater risk of development of breast tumor in old bitches, not castrated and before or
after puberty, as well as the use of contraceptives based on progesterone and estrogen
[12,24].
In relation to CMT location, our results are according with others works, which
reported a higher frequency of CMT in the abdominal and inguinal regions, in relation to
the thoracic region [23]. This is attributed to the high activity of the mammary glands to
63
hormonal stimuli, such as estrogen, in addition to having a greater amount of parenchyma
to be stimulated [18]. Our data reinforce that there is a relationship of tumor size with a
higher malignancy grade and with a worse prognosis [24].
Regarding the clinical staging, our data variated from I to IV stage, besides it was
identified metastasis. CMT was characterized according to World Health Organization
[20]. CMT with low clinical staging are associated with greater overall survival of
affected bitches [19]. One characteristic of cancer that provides worse clinical staging is
the metastasis formation, besides being linked directly to a poor prognosis. Bitches
identified with regional or distances metastasis present lower overall survival [19].
Therefore, clinical staging provides important data to aid in the treatment and prognosis
of affected animals, and that many CMT are classified with low staging, indicating a less
aggressive behavior of this histological type of carcinoma.
In the histological findings (Figure 1 and Table 1), CMT had predominantly grade
I. It has been reported that bitches with CMT presents a histological grade from low to
moderate tumor [7,10]. This indicates that much of the malignant mixed tumors will
hardly be of high grade. These data corroborate with research that evidences CMCMT as
a histological type with a better prognosis for the animals compared to other types of
mammary carcinoma [4].
Regarding the inflammatory infiltrate in CMT, it was observed that most of the
cells were distributed both in the peri and intratumoral regions and lymphocytes were the
major populations found in tumors (Table 1 and Figures 1, 2). Mammary tumors are often
infiltrated by heterogeneous populations of the immune system cells, whose lymphocytes
are the main cells found in the tumor microenvironment [16]. It has been reported that
CMT presents moderate-intensity multifocal inflammatory infiltrate consisting
64
predominantly of lymphocytes, and that there was no significant difference when
compared to the peripheral and intratumoral areas in relation to the morphological and
morphometric characteristics in the different inflammatory infiltrates [10,21]. The intense
lymphocytes infiltration into CMT was associated with histological alterations of
aggressiveness, as well as presenting higher (ρ = 0.045) lymphocyte infiltration in tumors
with a high histological grade compared to low grade [16]. Therefore, it is suggested that
tumor lymphocytosis may be necessary for malignant behavior of the tumor
microenvironment. The correlations between the neutrophil and macrophage population
with the CMT grade were negative. The macrophages and neutrophils in the neoplasias
play an important role in the stimulation of the immune system, and depending on the
cytokine profile produced, the tumor microenvironment may favor or inhibit the tumor
cells development [8].
When analyzing the animals overall survival with two intervals of the lymphocytic
infiltrate, it was observed that the CMT have worse overall survival, fortifying previously
studies who showed bitches with mammary tumor. Our data are according to results that
the carcinomas with high lymphocytic infiltrate exhibited shorter overall survival [2].
Besides, the high lymphocytic infiltrate can be associated with other poor prognostic
factors, such as high histological grade, lymphatic invasion, and necrosis [16].
In the present study, the immunostaining for CD4+ and CD8+ T lymphocytes was
evaluated in the inflammatory infiltrate of CMT. Both, CD4+ and CD8+ labeling were
distributed around the areas of carcinoma, but predominantly CD8+ labeling. Our data are
according with results which demonstrated that CD8+ T lymphocytes predominant
population in metastesis cases [10]. Furthermore, the composition of lymphocyte
infiltrate in CMT with high proportion of CD4+ T lymphocytes and low CD8+ T
lymphocytes have a shorter survival time [16]. These results reinforce those found in the
65
present study, suggesting the possibility of using these cells as prognostic biomarkers for
CMT, as well as being a potential target for anticancer therapies.
Another evaluated immunostaining was Forkhead box P3+ (FoxP3+). This marker
is a transcription factor that is closely linked to regulatory T lymphocytes (Treg) activity
and is responsible for characterize this subpopulation of T-lymphocytes in tissues [23].
In the present study, it was observed that FoxP3+ labeling were distributed in the
intratumoral region and in the inflammatory infiltrate. The increase of Treg in the tumor
microenvironment may be related to factors of poor prognosis of mammary carcinomas,
such as high histological grade, lymphatic invasion and necrosis, and lower survival rates
for animals [2,16]. In addition, it has been suggested that Treg play a key role in the
development of these tumors, since it would be linking immune suppression with tumor
angiogenesis, together in the same biological program [1]. Also has been reported the
increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors [14].
