RESUMO
A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.
Assuntos
Imuno-Histoquímica/métodos , Melanoma/imunologia , Melanoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo , Humanos , Ipilimumab/imunologia , Ipilimumab/uso terapêutico , Ativação Linfocitária , Linfócitos do Interstício Tumoral , Macrófagos/metabolismo , Melanoma/tratamento farmacológico , Metastasectomia , Pessoa de Meia-Idade , Estudos Prospectivos , Estatísticas não Paramétricas , Linfócitos T Reguladores/imunologia , Evasão TumoralRESUMO
The Ras oncogene is known to activate three major MAPK pathways, ERK, JNK, p38 and exert distinct cellular phenotypes, that is, apoptosis and invasion through the Ras-MKK3-p38-signaling cascade. We attempted to identify the molecular targets of this pathway that selectively govern the invasive phenotype. Stable transfection of NIH3T3 fibroblasts with MKK3(act) cDNA construct revealed similar p38-dependent in vitro characteristics observed in Ha-Ras(EJ)-transformed NIH3T3 cells, including enhanced invasiveness and anchorage-independent growth correlating with p38 phosphorylation status. To identify the consensus downstream targets of the Ras-MKK3-p38 cascade involved in invasion, in vitro invasion assays were used to isolate highly invasive cells from both, MKK3 and Ha-Ras(EJ) transgenic cell lines. Subsequently a genome-wide transcriptome analysis was employed to investigate differentially regulated genes in invasive Ha-Ras(EJ)- and MKK3(act)-transfected NIH3T3 fibroblasts. Using this phenotype-assisted approach combined with system level protein-interaction network analysis, we identified FOXM1, PLK1 and CDK1 to be differentially regulated in invasive Ha-Ras(EJ)-NIH3T3 and MKK3(act)-NIH3T3 cells. Finally, a FOXM1 RNA-knockdown approach revealed its requirement for both invasion and anchorage-independent growth of Ha-Ras(EJ)- and MKK3(act)-NIH3T3 cells. Together, we identified FOXM1 as a key downstream target of Ras and MKK3-induced cellular in vitro invasion and anchorage-independent growth signaling.