Advances in canine mammary cancer: a role for inflammatory infiltrate in tumor microenvironment
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2017
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A inflamação faz parte dos principais “hallmarks” associados ao cancro e tem um papel
relevante na carcinogénese mamária, relacionando-se com a agressividade tumoral e pior
prognóstico. Nos tumores mamários dos canídeos (TMC) os estudos que se debruçam
sobre o efeito das células imunitárias no prognóstico são escassos. Neste contexto, o
presente trabalho foi realizado com o objetivo de fomentar o conhecimento sobre o papel
das vias de sinalização compartilhadas pelo infiltrado de linfócitos T/macrófagos e
importantes biomarcadores tumorais na progressão, agressividade e prognóstico destes
tumores.
A imunossupressão associada ao infiltrado de linfócitos T no tumor tem sido explicada
pela capacidade inibitória das células T reguladoras (Treg) que impedem as atividades
citotóxicas anti-tumorais. Com base nestas evidências, caracterizámos por
imunohistoquímica as células Treg intratumorais (FoxP3+), bem como algumas
citoquinas relacionadas com elas: TGFβ e IL-35. Os nossos resultados demonstraram que
a maior imunorreactividade para o FoxP3 está presente em tumores com fenótipos mais
agressivos: elevado grau histológico de malignidade (GHM), presença de embolos
intravasculares e metástases nos linfonodos. Adicionalmente, mostrámos que as células
Treg FoxP3+ intratumorais estão associadas com uma menor sobrevida total (ST), por
análise univariada e multivariada. As células Treg secretam citoquinas inibidoras (TGFβ
e IL-35), induzindo a tolerância aos tumores. Os nossos resultados sugerem uma
associação entre o TGFβ e parâmetros de agressividade tumoral, refletindo o seu
envolvimento na transformação maligna. Demonstraram também uma correlação positiva
entre o FoxP3 intratumoral, os níveis de TGFβ, VEGF e CD31. Além disso, tumores com
abundante TGFβ e com elevada expressão simultânea de TGFβ/FoxP3, FoxP3/VEGF,
TGFβ/VEGF foram associados ao menor tempo de ST e a classe de tumores TGFβ/FoxP3
elevados revelou-se como um fator independente de mau prognóstico na análise
multivariada. A IL-35 é uma citoquina secretada pelas células Treg que inibe a
proliferação e função das células T. Demonstrámos que a sobre-expressão da IL-35 está
associada com o estádio clínico mais avançado e com um prognóstico desfavorável na
análise univariada e multivariada. Foi possível constatar, pela 1ª vez nos TMC, o valor
prognóstico independente das células FoxP3+ Treg e da classe de tumores TGFβ/FoxP3 elevados. A IL-35 evidenciou-se ainda como um novo biomarcador capaz de prever a
evolução clínica dos TMC.
No seguimento deste trabalho, realizámos um amplo estudo que incidiu sobre as vias de
sinalização compartilhadas pelo infiltrado de linfócitos T/macrófagos e importantes
biomarcadores tumorais no microambiente dos TMC. Com esse propósito estudámos
biomarcadores celulares importantes na carcinogénese mamária (COX-2, EGFR e c-kit),
que estão frequentemente sobre-expressos ou mutados nos TMC. Constatámos uma
associação significativa da imunoexpressão elevada da COX-2 com os linfócitos T CD3+
e macrófagos MAC387. Tumores com elevada expressão de COX-2/CD3 e COX-2/MAC
estão associados com variáveis de agressividade tumoral e menor ST. Demonstrámos
ainda que a expressão simultânea de COX-2+/EGFR+ está associada com maior número
de células T CD3+ intratumorais e foi observada uma associação significativa e uma
correlação positiva entre os linfócitos T CD3+ e a expressão de c-kit. Tumores com alta
expressão de c-kit apresentaram maior número de células T CD3+ estando associados a
um GHM elevado, presença de êmbolos intravasculares, metástases nos linfonodos e
menor ST. Os resultados demonstram que as vias de sinalização que envolvem a COX-2,
o EGFR e o c-kit são importantes, não só para a remodelação do microambiente do tumor
mamário, mas também como alvos para a terapia imunológica anti-tumoral.
Para complementar o trabalho, foi realizada uma experiência de co-cultura in vitro onde
se pretendia demonstrar a potencial participação bidirecional das células tumorais e
células imunitárias na regulação da COX-2. Os resultados mostraram que a co-cultura
entre a linha celular de carcinoma mamário de cão Sh1b e as células mononucleares de
sangue periférico de cão (PBMCs) induziram uma tendência para a sobre-expressão de
COX-2 nas células cancerígenas. Por sua vez, a expressão de COX-2 pelos PBMCs, entre
eles predominantemente macrófagos CD68+, foi significativamente atenuada pela cocultura
com Sh1b. Em conformidade, a co-cultura com CD68+ THP1 diferenciada
(dTHP1) provocou um aumento da produção intracelular de COX-2 pelas células Sh1b.
