Project description:Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target. Four primary melanomas (subtype SSMM) were dissociated into single cells. From each of these single cell suspensions, side population (SP) and bulk \main population\ (MP) cells were isolated based on their Hoechst-effluxing capability using fluorescence-activated cell sorting. Gene expression of SP cells is analyzed relative to the gene expression of the corresponding MP cells.

Project description:The crosstalk between tumor and immune cells has important implications for cancer treatment, in particular for immunotherapies. Pancreatic ductal adenocarcinoma (PDAC) generally displays a chemoresistant phenotype and is refractory to immune-based therapies. Highly tumorigenic and stem-like cells known as cancer stem cells (CSCs) within PDAC tumors have been demonstrated to contribute to its aggressiveness, metastatic capacity and chemoresistance. Here, we show that CSCs could be also relevant to establish an immunosuppresive environment. Using genetically engineered mouse models (GEMMs) of PDAC we isolated a triple positive (EpCAM+, Sca-1+ and CD133+) population of tumor cells, functionally validated these cells as bona fide CSCs and probed their transcriptional profile by microarray analysis. In addition to stem pathways, signatures related to tumor immune evasion, immune modulation, invasiveness, and innate immune response were enriched in this population. Peptidoglycan recognition protein 1 (Pglyrp1), an innate immune response anti-bacterial protein expressed primarily by neutrophils, was highly expressed in this triple-positive population and in CSC-enriched spheres from human cell lines. Interestingly, modulation of Pglyrp1 expression in murine PDAC primary cultures affected their immune evasive properties, specifically their ability to escape macrophage- and T-cell-mediated killing in vitro and to establish tumors in immunocompetent hosts. Pglyrp1 expression in tumor cells seemed to interfere with TNFα-mediated killing by T cells and to promote activated T-cell cytotoxicity. Importantly, these results could be validated in CSCs from human patient-derived xenografts, and PGLYRP1 protein levels were also significantly higher in PDAC patient samples versus healthy controls. Thus, our study demonstrates a hierarchical organization of cancer cells in a murine model of PDAC, validating the CSC concept, and identifies Pglyrp1 as a putative PDAC biomarker that confers immune privilege properties to CSCs which could have important therapeutic implications.

Dataset Information

Human pancreatic adenocarcinoma contains a side population resistant to gemcitabine

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Human pancreatic adenocarcinoma contains a side population resistant to gemcitabine.

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