<p>  </p> <p>Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is essentially the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, largely attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for the development of effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are extracellular vesicles (EVs). For the past years, the study of EVs released by tumor cells have gained attention, given their impact in modulating the tumor immunity, supporting tumorigenesis, and contributing to the development of metastasis. However, the mechanisms though which tumor EVs contribute to tumor development are still understudied. In this study, we isolated and characterized small EVs, also referred as exosomes, from NSCLC cell lines (H358, Calu6, H460, SKMES-1) and investigated their release, uptake, and impact in non-tumorigenic lung epithelial cells recipient cells (BEAS-2B and HBEC). Our results demonstrated that EVs from NSCLC can induce migration and invasion of non-tumorigenic epithelial cells, and impair epithelial barrier permeability, suggesting their role in supporting tumorigenesis and metastasis. Furthermore, we assessed the immunomodulatory effects of NSCLC EVs on anti-tumor immune cells, particularly T cells. Our findings revealed a suppressive effect of EVs derived from mutant KRASG12C NSCLC (H358) on T-cell proliferation, suggesting their contribution to immune evasion mechanisms in mutant KRAS tumors. To dissect the underlying mechanisms, we employed a dual approach utilizing genetic manipulation (shRNA knockdown) and a small molecule inhibitor (ARS-1620) targeting the oncogenic KRASG12C. Our data demonstrated that targeting KRASG12C impaired the EV-driven cancer phenotypes, highlighting the pivotal role of KRAS oncogenic signaling in tumorigenesis and immune suppression mediated by EVs. Overall, our study sheds light on the crucial involvement of tumor derived EVs in NSCLC progression, both in terms of promoting cellular migration and invasion, as well as dampening anti-tumor immune responses. By elucidating the mechanisms underlying EV-driven tumorigenesis and highlighting the therapeutic potential of targeting KRAS signaling, our findings pave the way for the development of novel and effective therapeutic agents for NSCLC.</p>

Câncer de Pulmão de Não-pequenas células (CPNPC) é o maior problema de saúde pública atualmente no mundo.Encontramos em nossas pesquisas que os níveis de mRNA da CFL-1 (cofilina) podem ser usados como um biomarcador prognóstico em biópsias de tumores de CPNPC. Em nossos estudos, estabelecemos e optimizamos um método simples de imunohistoquímica semiquantitativa (SQ-IHC) para quantificar em biópsias de tumores sua aplicação em uma coorte retrospectiva de pacientes diagnosticados com CPNPC, para explorar seu papel prognóstico. Tivemos acesso a uma coleção de arquivos médicos bem delineada de 50 tumores de pacientes, juntamente com informações relevantes clínicopatológicas de 5 anos de acompanhamento hospitalar. A análise imunoistoquímica e semiquantitativa da cofilina foi realizada em um estudo cego para confiabilidade do resultado clínico. A associação entre imunoconteúdo de cofilina e resultado clínico foi assegurando utilizando as curvas de mortalidade de Kaplan-Meier e o test log-rank. Pacientes foram agrupados pela expressão do biomarcador ou pelo seu estadiamento. Imunoconteúdo de cofilina (em densidade óptica) em biópsias de tumor foram capazes de determiner entre bom e mau prognóstico, onde altos níveis de cofilina foram correlacionados com baixa razão de sobrevida. Um método simples de imunoistoquímica semiquantitativa foi bem desenvolvido para avaliação quantitativa de cofilina em CPNPC. Nosso método mostrou boa sensibilidade/especificidade para indicar resultado em pacientes e por isso deve ser empregado em estudo prospectivo, de larga escala, com triagens clínicas randomizadas para estabelecer o valor prognóstico do imunoconteúdo de cofilina em CPNPC. / Nonsmall cell lung cancer (NSCLC) is a major public health problem worldwide. Previously we found that CFL1 (cofilin-1) mRNA levels can be used as a prognostic biomarker in NSCLC tumor biopsies. In this study, we established and optimized a simple semi-quantitative immunohistochemistry (SQ-IHC) method for cofilin quantification in tumor biopsies and applied it in a retrospective cohort of NSCLC patients, to exploit prognostic role. We accessed a well-defined archival collection of tumor samples from 50 patients with relevant clinicopathologic information and 5 years follow-up. Immunohistochemistry and semi-quantitative analysis of cofilin were performed blinded to clinical outcome. Association between cofilin immunocontent and clinical outcome was assessed using standard Kaplan-Meier mortality curves and the log-rank test. Patients were clustered according to either biomarker expression level or NSCLC stage grouping. Cofilin immunocontent (in optical densities) in tumor biopsies was able to discriminate between good and bad prognosis, where high cofilin levels are correlated with lower overall survival rate (P < .05). A simple semi-quantitative immunohistochemical method has been developed for quantitative evaluation of cofilin in NSCLC. Our method showed good sensitivity/specificity to indicate the outcome of patients and should be further employed in a prospective, large-scale, randomized clinical trial to establish the prognostic value of cofilin immunocontent in NSCLC.