A expressão intracelular de COX-2 pela dTHP1 diminuiu quando estas células foram
tratadas com meio condicionado das células Sh1b. Estes resultados representam um
avanço significativo na compreensão do papel da COX-2 na remodelação do
microambiente tumoral nos TMC, nomeadamente através da imunomodulação dos
macrófagos associados ao tumor.
Globalmente, o presente trabalho demonstrou que, à semelhança do cancro de mama da
mulher, também nos TMC as células inflamatórias no microambiente do tumor são
capazes de orquestrar programas facilitadores da progressão tumoral.
Cancer related inflammation is part of the major cancer hallmarks and has an important role in mammary carcinogenesis being involved in tumor aggressiveness and poor clinical outcome. In dogs only few studies have yet concentrated on the influence of immune cells in clinical outcome of dog mammary tumor patients. In this context, the present work was conducted with the main aim of up-to-date knowledge about the roles of the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in canine mammary tumors (CMT) progression, aggression and prognosis. Immunosuppression associated with tumor infiltrating T-lymphocytes (TILs) has been explained by the compartment of regulatory T-cells (Treg) that inhibit anti-tumor cytotoxic activities. Based on these evidences, first, we assessed, by immunohistochemistry, the characterization of intratumoral Treg cells, as well as some cytokines related by them: TGFβ and IL-35. Our results demonstrated that FoxP3 was present in tumors with more aggressive phenotypes: high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastasis. Additionally, also showed that intratumoral FoxP3+ Treg cells were associated with shorter overall survival (OS), both in univariate and multivariate analysis. Tregs can secrete inhibitory cytokines (TGFβ and IL-35), inducing tumors tolerance. Present work suggests a link between TGFβ and parameters of tumor aggressiveness, reflecting its involvement in CMT malignant transformation. Our data demonstrated also a positive correlation between intratumoral FoxP3, TGFβ levels, VEGF and CD31. Moreover tumors with abundant TGFβ and with concurrent high expression of TGFβ/FoxP3, FoxP3/VEGF, TGFβ/VEGF were associated with shorter OS time and the TGFβ/FoxP3 tumors class retained the association with worse survival in multivariate analysis, arising as an independent predictor of poor prognosis. IL-35 is a Treg cellsecreted cytokine that inhibits T-cells proliferation and function and to the best of our knowledge, this is the first study that investigates the role of IL-35 in CMT development and clinical outcome. Our results showed that IL-35 overexpression was significantly associated with advancement of tumor stage and unfavorable prognosis by univariate and multivariate survival analysis. Interestingly our findings indicate, for the first time in dog mammary tumors, the independent prognostic value of FoxP3+ Treg cells and TGFβ/FoxP3 tumors class. Additionally the IL-35 seems to be a new biomarker to predict the CMT clinical outcome. For a more comprehensive approach about the role of immune cells in dog mammary carcinogenesis, we perform a wide study focused on the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in mammary tumor microenvironment. We focused on relevant cell biomarkers in mammary carcinogenesis, the COX-2, EGFR and c-kit which are often overexpressed or mutated in this type of tumor. Present data demonstrated a significant association of high COX-2 immunoexpression with CD3+ T-lymphocytes and MAC387 macrophages. Tumors with concurrent high COX-2/CD3 and high COX-2/MAC expression were associated with variables of tumor aggressiveness and shorter OS. Current results also demonstrated that the concurrent COX-2+/EGFR+ expression was associated with higher numbers of intratumoral CD3+ T-cells. Furthermore a significant association and a positive correlation between CD3+ T-lymphocytes and c-kit expression were observed. Tumors with high c-kit expression showed higher counts of CD3+ T-cells and were associated with high HGM, presence of neoplastic intravascular emboli, presence of lymph node metastasis, angiogenesis and shorter OS. The findings indicate that COX-2, EGFR and c-kit pathways are important not only for the remodeling of mammary tumor microenvironment but also could be a very important targets for tumor immunological therapy. Finally, in an in vitro co-culturing experiment, was our aim to prove the potential bidirectional crosstalk between tumor cells and immune cells on COX-2 regulation. Our data showed that co-culturing of canine mammary carcinoma cell line Sh1b and canine peripheral blood mononuclear cells (PBMCs) induced a trend of COX-2 overexpression in mammary cancer cells. In turn, COX-2 expression by PBMCs, among them predominantly CD68+ macrophages, was significantly attenuated by co-culture with Sh1b. In accordance, co-culture with CD68+ differentiated THP1 (dTHP1) prompted an intracellular production of COX-2 in Sh1b cells. The intracellular COX-2 expression from dTHP1 decreased when they were treated with conditioned medium from cultured Sh1b cells. Present results represents a significant advance on understanding the possible role of COX-2 in inducing a cancer tolerogenic microenvironment in CMT namely through a cancer associated macrophages immunomodulation. Overall present work demonstrated that, similarly to human breast cancer, also in CMT the inflammatory responses in mammary cancer sites are able to orchestrate hallmark-facilitating programs in tumor microenvironment.
