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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2020.tde-20042022-145255
Document
Author
Full name
Gretel Rodríguez Rodríguez
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Lepique, Ana Paula (President)
Amano, Mariane Tami
Andrade, Luciana Nogueira de Sousa
Barbuto, Jose Alexandre Marzagao
Title in Portuguese
Estudo do papel do eixo IL-6/STAT-3 e G-CSF no microambiente tumoral e a nível sistêmico
Keywords in Portuguese
Cancer do colo do utero
Microambiente tumoral
Papilomavirus Humano
STAT-3. NF-B
Vias de sinalização
Abstract in Portuguese
A infecção por Papilomavírus Humano (HPV) de alto risco oncogênico é o principal fator etiológico do câncer cervical e uma porcentagem de outros cânceres anogenitais e orofaríngeos. Grupos de pesquisa mostraram que as células transformadas por HPV secretam IL-6 e G-CSF. O objetivo deste trabalho foi caracterizar o papel da IL-6 e G-CSF e a sinalização por STAT-3 em um modelo experimental associado ao HPV observando o microambiente tumoral e os efeitos sistêmicos do tumor sobre o sistema imune. Para isso, utilizamos a linhagem celular TC-1 que expressa os oncogenes E6 e E7 de HPV16 e Ha-ras. Este modelo experimental de tumor reproduz várias características do câncer do colo do útero. Animais C57BL/6 foram inoculados com células tumorais TC-1 por via subcutânea. Quando os tumores ficaram palpáveis, os camundongos foram tratados com anticorpos neutralizantes anti-G-CSF e/ou anti-IL-6 a cada 3 dias. Observamos que cada anticorpo neutralizante, de maneira independente, inibiu significativamente o crescimento do tumor, enquanto que a combinação de ambos foi ainda mais eficiente. Também, o anti-G-CSF ou combinação dos anticorpos reduziu a frequência das células mielóides na medula óssea e no baço. Houve um aumento na frequência de células T CD8+ nos tumores de camundongos tratados com anticorpos anti-IL-6 ou combinados. Observamos que a via NF-B apresentou maior fosforilação de p65 nas células apresentadoras de antígenos (APCs) e células T do baço dos camundongos tratados. Portanto, nós hipotetizamos que a via STAT-3 poderia interferir na sinalização de NF-B, de modo que após induzir os tumores, os animais foram tratados ou não com NSC 74859 (inibidor farmacológico de STAT-3) diariamente. Observamos que a inibição da STAT-3 reduziu significativamente o crescimento tumoral, reduziu a frequência de células mielóides no baço e houve o aumento dos linfócitos T CD8+ no infiltrado tumoral e promoveu um aumento na expressão de p65 NF-kB nos tumores e no baço. Além disso, promoveu um aumento na proliferação de linfócitos T CD8+-in vitro. Em seguida, após induzir os tumores em camundongos C57BL/6 foram tratados com NSC 74859 ou com o veículo ou com a combinação de NSC 74859 e JSH-23 (inibidor de NF-B). Observamos que o efeito do tratamento com NSC74859 de diminuição do crescimento do tumor foi parcialmente perdido nos animais tratados simultaneamente com o inibidor de NF-B. Finalmente, reconstituímos camundongos RAG1-/- que apresentaram tumor com linfócitos de doadores com tumor C57BL/6 tratados com inibidor STAT-3 ou controles. Observamos que os linfócitos transferidos adotivamente de doadores com tumor tratados com NSC 74859 protegiam os receptores contra o crescimento do tumor. Os linfócitos de ambos os doadores foram capazes de colonizar os linfonodos, e os dos doadores tratados pareciam ser mais eficientes na infiltração no tumor. Nossos dados indicam que IL-6 e G-CSF expressos no microambiente tumoral possuem efeitos locais e sistémicos que promovem o aumento nas populações de células mielóides com potencial fenótipo imunossupressor contribuindo com a diminuição de infiltrado de linfócitos T e a inibição de NF-B, provavelmente através da interferência da via STAT-3, fatores que em conjunto podem estar favorecendo a evasão imune tumoral.
Title in English
Study of the role of IL-6/STAT-3 and G-CSF axis in the tumor microenvironment and systemic effect
Keywords in English
Cervical cancer
Human papillomavirus
NF-B
Signaling pathways
STAT-3
Tumor microenvironment
Abstract in English
High-risk oncogenic human papillomavirus (HPV) infection is the main etiological factor of cervical cancer and a percentage of other anogenital and oropharyngeal cancers. Research groups showed that HPV-transformed cells secrete IL-6 and G-CSF. The objective of this work was to characterize the role of IL-6 and G-CSF and STAT-3 signaling in an HPV-associated experimental model observing the tumor microenvironment and the systemic effects of the tumor on the immune system. For this, we used the TC-1 cell line that expresses HPV16 and Ha-ras oncogenes E6 and E7. This experimental tumor model reproduces several characteristics of cervical cancer. C57BL/6 animals were inoculated with TC-1 tumor cells subcutaneously. When the tumors were palpable, mice were treated with neutralizing anti-G-CSF and/or anti-IL-6 antibodies every 3 days. We observed that each independently neutralizing antibody significantly prevented tumor growth, while the combination of both was even more efficient. In addition, anti-G-CSF or antibody combination reduced the frequency of myeloid cells in the bone marrow and in the spleen. There was an increase in CD8+T cell frequency in mouse tumors treated with anti-IL-6 antibodies or in combination. We observed that the NF-B pathway showed higher p65 phosphorylation in antigen presenting cells (APCs) and spleen T cells of the treated mice. Therefore, we hypothesized that the STAT-3 pathway could interfere with NF-B signaling, so that after inducing tumors, mice they were treated or not with NSC 74859 (STAT-3 pharmacological inhibitor) daily. We observed that STAT-3 inhibition significantly reduced tumor growth, reduced myeloid cell frequency in the spleen and increased CD8+T lymphocytes in the tumor infiltrate and promoted an increase in p65 NF-B expression in tumors and spleen. Moreover, promotes an increase in CD8+T lymphocyte proliferation -in vitro. Then, after inducing tumors in C57BL/6 mice they were treated with either NSC 74859 or the vehicle or the combination of NSC 74859 and JSH-23 (inhibitor of NF-B). We observed that the effect of NSC74859 treatment of tumor growth reduction was partially lost in animals treated simultaneously with the NF-B inhibitor. Finally, we reconstituted RAG1-/- mice that presented tumor with lymphocytes from STAT-3 inhibitor-treated C57BL/6 donors or controls. We observed that adoptively transferred lymphocytes from NSC 74859-treated tumor donors protected the receptors against tumor growth. Lymphocytes from both donors were able to colonize lymph nodes, and those from treated donors appeared to be more efficient at infiltrating the tumor. Our data indicate that IL-6 and G-CSF expressed in the tumor microenvironment have local and systemic effects that promote an increase in myeloid cell populations with potential suppressive phenotype contributing to decreased T lymphocyte infiltrate and NF-B inhibition. probably through the interference of the STAT-3 pathway, factors that together may be favoring tumor immune evasion.
 
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Release Date
2024-04-19
Publishing Date
2022-05-19
 
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