Protocol to characterize the melanoma tumor immune microenvironment in mice from single cell to flow cytometry analysis.

Here, we present a protocol to study and describe immune cells that surround or infiltrate tumor cells or get through the body of a melanoma syngeneic mice model. We describe steps for creating and establishing the syngeneic mouse model, euthanasia, and tumor or organ harvest. We then detail procedures to rapidly achieve a single-cell suspension from different tissue samples to further quantify and analyze the phenotype of the immune cell population (lymphocytes T and B, tumor-associated macrophages, and myeloid-derived suppressor cells) by flow cytometry.

ARID1A-deficient cells require HDAC6 for progression of endometrial carcinoma.

AT-rich interactive domain-containing protein 1A (ARID1A) loss-of-function mutation accompanied by a loss of ARID1A protein expression is frequently observed in endometrial carcinomas. However, the molecular mechanisms linking these genetic changes to the altered pathways regulating tumour initiation, maintenance and/or progression remain poorly understood. Thus, the main aim of this study was to analyse the role of ARID1A loss of function in endometrial tumorigenesis. Here, using different endometrial in vitro and in vivo models, such as tumoral cell lines, 3D primary cultures and metastatic or genetically modified mouse models, we show that altered expression of ARID1A is not enough to initiate endometrial tumorigenesis. However, in an established endometrial cancer context, ARID1A loss of function accelerates tumoral progression and metastasis through the disruption of the G2/M cell cycle checkpoint and ATM/ATR-mediated DNA damage checkpoints, increases epithelial cell proliferation rates and induces epithelial mesenchymal transition through the activation of histone deacetylase 6 (HDAC6). Next, we demonstrated that the inhibition of HDAC6 function, using the HDAC6-specific inhibitor ACY1215 or by transfection with HDAC6 short hairpin RNA (shRNA), can reverse the migratory and invasive phenotype of ARID1A-knockdown cells. Further, we also show that inhibition of HDAC6 activity causes an apoptotic vulnerability to etoposide treatments in ARID1A-deficient cells. In summary, the findings exposed in this work indicate that the inhibition of HDAC6 activity is a potential therapeutic strategy for patients suffering from ARID1A-mutant endometrial cancer diagnosed in advanced stages.

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics.

BACKGROUND: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. METHODS: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. RESULTS: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. CONCLUSIONS: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

T-Type Calcium Channels: A Potential Novel Target in Melanoma.

T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAFV600E-mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic option.

T-Type Calcium Channels as Potential Therapeutic Targets in Vemurafenib-Resistant BRAFV600E Melanoma.

Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common BRAFV600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAFV600E-mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAFV600E-mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAFV600E-mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.

In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.

Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.