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Regulation of breast cancer extracellular matrix to boost breast cancer immunity
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O cancro da mama é um dos tipos de cancro mais frequentemente detetados em mulheres. Os cancros luminais são os cancros de mama mais comuns, onde o luminal B apresenta alta agressividade e genes relacionados à proliferação tumoral.
O desenvolvimento do cancro da mama está associado aos fatores celulares e solúveis da matriz extracelular (ECM). A ECM promove a migração celular conectando o citoesqueleto das células com aderências focais (FA). A presença de FA está relacionada com a fosforilação da tirosina, sendo a quinase de adesão focal (FAK) uma das mais prontamente fosforiladas no cancro. A superexpressão de FAK está associada à proliferação de células tumorais, contribuindo também para a ativação das células endoteliais, que expressam a P-selectina na sua superfície. A ação da P-selectina é desencadeada pela sua interação com o ligando-1 da glicoproteína P-selectina (PSGL-1) expresso nas células tumorais, promovendo a circulação de células cancerígenas e metástase.
Como a interação entre FAK e P-selectina é pouco compreendida, este projeto tem como objetivo avaliar o efeito antitumoral sinérgico da interação entre os inibidores da FAK e da P-selectina e a nanovacina direcionadas a DC que entregam antígenos associados a tumores e adjuvantes, em modelos esferóides 3D.
A expressão de FAK, FAK fosforilada (pFAK), P-selectina e PSGL-1 foram avaliadas em células EO771 e do microambiente tumoral (TME) recolhidas de ratinhos inoculados com EO771. Verificou-se que FAK, pFAK e P-selectina estavam superexpressas em células EO771 TME (> 97%). Para modular o TME em direção a um microambiente efetor imunológico, antes de avaliar o papel do defactinib (inibidor da FAK) e do KF-38789 (inibidor da P-selectina) na função e frequência das células imunes dentro do TME, avaliámos o impacto do defactinib e do KF-38789 na viabilidade das células EO771 e TME EO771. Consequentemente, o defactinib e o KF-38789 induziram um efeito citotóxico significativo nas células EO771 para concentrações acima de 1 e 0,1 μM, respetivamente. Além disso, foi obtida uma diminuição significativa na viabilidade das células EO771 TME para concentrações de defactinib e KF-38789 acima de 0,5 e 0,05 µM, respetivamente. Finalmente, para avaliar o impacto do efeito sinérgico entre a inibição da FAK e da P-selectina combinada com a nanovacina, foi realizada a co-cultura dos esferóides 3D de tumores EO771 (EO771 TME) e esplenócitos. De acordo com as diferentes condições, o tratamento de esferóides 3D do cancro da mama com KF-38789 e a nanovacina isoladamente ou em combinação com o tratamento com defactinib mostrou-se promissor na modulação da invasão tumoral. Finalmente, uma inibição completa do sprouting resultando na morte de células esferóides 3D foi alcançada com a combinação de defactinib e esplenócitos estimulados seguido de KF-38789, representando o efeito sinérgico mais promissor entre os tratamentos testados. Considerando a estratégia terapêutica mais promissora e eficaz, levanta-se a hipótese de que o defactinib primeiro modula o TME para permitir uma melhor infiltração imunológica desencadeada pela vacinação, enquanto a P-selectina previne a proliferação de células cancerígenas.
