Characterization of Human Melanoma Skin Cancer Models: Paving the Way for Model-Based Melanoma Research and Drug Discovery
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PROVIDER: PRJNA1070660 | ENA |
REPOSITORIES: ENA
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Project description:The need for advanced tissue models in medical research and drug development is more critical than ever. Recent changes in EU law to reduce animal testing and U.S. regulation allowing the FDA to approve drugs without animal trials highlight the importance of these models in advancement of medical science. For the models to be applicable in preclinical studies, it is essential that models are thoroughly characterized so as to understand their applicability and limitations to avoid misinterpretation caused by their limited replication of the biological functions. In this study, we applied bioimaging and RNA sequencing to assess and contrast the architecture and gene expression profiles of various skin models, including melanoma skin models. Our results indicate that these models closely emulate actual skin morphology and gene expression, with the full-thickness (FT) models exhibiting superior resemblance to human skin, especially in the formation of the basement membrane and cellular interactions. These characteristics make the FT model particularly useful for research that focuses on these aspects. Furthermore, the integrity of the skin-like properties and genetic signatures, of both skin and melanoma cell lines, was maintained upon integration of melanoma cells, establishing these models as robust platforms for cancer research and pharmacological development. The responsiveness of the FT melanoma models to vemurafenib was successfully monitored through live imaging, demonstrating their validity as a reliable, reproducible, and humane tool for cancer research and drug development.
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Project description:A robust system using disease relevant cells to systematically evaluate the role in diabetes for loci identified through genome wide association studies (GWAS) is urgently needed. Toward this goal, we created isogenic mutant human embryonic stem cell (hESC) lines in GWAS-identified candidate diabetes genes including CDKAL1, KCNQ1 and KCNJ11, and used directed differentiation to evaluate the function of derivative human beta-like cells. The mutations did not affect the generation of insulin+ cells, but impaired insulin secretion both in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1-/- insulin+ cells also displayed hypersensitivity to lipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-/--specific defects by inhibiting the AP1 (FOS/JUN) pathway. These studies establish a platform using isogenic hESCs to evaluate the function of GWAS-identified loci, and identify a drug candidate that rescues gene-specific defects, paving the way to precision therapy of metabolic diseases.A robust system using disease relevant cells to systematically evaluate the role in diabetes for loci identified through genome wide association studies (GWAS) is urgently needed. Toward this goal, we created isogenic mutant human embryonic stem cell (hESC) lines in GWAS-identified candidate diabetes genes including CDKAL1, KCNQ1 and KCNJ11, and used directed differentiation to evaluate the function of derivative human beta-like cells. The mutations did not affect the generation of insulin+ cells, but impaired insulin secretion both in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1-/- insulin+ cells also displayed hypersensitivity to lipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-/--specific defects by inhibiting the AP1 (FOS/JUN) pathway. These studies establish a platform using isogenic hESCs to evaluate the function of GWAS-identified loci, and identify a drug candidate that rescues gene-specific defects, paving the way to precision therapy of metabolic diseases.
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Project description:Identify miRNAs enriched or overexpressed in melanoma-derived exosomes compared to melanoma cells. We analyzed the 2,578 human miRNAs located in the array according to two ways. The first one identified the differentially expressed miRNAs between melanoma-derived exosomes and their parent cells. In this way, we found 198 miRNAs up-regulated in melanoma cell lines compared to their exosomes and 206 miRNAs up-regulated in exosomes compared to their donor cells. The second way of analysis identifies the most expressed miRNAs in the melanoma-derived exosomes without assumption about their expression in parent cells. We defined two criteria; the first one is a RMA (Robust Multi-Array Average, a good alternative to gene expression value) above 5, corresponding to the mean expression level of the array and a SD ≤ 0.2 between all samples to discover miRNAs expressed uniformly between melanoma-derived exosomes M113 and M117. We identified 44 miRNAs under these criteria.