Câncer de Pulmão de Não-pequenas células (CPNPC) é o maior problema de saúde pública atualmente no mundo.Encontramos em nossas pesquisas que os níveis de mRNA da CFL-1 (cofilina) podem ser usados como um biomarcador prognóstico em biópsias de tumores de CPNPC. Em nossos estudos, estabelecemos e optimizamos um método simples de imunohistoquímica semiquantitativa (SQ-IHC) para quantificar em biópsias de tumores sua aplicação em uma coorte retrospectiva de pacientes diagnosticados com CPNPC, para explorar seu papel prognóstico. Tivemos acesso a uma coleção de arquivos médicos bem delineada de 50 tumores de pacientes, juntamente com informações relevantes clínicopatológicas de 5 anos de acompanhamento hospitalar. A análise imunoistoquímica e semiquantitativa da cofilina foi realizada em um estudo cego para confiabilidade do resultado clínico. A associação entre imunoconteúdo de cofilina e resultado clínico foi assegurando utilizando as curvas de mortalidade de Kaplan-Meier e o test log-rank. Pacientes foram agrupados pela expressão do biomarcador ou pelo seu estadiamento. Imunoconteúdo de cofilina (em densidade óptica) em biópsias de tumor foram capazes de determiner entre bom e mau prognóstico, onde altos níveis de cofilina foram correlacionados com baixa razão de sobrevida. Um método simples de imunoistoquímica semiquantitativa foi bem desenvolvido para avaliação quantitativa de cofilina em CPNPC. Nosso método mostrou boa sensibilidade/especificidade para indicar resultado em pacientes e por isso deve ser empregado em estudo prospectivo, de larga escala, com triagens clínicas randomizadas para estabelecer o valor prognóstico do imunoconteúdo de cofilina em CPNPC. / Nonsmall cell lung cancer (NSCLC) is a major public health problem worldwide. Previously we found that CFL1 (cofilin-1) mRNA levels can be used as a prognostic biomarker in NSCLC tumor biopsies. In this study, we established and optimized a simple semi-quantitative immunohistochemistry (SQ-IHC) method for cofilin quantification in tumor biopsies and applied it in a retrospective cohort of NSCLC patients, to exploit prognostic role. We accessed a well-defined archival collection of tumor samples from 50 patients with relevant clinicopathologic information and 5 years follow-up. Immunohistochemistry and semi-quantitative analysis of cofilin were performed blinded to clinical outcome. Association between cofilin immunocontent and clinical outcome was assessed using standard Kaplan-Meier mortality curves and the log-rank test. Patients were clustered according to either biomarker expression level or NSCLC stage grouping. Cofilin immunocontent (in optical densities) in tumor biopsies was able to discriminate between good and bad prognosis, where high cofilin levels are correlated with lower overall survival rate (P < .05). A simple semi-quantitative immunohistochemical method has been developed for quantitative evaluation of cofilin in NSCLC. Our method showed good sensitivity/specificity to indicate the outcome of patients and should be further employed in a prospective, large-scale, randomized clinical trial to establish the prognostic value of cofilin immunocontent in NSCLC.