Heat shock proteins (HSP) belong to a large family of molecular chaperones with
the ability to interact reversibly with other proteins, aiding in the formation, folding and
trans-membrane transport, besides assisting in the apoptosis process [5]. In our work,
HSP60+ immunostaining was observed in the inflammatory infiltrate, in mammary
alveoli cells, not being observed in myoepithelial cells or lobular ducts cells. It has been
demonstrated that HSP60 participates as a potent activator of the immune system through
its peptides [11]. Thus, HSP60 together with their peptides could somehow induce a better
response of lymphocytes to CMT. On the other hand, other HSP types, such as HSP27,
HSP72 and HSP90 were studied in mammary carcinomas in bitches and presenting results
that indicate the association of these proteins with the carcinogenesis process [22].
However, more studies are needed to understand the involvement of HSP60 in CMT.
66
CONCLUSIONS
The present study characterized the inflammatory infiltrate in CMT,
demonstrating T-lymphocytes as the predominant population. In addition, it was observed
that the amount of lymphocytes may be associated with tumor malignancy criteria. CD4+,
CD8+ and Foxp3+ markers are present in CMT and their distribution may be associated
with the prognosis of bitches. This is the first work that presents results about the HSP60+
participation in CMT, however this data needs further investigation.
MANUFACTURERS
1Nikon, Minato, Tokyo, Japan.
2Dako® Denmark AS. Glostrup, Denmark.
3Santa Cruz Biotechnology®. St. Louis, MO, EUA
4GraphPad Prism 5.0®, San Diego, California, USA.
Funding. We would like to thank the CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico, Brazil) and FINEP (Empresa Financiadora de Estudos e
Projetos, Brazil) for financial support.
Ethical approval. All procedures and animal care were approved by the Ethical
Committee in Animal Use of the State University of Ceará, Brazil (CEUA, UECE),
Protocol no. 12247080-2.
Declaration of interest. The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
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67
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8 CAPÍTULO 3
Relationship between PD-L1 expression and tumor-infiltrating lymphocytes in
canine mammary tumor
Relação entre a expressão de PD-L1 e infiltrado linfocítico tumoral no tumor de mama
em cadelas
Periódico: BMC Cancer (Submetido em Nov 2017)
ISSN: 1471-2407/QUALIS A1
71
Relationship between PD-L1 expression and tumor-infiltrating lymphocytes in
canine mammary tumor
Belarmino Eugênio Lopes-Neto1,2*, Diana Célia Sousa Nunes-Pinheiro1, Júlio Gil Vale
Carvalheira2,3, Fernando Schmitt4,5,6, Maria de Fátima Gärtner2,5,6
1Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e
Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do
Ceará. 2Instituto de Ciências Biomédicas Abel Salazar, da Universiade do Porto (ICBAS-
UP). 3CIBIO-InBIO, Universidade do Porto. 4Faculdade de Medicina da Universidade do
Porto.5Instituto de Patologia e Imunologia Molecular da Universidade do Porto
(IPATIMUP). 6Instituto de Investigação e Inovação em Saúde (i3S), Universidade do
Porto.
*Corresponding author: [email protected]
Diana Célia Sousa Nunes-Pinheiro email address: [email protected]
Júlio Gil Vale Carvalheira email address: [email protected]
Fernando Schmitt email address: [email protected]
Maria de Fátima Gärtner: [email protected]
Fernando Schmitt and Maria de Fátima Gärtner co-supervised this work.
Diana C. Nunes-Pinheiro and Júlio G. Carvalheira contributed equally to this work.
Full list of author information is available at the end of the article
Abstract
Background: Breast cancer is often diagnosed in both dogs and human. Programmed
death 1 ligand 1 (PD-L1) is an immune regulatory molecule that limits lymphocytes
activity. This study investigates the relationship of PD-L1 expression and tumor-
infiltrating lymphocytes (TILs) in canine mammary tumor (CMT).
Methods: PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The
tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n
= 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50%
of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area.
Clinicopathologic data of CMT was characterized according to key parameters, (tumor
size; tumor ulceration; vascular invasion; presence of lymph node metastasis; clinical
stage; histological grade) as well as survival rate.
Results: TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had
TILs-Low. TILs-High had higher (P = 0.002) percentage of stromal TILs compared with
72
percentage of TILs-Low. PD-L1 expression and stromal TILs were significantly
associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of
which 17, 4% (n = 4) were from CA group and 21, 7% (n = 5) were from CC group. PD-
L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in
malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was
associated with involvement of regional lymph nodes (P = 0.034). Survival curves
demonstrated TILs-Low had higher (P = 0.010) overall survival (OS) compared with
TILs-High, and PD-L1+ and PD-L1– (P = 0.06) did not differed. The clinicopathologic
variables significantly correlated with OS by univariate analysis were histological grade
(P = 0.009), lymph node involvement (P = 0.004), stromal TILs (P = 0.016), and PD-
L1+/TILs-High vs. PD-L1–/TILs-Low (P = 0.010). Multivariate analysis revealed that
group of tumors with grade II-III was independent and negative prognostic factors for
OS.