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Project description:Metastatic melanoma is difficult to treat and is resistant to most current therapies. We hypothesized that stem cell related pathways may play a role in melanoma growth since stem cells and cancer cells share essential properties. Lately, the ABC transporter, ABCB5, as well as Cripto-1, CD20, CD133 and ABCG2 were used to isolate tumorigenic cells. Melanoma cells expressing MDR1 (ABCB1) were found in our lab to be enriched with ABCB5, Nanog, and Oct3/4 expression and were characterized by increased self-renewal capacity and anchorage independence. Melanoma significant heterogeneity was further demonstrated by Quintana et al. who found high prevalence of melanoma initiating cells when implanted into severely immunocompromised mice. Therefore, we took a more general approach in order to unravel pathways that are important for self-renewal and tumorigenicity of melanoma. We enriched for melanoma precursor cells (MPC) using culture conditions shown to enrich for normal melanocyte precursors. MPCs expressed higher levels of nanog and Oct3/4, as well as ABCB1 (MDR1) and ABCB5. Microarray analysis revealed deregulation of genes related to development, invasion, and cell cycle, in addition to upregulation of genes involved in Wnt signaling such as DVL3, DKK1, ID3, JAG1 and Wnt receptor, FZD7. We focused on Wnt signaling and found enrichment of FZD7+ cells in MPCs. Furthermore, FZD7+ cells co-expressed MDR1 and were enriched in highly tumorigenic xenograft parts when tested in mice. Inhibition of the Wnt pathway using secreted frizzled related protein-1 (sFRP1), resulted in attenuation of melanoma proliferation in a dose dependent manner demonstrating the important role of the Wnt pathway in melanoma growth and suggesting this pathway as a target for rational therapy. The canonical Wnt/beta-catenin pathway was not activated in MPC, as revealed using the Tcf Reporter assay. In contrast, we found increased of phosphorylated CAMKII, suggesting activation of the non-canonical Wnt pathway. Our data indicate that precursors within the melanoma tumor may acquire increased survival abilities that should be therapeutically targeted. In particular, we found that the non canonical wnt pathway and the wnt receptor FZD7 play a role in melanoma proliferation and tumorigenicity. We used specialized culture conditions that were found to enrich for normal melanoblasts, the precursors of melanocytes [7]. We found previously that these conditions result in a typical dedifferentiated unpigmented phenotype of melanoma cells in addition to enrichment of self-renewing cells [2]. Further characterization of these precursor cultures prepared from 6 human metastatic melanoma lesions revealed increased expression of stemness genes such as nanog and Oct3/4, as well as two recently reported melanoma precursor markers, MDR1 and ABCB5. Consistent with the normal melanoblast cultures we found increased expression of the melanoblast marker POU3F2 (BRN2, DCT). In contrast, the expression of MITF, a melanocyte differentiation marker was reduced by 85% in MPCs when compared with the primary culture (Figure 1a, relative expression of MPC vs. cells obtained from the same patient and cultured in regular conditions). Cell cycle analysis excluded death of differentiated cells as the cause of the precursor enrichment, as no apoptosis was evident (Figure 1b, c). Interestingly, there was an increase in S phase cells, indicating that these cells are undergoing divisions. Similarly, Wang et al. reported that CD133+ glioma cells have higher percentage of S phase cells as compared to CD133- cells [8]. We concluded that these culture conditions cause dedifferentiation of melanocytes in addition to enrichment by increased divisions of melanoma precursor cells. Therefore, we defined these cells as melanoma precursor cells (MPC). To reveal pathways of self-renewal in melanoma we have used two single cell clones that were prepared from primary human melanoma cultures obtained from a primary lesion. These cell clones differed in the self-renewal capacity (in the limiting dilution assay) and in their morphology: high self-renewal/polygonal vs low self-renewal/spindle shaped. We compared the two cell types described above, cultured in either regular RPMI-based media or culture conditions developed to enrich for normal melanocytic precursors (Cook AL, Donatien PD, Smith AG, Murphy M, Jones MK, et al. (2003) Human melanoblasts in culture: expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3. J Invest Dermatol 121: 1150-1159.), designated MPC (melanoma precursor cells) conditions, for two weeks.
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