Breast cancer is one of the most frequently detected cancer types in women. Luminal cancers are the most common breast cancers, where luminal B presents high aggressiveness and genes related to tumor proliferation. Breast cancer development is associated with the extracellular matrix (ECM) cellular and soluble factors. The ECM promotes cell migration by connecting the cells' cytoskeleton with focal adhesions (FA). FA presence is related to tyrosine phosphorylation, being the focal adhesion kinase (FAK) one of the most promptly phosphorylated in cancer. FAK overexpression is associated with the proliferation of tumor cells, contributing also to the endothelial cell’s activation, which expresses P-selectin on their surface. The P-selectin action is triggered by its interaction with the P-selectin glycoprotein ligand-1 (PSGL-1) expressed on tumor cells, promoting the circulation of cancer cells and metastasis. Since the interaction between FAK and P-selectin is poorly understood, this project aims to evaluate the synergic anti-tumor effect of the interaction between FAK and P-selectin inhibitors and the DC-targeted nanovaccine delivering tumor-associated antigens and adjuvants, in 3D spheroid models. FAK, phosphorylated FAK (pFAK), P-selectin, and PSGL-1 expression were assessed in EO771 and tumor microenvironment (TME) cells collected from EO771-bearing mice. FAK, pFAK, and P-selectin were found to be overexpressed at EO771 TME cells (> 97%). To modulate the TME toward an immune effector microenvironment, before evaluating the role of defactinib (FAK inhibitor) and KF-38789 (P-selectin inhibitor) on immune cells’ function and frequency within the TME, we evaluated defactinib and KF-38789 impact on the viability of EO771 and TME EO771 cells. Accordingly, defactinib and KF-38789 induced a significant cytotoxic effect on EO771 cells for concentrations above 1 and 0.1 μM, respectively. Moreover, a significant decrease in EO771 TME cell viability was obtained for concentrations of defactinib and KF-38789 above 0.5 and 0.05 µM, respectively. Finally, to evaluate the impact of the synergistic effect between the FAK and P-selectin inhibition combined with a nanovaccine, the co-culture of the 3D spheroids with the EO771 tumors (EO771 TME) and splenocytes was performed. According to the different conditions, the treatment of 3D spheroids of breast cancer with KF-38789 and the nanovaccine alone or in combination with defactinib treatment showed to be promising in modulating the tumor invasion. Finally, a complete sprouting inhibition resulting in the death of 3D spheroid cells was attained with the combination of defactinib and stimulated splenocytes followed by KF-38789, representing the most promising synergistic effect among the tested treatments. Considering the most promising and effective therapeutic strategy, we hypothesize that defactinib first modulates the TME to enable a better immune infiltration triggered by vaccination, while the P-selectin prevents cancer cell proliferation.
Breast cancer is one of the most frequently detected cancer types in women. Luminal cancers are the most common breast cancers, where luminal B presents high aggressiveness and genes related to tumor proliferation. Breast cancer development is associated with the extracellular matrix (ECM) cellular and soluble factors. The ECM promotes cell migration by connecting the cells' cytoskeleton with focal adhesions (FA). FA presence is related to tyrosine phosphorylation, being the focal adhesion kinase (FAK) one of the most promptly phosphorylated in cancer. FAK overexpression is associated with the proliferation of tumor cells, contributing also to the endothelial cell’s activation, which expresses P-selectin on their surface. The P-selectin action is triggered by its interaction with the P-selectin glycoprotein ligand-1 (PSGL-1) expressed on tumor cells, promoting the circulation of cancer cells and metastasis. Since the interaction between FAK and P-selectin is poorly understood, this project aims to evaluate the synergic anti-tumor effect of the interaction between FAK and P-selectin inhibitors and the DC-targeted nanovaccine delivering tumor-associated antigens and adjuvants, in 3D spheroid models. FAK, phosphorylated FAK (pFAK), P-selectin, and PSGL-1 expression were assessed in EO771 and tumor microenvironment (TME) cells collected from EO771-bearing mice. FAK, pFAK, and P-selectin were found to be overexpressed at EO771 TME cells (> 97%). To modulate the TME toward an immune effector microenvironment, before evaluating the role of defactinib (FAK inhibitor) and KF-38789 (P-selectin inhibitor) on immune cells’ function and frequency within the TME, we evaluated defactinib and KF-38789 impact on the viability of EO771 and TME EO771 cells. Accordingly, defactinib and KF-38789 induced a significant cytotoxic effect on EO771 cells for concentrations above 1 and 0.1 μM, respectively. Moreover, a significant decrease in EO771 TME cell viability was obtained for concentrations of defactinib and KF-38789 above 0.5 and 0.05 µM, respectively. Finally, to evaluate the impact of the synergistic effect between the FAK and P-selectin inhibition combined with a nanovaccine, the co-culture of the 3D spheroids with the EO771 tumors (EO771 TME) and splenocytes was performed. According to the different conditions, the treatment of 3D spheroids of breast cancer with KF-38789 and the nanovaccine alone or in combination with defactinib treatment showed to be promising in modulating the tumor invasion. Finally, a complete sprouting inhibition resulting in the death of 3D spheroid cells was attained with the combination of defactinib and stimulated splenocytes followed by KF-38789, representing the most promising synergistic effect among the tested treatments. Considering the most promising and effective therapeutic strategy, we hypothesize that defactinib first modulates the TME to enable a better immune infiltration triggered by vaccination, while the P-selectin prevents cancer cell proliferation.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Breast cancer Focal adhesion kinase P-selectin 3D models tumor microenvironment Teses de mestrado - 2023