Conclusions: We conclude that PD-L1 expression was associated with stromal TILs, and
both may represent important prognostic biomarkers for canine mammary tumors.
Keywords: Breast cancer, Canine Mammary Tumors, PD-L1, Prognosis
Background
Breast cancer (BC) is the most frequently diagnosed malignancy among women
[1], and similarly canine mammary tumors (CMT) are the major cancer in female dogs,
and malignant types can achieve 74% of tumors [2]. The involvement of the immune
system in cancer has been widely studied in medicine, and dogs have been used as a
model to understand new strategies in the fight against BC since the CMT have shown
biological and molecular similarities [3–5].
Several factors are involved in cancer progression, including genetic mutations,
epigenetic elements as well as the tumor microenvironment components, such as
inflammatory cells, and extracellular matrix [6,7]. The role of innate and adaptive
immunity has presented paradoxical results in the control of CMT since cytotoxic T
lymphocytes, and natural killer cells participated in controlling and elimination of tumor
cells, unlike tumor-associated macrophages, B cells and lymphocytes T regulatory in
tumor microenvironment were related to tolerance of neoplastic cell [7–9].
In this way, recently studies pointed the relationship between lymphocytes and
CMT, and showed that T lymphocytes in the peripheral blood and tumor
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microenvironment of dogs with cancer have been linked with prognosis [10,11]. Thereby,
similar to BC, the tumor-infiltrating lymphocytes (TILs) have considerable importance in
CMT, and TILs increased, especially T lymphocytes having a positive relationship with
features of malignancy and with a shorter overall survival of the dogs. Besides, CD4+
and regulatory T cells might play a key role in canine mammary tumors progression [12].
Although the search of TILs in CMT has been enhanced, the link between immune cells
and cancer progress still unknown.
Cancer cells sometimes find ways to use these immune checkpoint proteins as a
shield to avoid being identified and attacked by the immune system. Recent studies have
indicated that checkpoint molecules such as PD-L1 may played an important role in
canine cancer mediated immune modulation [13,14]. PD-L1 is the ligand for the T cell
inhibitory receptor PD-1 and is expressed on epithelial cells and various immune cells, in
particular dendritic cells, macrophages and B cells [15]. However, the interaction of
stromal TILs and PD-L1 expression in CMT remains unclear.
In this study, we investigated the relationship of PD-L1 expression and stromal
TILs in CMT. We also explored the association with clinical and pathological
characteristics of the tumors.
Methods
Animals
Twenty-three female dogs with CMT admitted at the Veterinary Hospital of the
Universidade Estadual do Ceará (UECE) between January and December of 2016 were
enrolled in the experiment. The animals were diagnosed by clinical and pathological
evaluation. All the dogs´ owners were informed about the study procedures and follow-
up appointment, signing a free and informed consent form. This study was approved by
the Committee for Ethics in Research using Animals (CEUA-UECE), protocol
8141226/2014.
Animals with clinical or laboratorial evidence of any other disease were excluded
from study and those who had been previous treated for mammary pathology. The search
for distant metastasis was accessed through chest radiographs and abdominal ultrasound.
Sentinel lymph node was detected to search for regional metastasis. After surgery, tumor
samples and lymph nodes were fixed in 10% buffered formalin and paraffin wax-
embedded. The follow-up period lasted 365 days after surgery in each case. The
clinicopathologic parameters evaluated in CMT-bearing dogs was tumor size, ulceration,
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necrosis, vascular invasion, lymph nodes involvement, metastasis, clinical stage,
histological grade, TILs intensity expression (Table 1).
Sample selection and histological analysis
The tumor samples were stained with hematoxylin-eosin (H&E) and sections (3
µm) were classified according to the veterinary histological classification [16]. Two
veterinary pathologists histologically examined a minimum of 3 sections of the mammary
tumors, and after classification, the tumors were grouped into simple carcinoma (CA, n
= 8) which was composed of one type of malignant cell either resembling luminal
epithelial or myoepithelial cells (Figure 1A). Tumors with foci of malignant-appearing
cells or distinct nodules of such cells occurring in complex adenomas or benign mixed
tumors (Figure 1B) were grouped as complex carcinoma (CC, n = 15).
The histological grade was assessed by classifying the carcinomas according to
three different prognostic features such as tubular formation, nuclear pleomorphism, and
mitotic counts [17,18]. Mitotic activity was assessed in 10 high-power fields (HPFs) in
the most mitotically areas and considering one HPFs should be a field area of 2.37 mm2,
in agreement with previous study [19]. According to the grading result, the tumors were
sorted as grade I (well differentiated), grade II (moderately differentiated), and grade III
(poorly differentiated).
