E-MTAB-9216
Single-cell RNA-seq (10x Chromium V3) of heterogeneous spheres consisting of melanoma cell line and two types of human primary dermal fibroblasts
Photo-damage represents an important aspect of cutaneous carcinogenesis. The photo-damaged microenvironment significantly facilitates progression of malignant melanoma. Therefore human primary dermal fibroblasts from photo-damaged adult skin and juvenile sun-protected skin were isolated and expanded as described by Dvorankova et al. (PMID: 29675784). Low passage fibroblasts were mixed in suspension (in ratio 1:1) with G361 malignant melanoma cell line (CVCL_1220). Using the hanging drop technique (PMID: 29786551), the melanoma/fibroblast spheres containing 50,000 cells per sphere were formed during the next 72 hours. The organoids were consequently maintained for another 2 days in non-adhesive culture wells in complete DMEM culture medium. At this time, heterogeneous spheres were harvested for immunohistochemical analysis or for further invasion studies. Single-cell suspensions from heterogeneous spheres were analysed by single-cell RNA sequencing (10x Chromium V3) to reveal the transcriptional heterogeneity of model cell populations under given conditions with emphasis on the features of dermal fibroblasts exposed to the influence of malignant melanoma.
Photo-damage represents an important aspect of cutaneous carcinogenesis. The photo-damaged microenvironment significantly facilitates progression of malignant melanoma. Therefore human primary dermal fibroblasts from photo-damaged adult skin and juvenile sun-protected skin were isolated and expanded as described by Dvorankova et al. (PMID: 29675784). Low passage fibroblasts were mixed in suspension (in ratio 1:1) with G361 malignant melanoma cell line (CVCL_1220). Using the hanging drop technique (PMID: 29786551), the melanoma/fibroblast spheres containing 50,000 cells per sphere were formed during the next 72 hours. The organoids were consequently maintained for another 2 days in non-adhesive culture wells in complete DMEM culture medium. At this time, heterogeneous spheres were harvested for immunohistochemical analysis or for further invasion studies. Single-cell suspensions from heterogeneous spheres were analysed by single-cell RNA sequencing (10x Chromium V3) to reveal the transcriptional heterogeneity of model cell populations under given conditions with emphasis on the features of dermal fibroblasts exposed to the influence of malignant melanoma.