Project description:Summary: Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function Background: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi subpopulations of cancer cells some of which may possess stem cell-like properties supporting the notion of plasticity. Although useful for certain tumors, the use of the sphere-formation assay to identify stem cell-like or tumor initiating cells subpopulations in human melanoma has been recently challenged. Our study reveals that this assay predicts a functional phenotype associated with aggressive behavior of tumor cells. Methodology/Principal Findings: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic (SLM8) or advanced primary (Mela1) tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages, and showed enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not extensive self-renewal or enhanced tumorigenicity when compared to their adherent counterparts. To determine whether melanoma spheroids in our model could predict a molecular or functional phenotype, we performed gene expression profiling experiments using Affymetrix microarrays. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that these spheroids are endowed with enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Thus, our findings reveal novel immune-modulator function of melanoma spheroid cells and suggest specific roles for these spheroids in invasion and in evasion of antitumor immunity. Conclusion/Significance: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroid cells reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and could therefore, be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability. 12 Total samples were analyzed: SLM8 adherent (SLMA) and spheroids (SLMS) cells, and Mela1 adherent (MelaA) and spheroid (MelaS) cells, all performed in triplicates. Paired statistical analyses were performed using Student's paired t-test on the gene signal intensities (gene level) and results were considered statistically significant at p-values <=0.05 and fold-change >=1.5.

Project description:Summary: Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function Background: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi subpopulations of cancer cells some of which may possess stem cell-like properties supporting the notion of plasticity. Although useful for certain tumors, the use of the sphere-formation assay to identify stem cell-like or tumor initiating cells subpopulations in human melanoma has been recently challenged. Our study reveals that this assay predicts a functional phenotype associated with aggressive behavior of tumor cells. Methodology/Principal Findings: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic (SLM8) or advanced primary (Mela1) tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages, and showed enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not extensive self-renewal or enhanced tumorigenicity when compared to their adherent counterparts. To determine whether melanoma spheroids in our model could predict a molecular or functional phenotype, we performed gene expression profiling experiments using Affymetrix microarrays. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that these spheroids are endowed with enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Thus, our findings reveal novel immune-modulator function of melanoma spheroid cells and suggest specific roles for these spheroids in invasion and in evasion of antitumor immunity. Conclusion/Significance: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroid cells reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and could therefore, be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

Project description:Full title: Comprehensive Characterization of Three-Dimensional Models for Prostate Cancer Growth and Invasion in Laminin-rich Extracellular Matrix Prostate Cancer (PrCa) cells undergo acinar morphogenesis and spheroid formation in three-dimensional (3D) culture, supported by laminin-rich extracellular matrix (lrECM, Matrigel). We developed miniaturized 3D model systems that facilitate investigation of morphogenesis and invasion of normal and PrCa cell lines in lrECM. Primary and non-transformed cell lines formed round structures with strong cell-cell contacts and epithelial polarization, lumen and a complete basal lamina (BL). In contrast, most PrCa cell lines formed either defective, “mass” spheroids with incomplete BL, or invasive “stellate” structures. The bioinformatic analyses of genome-wide mRNA expression data revealed massive alteration of key functional and signaling pathways in 3D cultures, with lipid and steroid metabolism, epigenetic reprogramming, and differentiation-related transcription factors induced across all cell lines by lrECM. In invasive cells, AKT, PI3Kinase, mTOR, and hedgehog signaling pathways were most highly activated, validated by small molecule inhibitors compounds specifically targeting key regulatory molecules. Compounds against AKT and PI3kinase pathways were significantly more effective in invasive cells, compared to mass or round/normal phenotype spheroids, and monolayer culture. A severe morphologic conversion was observed in PC-3 and PC-3M cells, transforming initially round, normal-appearing epithelial spheroids into rapidly invading cell masses. Markers for EMT (epithelial-mesenchymal transition) were highly expressed already in early stage, round spheroids prior to invasive conversion, and were not further increased in invasive cells. This indicates that PrCa cells can display extraordinary plasticity. EMT may be involved in providing a metastable genotype that allows morphological transformation, but is not be required for invasive processes themselves. Total RNA was obtained from non-transformed prostate epithelial cells and prostate cancer cells cultured in monolayer and three-dimensional laminin-rich extracellular matrix (growth factor-reduced Matrigel).

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Kidney spheroids generate a functional quiescent niche leading to enhanced tubulogenic potency of human kidney-derived cells

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