Lymph node samples were sectioned and included following recommended
criteria [20]. Aiming to confirm the absence of metastasis, the sections were analyzed in
H&E–stained and immunostaining with antibodies specific for cytokeratin AE1/AE3
performed to detect macro and micrometastasis (Figure 1I).
Clinical staging
Clinical stage was attended following modified criteria [21,22]. The clinical stage
classification consists of five stages (I to V) determined by the size of primary tumor (T),
involvement of regional lymph nodes (N) and presence or absence of distant metastases
(M). Tumor size was assessed by measuring the greater diameter of the largest malignant
tumor present. The presence of lymph node involvement or distant metastasis was carried
out as described above.
Quantification of TILs
Histopathologic analyses of TILs were performed on H&E–stained sections from
twenty-three CMT from female dogs describes above. The analysis was conducted by
two pathologists who were blinded to the clinical parameters and using an Nikon
ECLIPSE E600 microscope fitted to a 10× eyepiece and a 40× objective. TILs were
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quantified as a percentage estimate of the stromal area adjacent to the tumor that
contained lymphocytic and plasm cells infiltrate, using semi-quantitative evaluation as
described in the literature [23]. Because heterogeneous histological features of CMT, the
TILs-assessment was done in different regions and reported the average.
Table 1. Clinicopathologic data of CMT.
Groups CMT
Histological Classification
CA CC
nº (%) 8 (35%) 15 (65%)
Age 10.25 (± 2.76) 9.34 (± 1.94)
Skin ulceration (%)
No
Yes
(5%)
(30%)
(17%)
(48%)
Tumor necrosis (%)
No
Yes
(5%)
(20%)
(30%)
(45%)
Vascular invasion (%)
No
Yes
(20%)
(20%)
(65%)
(0%)
T (cm) 3.64 (± 1.91) 5.12 (± 2.07)
N (%)
No
Yes
(20%)
(20%)
(55%)
(5%)
M (%)
No
Yes
(25%),
(10%)
(65%),
(0%)
TNM clinical stage (%)
I
II
III
IV
V
(5%)
(12%)
(5%)
(5%)
(8%)
(17%)
(12%)
(17%)
(19%)
(0%)
Tumor grade (%)
I
II
III
(12%)
(17%)
(6%)
(40%)
(25%)
(0%) T: size of the primary tumor; N: involvement of regional lymph nodes; M: presence of distant metastases;
CA: simple carcinoma; CC: complex carcinoma; n°: numbers
Thereby, two thresholds for TILs were reported as TILs-Low representing < 50%
(Figure 1A) of infiltrate within stromal area and TILs-High representing ≥ 50% of
analyses area (Figure 1B).
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Immunohistochemistry
For immunohistochemistry staining, 3 µm sections from paraffin wax-embedded
TMC and lymph node, were dewaxed and gradually hydrated through increasing
concentrations of alcohol. Antigen unmasking was performed in citrate (PD-L1) or
retrieval (AE1/AE3) buffer by microwave heating for 10 min. Endogenous peroxidase
activity was blocked by incubation the section in methanol containing hydrogen peroxide
(3%) for 10 min. Each section was blocked for 10 min at slide moisture chamber. Primary
antibody incubation was performed overnight at 4º C using rabbit PD-L1 monoclonal
antibody (1:100; E1L3N, Cell Signaling Technology, Beverly, MA), or mouse
cytokeratin AE1/AE3 monoclonal antibody (1:300, MP-011-CM01, A.Mirini). The
sections were rinsed in TBS and incubated with post-primary at room temperature for 30
min and followed by polymer incubation at room temperature for 30 min. Finally, positive
staining was visualized with 3-diaminobenzidine tetrahydrochloride (DAB). Sections
were counterstained with Harris Hematoxylin solution. The sections were observed under
an optical microscope. Section of human placenta were used as positive controls of PD-
L1 expression.
PD-L1 evaluated
The immunostaining was considered positive when 1% of tumor cells showed
membranous as well as cytoplasmic staining since PD-L1 was expressed on the cell
membrane and endomembrane system [24].
Statistical analysis
Statistical analyses were performed using SPSS version 24.0 (IBM Corp, Armonk,
NY). Pearson´s χ2 tests were used to compare categorical variables and Mann-Whitney
U test for quantitative variables. The relationship between clinicopathologic
characteristics and survival was examined using Kaplan-Meier log-rank survival analysis
and univariate Cox proportional hazards regression to calculate hazard ratios (HR) for
95% confidence intervals (95% CIs). Variables statistically significant on univariate
analysis were subsequently entered into a multivariate model using a backwards
conditional method. A P ≤ 0.05 was considered statistically significant.