Cancer related inflammation is part of the major cancer hallmarks and has an important role in mammary carcinogenesis being involved in tumor aggressiveness and poor clinical outcome. In dogs only few studies have yet concentrated on the influence of immune cells in clinical outcome of dog mammary tumor patients. In this context, the present work was conducted with the main aim of up-to-date knowledge about the roles of the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in canine mammary tumors (CMT) progression, aggression and prognosis. Immunosuppression associated with tumor infiltrating T-lymphocytes (TILs) has been explained by the compartment of regulatory T-cells (Treg) that inhibit anti-tumor cytotoxic activities. Based on these evidences, first, we assessed, by immunohistochemistry, the characterization of intratumoral Treg cells, as well as some cytokines related by them: TGFβ and IL-35. Our results demonstrated that FoxP3 was present in tumors with more aggressive phenotypes: high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastasis. Additionally, also showed that intratumoral FoxP3+ Treg cells were associated with shorter overall survival (OS), both in univariate and multivariate analysis. Tregs can secrete inhibitory cytokines (TGFβ and IL-35), inducing tumors tolerance. Present work suggests a link between TGFβ and parameters of tumor aggressiveness, reflecting its involvement in CMT malignant transformation. Our data demonstrated also a positive correlation between intratumoral FoxP3, TGFβ levels, VEGF and CD31. Moreover tumors with abundant TGFβ and with concurrent high expression of TGFβ/FoxP3, FoxP3/VEGF, TGFβ/VEGF were associated with shorter OS time and the TGFβ/FoxP3 tumors class retained the association with worse survival in multivariate analysis, arising as an independent predictor of poor prognosis. IL-35 is a Treg cellsecreted cytokine that inhibits T-cells proliferation and function and to the best of our knowledge, this is the first study that investigates the role of IL-35 in CMT development and clinical outcome. Our results showed that IL-35 overexpression was significantly associated with advancement of tumor stage and unfavorable prognosis by univariate and multivariate survival analysis. Interestingly our findings indicate, for the first time in dog mammary tumors, the independent prognostic value of FoxP3+ Treg cells and TGFβ/FoxP3 tumors class. Additionally the IL-35 seems to be a new biomarker to predict the CMT clinical outcome. For a more comprehensive approach about the role of immune cells in dog mammary carcinogenesis, we perform a wide study focused on the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in mammary tumor microenvironment. We focused on relevant cell biomarkers in mammary carcinogenesis, the COX-2, EGFR and c-kit which are often overexpressed or mutated in this type of tumor. Present data demonstrated a significant association of high COX-2 immunoexpression with CD3+ T-lymphocytes and MAC387 macrophages. Tumors with concurrent high COX-2/CD3 and high COX-2/MAC expression were associated with variables of tumor aggressiveness and shorter OS. Current results also demonstrated that the concurrent COX-2+/EGFR+ expression was associated with higher numbers of intratumoral CD3+ T-cells. Furthermore a significant association and a positive correlation between CD3+ T-lymphocytes and c-kit expression were observed. Tumors with high c-kit expression showed higher counts of CD3+ T-cells and were associated with high HGM, presence of neoplastic intravascular emboli, presence of lymph node metastasis, angiogenesis and shorter OS. The findings indicate that COX-2, EGFR and c-kit pathways are important not only for the remodeling of mammary tumor microenvironment but also could be a very important targets for tumor immunological therapy. Finally, in an in vitro co-culturing experiment, was our aim to prove the potential bidirectional crosstalk between tumor cells and immune cells on COX-2 regulation. Our data showed that co-culturing of canine mammary carcinoma cell line Sh1b and canine peripheral blood mononuclear cells (PBMCs) induced a trend of COX-2 overexpression in mammary cancer cells. In turn, COX-2 expression by PBMCs, among them predominantly CD68+ macrophages, was significantly attenuated by co-culture with Sh1b. In accordance, co-culture with CD68+ differentiated THP1 (dTHP1) prompted an intracellular production of COX-2 in Sh1b cells. The intracellular COX-2 expression from dTHP1 decreased when they were treated with conditioned medium from cultured Sh1b cells. Present results represents a significant advance on understanding the possible role of COX-2 in inducing a cancer tolerogenic microenvironment in CMT namely through a cancer associated macrophages immunomodulation. Overall present work demonstrated that, similarly to human breast cancer, also in CMT the inflammatory responses in mammary cancer sites are able to orchestrate hallmark-facilitating programs in tumor microenvironment.
Descrição
Tese de Doutoramento em Ciências Veterinárias
Palavras-chave
Neoplasias mamárias animais , Macrófagos , Prognóstico , Linfócitos T , Linfócitos T reguladores , Microambiente tumoral , Células Treg