Câncer de Pulmão de Não-pequenas células (CPNPC) é o maior problema de saúde pública atualmente no mundo.Encontramos em nossas pesquisas que os níveis de mRNA da CFL-1 (cofilina) podem ser usados como um biomarcador prognóstico em biópsias de tumores de CPNPC. Em nossos estudos, estabelecemos e optimizamos um método simples de imunohistoquímica semiquantitativa (SQ-IHC) para quantificar em biópsias de tumores sua aplicação em uma coorte retrospectiva de pacientes diagnosticados com CPNPC, para explorar seu papel prognóstico. Tivemos acesso a uma coleção de arquivos médicos bem delineada de 50 tumores de pacientes, juntamente com informações relevantes clínicopatológicas de 5 anos de acompanhamento hospitalar. A análise imunoistoquímica e semiquantitativa da cofilina foi realizada em um estudo cego para confiabilidade do resultado clínico. A associação entre imunoconteúdo de cofilina e resultado clínico foi assegurando utilizando as curvas de mortalidade de Kaplan-Meier e o test log-rank. Pacientes foram agrupados pela expressão do biomarcador ou pelo seu estadiamento. Imunoconteúdo de cofilina (em densidade óptica) em biópsias de tumor foram capazes de determiner entre bom e mau prognóstico, onde altos níveis de cofilina foram correlacionados com baixa razão de sobrevida. Um método simples de imunoistoquímica semiquantitativa foi bem desenvolvido para avaliação quantitativa de cofilina em CPNPC. Nosso método mostrou boa sensibilidade/especificidade para indicar resultado em pacientes e por isso deve ser empregado em estudo prospectivo, de larga escala, com triagens clínicas randomizadas para estabelecer o valor prognóstico do imunoconteúdo de cofilina em CPNPC. / Nonsmall cell lung cancer (NSCLC) is a major public health problem worldwide. Previously we found that CFL1 (cofilin-1) mRNA levels can be used as a prognostic biomarker in NSCLC tumor biopsies. In this study, we established and optimized a simple semi-quantitative immunohistochemistry (SQ-IHC) method for cofilin quantification in tumor biopsies and applied it in a retrospective cohort of NSCLC patients, to exploit prognostic role. We accessed a well-defined archival collection of tumor samples from 50 patients with relevant clinicopathologic information and 5 years follow-up. Immunohistochemistry and semi-quantitative analysis of cofilin were performed blinded to clinical outcome. Association between cofilin immunocontent and clinical outcome was assessed using standard Kaplan-Meier mortality curves and the log-rank test. Patients were clustered according to either biomarker expression level or NSCLC stage grouping. Cofilin immunocontent (in optical densities) in tumor biopsies was able to discriminate between good and bad prognosis, where high cofilin levels are correlated with lower overall survival rate (P < .05). A simple semi-quantitative immunohistochemical method has been developed for quantitative evaluation of cofilin in NSCLC. Our method showed good sensitivity/specificity to indicate the outcome of patients and should be further employed in a prospective, large-scale, randomized clinical trial to establish the prognostic value of cofilin immunocontent in NSCLC.

Chemoresistance is a major obstacle in successful chemotherapy. This research explored several genetic and epigenetic factors involved in chemoresistance, angiogenesis and metastasis in the human non-small cell lung cancer cell line A549 and its paclitaxel resistant subclone A549-T24. We characterized various morphological and biochemical differences (with a special focus on the Bcl-2 family of apoptotic regulators) between the two cell lines. Although paclitaxel induced apoptosis in both the cell lines, the subclone was 10 fold more resistant to this drug. Our immunocytochemistry data indicated that VEGF (a potent inducer of angiogenesis) and VEGF receptor-2 mRNA expression levels were higher in A549-T24 cells compared to those in A549 cells. We also observed a higher angiogenic potential in A549-T24 cells as determined by the effect of these cells on endothelial cell growth and cell sprouting using EA.hy926 human umbilical vein endothelial cells and rat aortic ring models, respectively. Our data suggested that tumor cell-induced angiogenesis may involve activation of nitric oxide, calcium and PI3K signaling pathways. Intracellular calcium [Ca 2+ ] i plays a critical role in cellular growth and apoptosis. We characterized alterations in the regulatory pathways of [Ca 2+ ] i handling in our cell lines. While the endoplasmic reticulum calcium store ([Ca 2+ ] er ) was significantly lower, calcium influx pathways were considerably inhibited in A549-T24 cells compared to A549 cells. We investigated the actions of 2-aminoethoxydiphenyl borate to release [Ca 2+ ] er and to block store operated Ca 2+ channels. In addition, we studied the role of Ca 2+ in thapsigargin-induced apoptosis in A549 cells. Integrins, a family of cell adhesion proteins, inhibit apoptosis via activation of survival signals. Integrin (mainly β 3 and α 5 ) gene expression patterns and functions differed between A549 and A549-T24 cell lines, suggesting that adhesion to matrix elements may modulate the response to paclitaxel. Indeed, adhesion to extracellular matrix proteins via integrins resulted in a further decrease in chemosensitivity in A549-T24 cells with simultaneous inactivation of BAD (a pro-apoptotic protein). Cell adhesion mediated drug resistance was successfully reversed using integrin blockers (GRGDS and LM609). In summary, our data suggested that chemoresistance is multifactorial. Understanding the molecular mechanisms of chemoresistance will enable the design of better anticancer agents.