Results
Clinicopathologic characteristics
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The study includes 23 CMT-bearing dogs with clinical and pathological data
summarized in Table 1. The mean age of CA and CC groups were 9.48 ± 1.35 and 9.89
± 2.87 years old, respectively. Macroscopically, most of the tumors had a size between 3
and 5 cm, and 78% (n = 18) had ulceration. Histopathological diagnoses were grouped as
eight CA (1 tubular, 2 papillary, 3 tubular-papillary, 2 solids) and fifteen CC (9 complex
carcinomas, 6 carcinomas in benign mixed tumors). Of all CMT, 52% (n = 12) of the
cases had a histological grade I, 42% (n = 10) had grade II, and only 6% (n = 1) had grade
III. It was also observed that 65% (n = 15) of the cases had areas of tumor necrosis.
Vascular invasion was identified in 20% (n = 5) of all samples, 25% (n = 6) had lymph
node metastasis, and in 10% (n = 2) was reported the presence of distant metastases.
TILs assessment and associations
Evaluation of the TILs within tumor stroma revealed that 65% (n = 15) of all
samples had TILs-Low as indicated in figure 1A. Most tumors showed stromal TILs
distributed multifocal or diffuse inflammation. TILs assessment was reported according
to the percentage of stromal TILs, in this way percentage of TILs-High was higher (P =
0.002) compared with percentage of TILs-Low (Figure 2B). However, TILs did not have
significant association between different clinicopathologic parameter (Table 2).
Expression of PD-L1 and associations
The PD-L1 immunostaining was observed in the cytoplasm and in the
cytoplasmatic membrane of malignant epithelial cells (Figure 1F,G), and present in 39%
(n = 9) of the cases. The myoepithelial cells and metaplastic component was also
evaluated in CC group and no PD-L1 expression was detect (Figure 1C,E). Furthermore,
we reported PD-L1 expression in malignant epithelium present in all lymph node
metastasis (n = 5), and only a few lymphocytes were immunostained (Figure 1H). PD-L1
expression in stromal TILs (TILsPDL1) has shown immunostaining in 39% (n = 9) of cases.
(Figure 1C,D).
The clinicopathologic features that shown significantly association with PD-L1
was regional lymph nodes metastasis (P = 0.034). PD-L1 expression and TILs were
significantly associated between each other (P = 0.009). In addition, PD-L1+ had higher
(P = 0.033) percentage of stromal TILs then PD-L1– (Figure 2C).
Table 2. Association between PD-L1expression, stromal TILs and Clinicopathologic data
of CMT.
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Clinicopathologic
parameter PD-L1– (%) PD-L1+ (%) P- value TILs-Low (%) TILs-High (%) P- value
Histologic type
CA 13,0% 17,4% 0.094 17,4% 17,4% 0.267
CC 47,8% 21,7% 47,8% 17,4%
Grade of tumor
I 17.4% 4,3% 17,4% 4,3%
II 21,7% 17.4% 0.740 26,1% 13,0% 0.640
III 21,7% 17.4% 21,7% 17,4%
Skin ulceration
Absent 8.7% 13% 0.285 8,7% 13,0% 0.190
Present 52,2% 26,1% 56,5% 21,7%
Tumor necrosis
Absent 26,1% 17,4% 0.940 21,7% 21,7% 0.178
Present 34,8% 21,7% 43,5% 13%
T
T1 < 3 cm 34,8% 17,4% 34,8% 17,4%
T2 3-5 cm 26,1% 17,4% 0.358 26,1% 17,4% 0.612
T3 > 5 cm 0% 4,3% 0 4,3%
N
Negative 56,5% 21,7% 0.034 56,5% 21,7% 0.190
Positive 4,3% 17,4% 8,7% 13%
M
Negative 56,5% 34,8% 0.744 60,9% 30,4% 0.644
Positive 4,3% 4,3% 4,3% 4,3%
TNM clinical stage
I 17,4% 4,3% 17,4% 4,3%
II 21,7% 13,0% 26,1% 8,7%
III 13,0% 8,7% 0.758 13% 8,7% 0.663
IV 4,3% 8,7% 4,3% 8,7%
V 4,3% 4,3% 4,3% 4,3%
TILs
TILs-Low 52,2% 13% 0.009 - - -
TILs-High 8,7% 26,1% - - - T: size of the primary tumor; N: involvement of regional lymph nodes; M: presence of distant metastases;
CA: simple carcinoma; CC: complex carcinoma
Survival analyses
The median survival was 272 days, and 8 animals (35%) had died at the endpoint
of follow-up. In addition, survival curves comparison stratified based on the TILs density
demonstrated significant difference between TILs-Low and TILs-High (P = 0.01) in
overall survival (OS). PD-L1– has high OS compared to PD-L1+ but did not signicantly
(P = 0.06). The animals were divided into four subgroups: PD-L1+/TILs-High, PD-
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L1+/TILs-Low, PD-L1–/TILs-High and PD-L1–/TILs-Low. In this way, Kaplan-Meier
graphical analysis demonstrated that PD-L1–/TILs-Low had high OS compared PD-
L1+/TILs-High (P = 0.01), but did not showed with other subgroups (Figure 3C).
The clinicopathologic variables significantly correlated with OS by univariate
analysis using Cox proportional hazard model, the multivariate analysis revealed that
group of tumors with grade II-III was independent and negative prognostic factors for OS
(Table 3).
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Table 3. Univariate and multivariate cox proportional hazard model for overall survival
Univariate Multivariate
95% CI 95% CI Variable HR Low Upper P-value HR Low Upper P-value
Histologic type Simple vs. Complex 0.805 0.192 3.37 0.766
Grade of tumor II-III vs. I 9.87 1.20 65.58 0.009 14.77 1.15 88.36 0.038
T (cm) ≥ 3 cm vs. < 3 cm 2.03 0.25 16.55 0.495 N pos vs neg 6.33 1.50 23.69 0.004 1.22 1.19 7.794 0.833
M pos vs neg 1.46 0.18 11.99 0.719 TNM clinical stage
IV-V vs. I-III 2.84 0.67 12.01 0.138 PD-L1 status pos vs neg 3.60 0.85 15.28 0.063 TILsPDL1
pos vs neg 1.43 0.28 7.12 0.658
Stromal TILs TILs-high vs TILs-Low 4.99 1.17 21.22 0.016 8.22 1.42 60.32 0.165
PD-L1*TILs (PD-L1+/TILs-High vs.
PD-L1+/TILs-Low) 5.01 0.45 25.30 0.871 (PD-L1+/TILs-High vs.
PD-L1–/TILs-High) 2.46 0.22 27.30 0.420 (PD-L1+/TILs-High vs.
PD-L1–/TILs-Low) 6.67 1.98 37.14 0.010 2.026 0.180 22.78 0.567
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Figure 1. TILs assessment and PD-L1 expression within CMT and stromal TILs. A.
Carcinoma in benign mixed tumor with few stromal TILs (H&E, 200x); B. Solid
carcinoma with high stromal TILs (H&E, 200x); C. Complex carcinoma with few
infiltrate cells expressing PD-L1 (200x); D. Tubular carcinoma with high infiltrate cells
expressing PD-L1 (200x); E. Complex carcinoma with negative PD-L1 expression
(100x); F. Tubular carcinoma with positive PD-L1 expression (100x); Malignant
epithelium with strong PD-L1 expression (400x); Lymph node metastasis with PD-L1
expression (200x). I. Lymph node metastasis with AE1/AE3 positive expression (100x)
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Figure 2. Relationship between PD-L1 expression and stromal TILs. A. Figures
within this bar graph depict percentage of cases with PD-L1 expression and stromal TILs
intensity. B. Analysis of TILs status and stromal TILs(%). C. Analysis of PD-L1 status
and stromal TILs(%). Significant differences at P < 0.05 are highlighted by asterisk.
52,20%
13%
8,70%
26,10%
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
70,00%
PDL1- PDL1+
TIL-Low TIL-High
A
B C
83
A
B
C
Figure 3. Overall survival for stromal TILs (A), PD-L1 expression (B), and PD-L1*
TILs.
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Discussion
Understanding the functions and interactions of different tumor-infiltrating
lymphocytes is crucial for the development of antitumor immunotherapy. Inflammation
seems to promote tumorigenesis under certain conditions, while in other situations,
infiltration of lymphocytes were shown to suppress tumor growth, likely seen when PD1
bind PD-L1 in T cells triggering the immune editing to support the tumor progression
[15].
In this study, we evaluated the clinical relevance of PD-L1 expression in CMT
and the relationship with TILs. PD-L1 was differently expressed according to the
histologic subtypes of TMC. Besides, TILs were associated with PD-L1 expression and
showed poor prognosis when two parameters were correlated. In recent studies, PD-L1
expression has been demonstrated in a variety of solid tumor types in humans, including
lung, melanoma, ovarian, colon, and BC [25]. Currently, has been showed the presence
of PD-L1 in several canine cancer, such melanoma, mast cell tumor, renal cancer, and
CMT [26].
The evaluation of PD-L1 expression is challenging due to heterogeneity in
expression and non-reproducibility of antibody reagents [27]. Only a few studies have
described PD-L1 protein expression in canine tumors, and showed PD-L1 expression in
canine tumor cell lines, although the levels of basal expression were very variable.
Significant upregulation of PD-L1 expression by all tumor cell lines was observed
following IFN-γ exposure and by exposure to a TLR3 ligand. Canine monocytes and
monocyte-derived macrophages did not express PD-L1, but the expression was
significantly upregulate following treatment with IFN-γ [28]. Research employing bovine
lymphocytes reported that crosslinking of PD-L1 by PD-1-Ig increased cell death and
decreased cytokine production in PD-L1 expressing cells, suggesting a role for PD-L1 in
inducing immunosuppression and lymphocyte cell death [29].
Coy et al (2017) using flow cytometry described the PD-1 expression by CD4+
and CD8+ T cells from canine peripheral blood with various types of cancer. The
percentage of PD-1+ CD4 T cells was significantly higher in dogs with cancer than in
control dogs, whereas the increase in PD-1 expression by CD8+ T cells did not reach the
level of statistical significance. In addition, the study showed that both CD4+ and CD8+
T cells, as well as neutrophils and monocytes, were negative for PD-L1expression. Unlike
85
this data, the present study found a strong expression of PD-L1 by canine lymphocytes
and plasm cells within TILs (Figure 1C,D).
Increased expression of PD1 and PD-L1 was shown in CD3+ T cells and CD21+
B lymphocytes within the peripheral blood and splenic mononuclear cells from dogs with
canine visceral leishmaniosis [30]. Moreover, the authors demonstrate PD-1/PD-L1 were
involved in the induction of T lymphocyte apoptosis and in regulating the production of
nitric oxide, TNF-, and IL-4, as well as reducing the parasitic load in dogs with
leishmaniosis. The increase PD-L1 cell surface expression by tumor cells induced by
PTEN loss can lead to decreased T-cell proliferation and increased apoptosis. Besides,
agents targeting the PI3K pathway may enhance the antitumor adaptive immune
responses once PTEN loss is one mechanism regulating PD-L1 expression [31].
The relationship of PD-L1 expression and TILs in this study have strong
association (P = 0.009), and TILs were frequently immunostaining for anti-PD-L1. The
expression of PD-L1 was described in 50% of BC and was not restricted to the tumor
epithelium, but was also expressed by TILs [32]. Recently was reported the association
of stromal TILs and PDL1 expression with aggressive types of BC and that both are
already found in in situ stages [33]. PD-L1 overexpression was significantly associated
with a series of clinicopathological parameters, such as large tumor size, lymph node
metastasis, estrogen receptor-negativity, and triple-negative breast cancer [24,34]. We
found in this study an association of lymph node metastasis and PD-L1 expression (P =
0.034) and the univariate analysis showed high risk for OS in animals with lymph node
involvement. It can be supported by presence of PD-L1 in malignant epithelium in all
lymph node metastasis (Figure 1H).
Along with PD-L1 expression, decreased TILs were associated with a poor
prognosis in triple-negative BC. Additionally, multivariate Cox proportional hazards
model analysis showed that PD-L1+/TILs-Low was an independent negative prognostic
factor for both recurrence-free survival and overall survival [35]. Interestingly,
contrasting the findings in BC, we demonstrated that PD-L1+/TILs-High had higher risk
for OS than another group’s interaction. High PD-L1 expression may be a prognostic
indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free
survival, although it was not significantly associated with overall survival [34].
86
Presence of TILs has been shown to be potentially predictive and a prognostic
factor in BC subtypes. Specifically, in patients with human epidermal growth factor
receptor 2 (HER2) positive and triple-negative breast cancer [36]. In canine mammary
tumors, has been reported that high presence of TILs were correlated to several
malignancy characteristics [10,37]. In addition, when the subpopulation of lymphocytes
was assessed as CD4+ T, CD8 + T, and regulatory T cells present in TILs were shown
low OS associated with high cell CD4+/CD8+ ratio, and a worse prognosis was associated
with regulatory T lymphocytes [38]. Our data supports the worse prognosis associated
with high stromal TILs in CMT, since TILs-High showed low OS (P = 0.01) compared
with TILs-Low group.
Recently, a pilot clinical study was performed using monoclonal antibodies
against PD-L1 in dogs with oral malignant melanoma and undifferentiated sarcoma
demonstrating a favorable antitumor activity [39]. It is particularly important since
PDL/PD-L1 inhibition has been used to several cancer treatments in humans [40].
Unfortunately, the immunohistochemistry criteria for PD-L1 expression have not yet
been standardized. Indeed, different thresholds for positivity PD-L1 expression have been
used in the clinical trials and more studies are needed to achieve as a potential biomarker
for CMT.
Conclusions
In conclusion, we have confirmed the association of PD-L1 expression and
stromal TILs in CMT with relation to higher grade of tumor and lymph node involvement.
Thereby suggesting the immune checkpoint PD-L1 as a potential therapeutic target for
the treatment of CMT, however further research are necessary.
Abbreviations
BC: Breast cancer; CA: Simple carcinoma; CC: Complex carcinoma; CMT: Canine
mammary tumor; DAB: 3-diaminobenzidine tetrahydrochloride; H&E: Hematoxylin-
eosin; HPFs: High-power fields; HR: Hazard ratios; OS: overall survival; PD-L1:
Programmed death 1 ligand 1; TBS: Tris buffered saline; TILs: Tumor-infiltrating
lymphocytes
Acknowledgements
87
This study was supported in part by Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) and the research scholarship by Sandwich Doctorate
(PDSE/CAPES). The antibodies for immunohistochemistry were provided by
Department of Molecular Pathology and Immunology (ICBAS), University of Porto.
Funding
This study was supported by the grant No.88881.132917/2016-01 from the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (Brazil). The authors declare that the
funding body has had no role in the design of the study and collection, analysis, and
interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets who support the results and conclusions of this article are available from the
corresponding author.
Authors’ contributions
BELN and MFG participated in the design of the study. BELN performed the
experiments. BELN and JGVC carried out statistical analysis and interpretation of data.
FS: participated in the study design and revised the manuscript. MFG and DCSNP
participated in coordination of the study and revised the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
The Committee for Ethics in Animal Research of the Universidade Estadual do Ceará,
Brazil, certified the research project under the protocol number 8141226/2014 and which
involves use animals and biological samples with the purpose to scientifically
investigation. The dogs’ owners in Brazil were informed about the study procedures and
follow-up appointment, signing a free and informed consent form.
Consent for publication
Not Applicable
Competing interests
The authors declare that they have no competing interests.
Author details
88
1Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e
Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do
Ceará, 60714-903 Fortaleza, Brasil. 2Instituto de Ciências Biomédicas Abel Salazar, da
Universiade do Porto (ICBAS-UP), 4050-013 Porto, Portugal. 3Centro de Investigação
em Biodiversidade e Recursos Genéticos (CIBIO-InBIO), Universidade do Porto, 4485-
661 Vairão, Portugal. 4Faculdade de Medicina da Universidade do Porto, 4200-319 Porto,
Portugal. 5Instituto de Patologia e Imunologia Molecular da Universidade do Porto
(IPATIMUP), 4200-135 Porto, Portugal. 6Instituto de Investigação e Inovação em Saúde
(i3S), Universidade do Porto, 4200-135 Porto, Portugal.
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9 CONCLUSÕES
A exacerbação sistêmica da peroxidação lipídica demonstrado pelo aumento nos
níveis séricos de MDA juntamente com o desequilíbrio das moléculas antioxidantes não
enzimáticos podem contribuir para a instabilidade da homeostase em animais com tumor
mamário canino (TMC).
A caracterização do infiltrado inflamatório no TMC demostrou que a população
predominante foi de linfócitos. Além disso, observou-se que a quantidade de linfócitos
pode estar associada a critérios de malignidade tumoral. Os linfócitos que expressão CD4,
CD8 e Foxp3 estão presentes no TMC e sua distribuição pode estar associada ao
prognóstico das cadelas. Este trabalho apresentou resultados sobre a presença da proteína
de choque térmico HSP60 no TMC.
Também confirmamos a associação da expressão de PD-L1 e o infiltrado
linfocítico tumoral no TMC, podendo esta associação está relacionada ao grau do tumor
e a metástase do linfonodo regional. Assim, sugerimos a molécula PD-L1 como potencial
alvo terapêutico para o tratamento do tumor de mama em cadelas.
94
10 PERSPECTIVAS
Os resultados obtidos no presente estudo evidenciam algumas etapas da
inflamação ocorrida no organismo e no próprio microambiente tumoral de cadelas com
tumor de mama. O estresse oxidativo pode contribui para um maior processo inflamatório
em cadelas com TMC, podendo alguns subtipos do tumor desencadear uma maior
resposta oxidativa. Além disso, a presença de proteínas de choque térmico HSP60 no
TMC evidencia a participação do estresse oxidativo nos TMC. Dessa forma, torna-se
necessário mais pesquisas avaliando o metabolismo oxidativo das células tumorais no
intuito de estabelecer o verdadeiro papel patogênico do estresse oxidativo no câncer e,
com isso, tornar-se alvo de terapias anticâncer.
Outro ponto da inflamação abordado foi a presença no infiltrado inflamatório no
TMC. O aumento do infiltrado linfocítico tumoral parece estar relacionado com o pior
prognóstico dos TMC. Porém, é necessário mais estudo para melhor entender a influência
da subpopulação de linfócitos e tanto no microambiente tumoral como na própria
neoplasia. Da mesma forma, mais investigações são necessárias para estabelecer a função
do eixo das moléculas de controle imunológico PD-1/PD-L1 no controle e promoção do
TMC. Por fim, este estudo amplia as pesquisas envolvendo os eventos inflamatórios e
imunológicos presentes nos tumores da glândula mamária canina.
95
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APÊNDICE
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APÊNDICE A – CERTIFICADO DO COMITÊ DE ÉTICA PARA O USO DE
ANIMAIS /UECE
