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Article

Single-Cell RNA Sequencing Unravels Heterogeneity of the Stromal Niche in Cutaneous Melanoma Heterogeneous Spheroids

1
Laboratory of Genomics and Bioinformatics, Institute of Molecular Genetics of the Czech Academy of Sciences, 142 20 Prague, Czech Republic
2
Department of Informatics and Chemistry, Faculty of Chemical Technology, University of Chemistry and Technology, 160 00 Prague, Czech Republic
3
Institute of Anatomy, First Faculty of Medicine, Charles University, 128 00 Prague, Czech Republic
4
BIOCEV, First Faculty of Medicine, Charles University, 25250 Vestec, Czech Republic
5
Institute of Pathology, First Faculty of Medicine, Charles University, 128 00 Prague, Czech Republic
6
Department of Dermatovenereology, First Faculty of Medicine, Charles University and General University Hospital, 128 00 Prague, Czech Republic
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Cancers 2020, 12(11), 3324; https://doi.org/10.3390/cancers12113324
Received: 15 September 2020 / Revised: 3 November 2020 / Accepted: 8 November 2020 / Published: 10 November 2020
(This article belongs to the Special Issue Tumor Evolution: Progression, Metastasis and Therapeutic Response)
Cutaneous malignant melanoma is one of the most dangerous forms of skin cancer affecting humans. Frequently, it is linked to DNA damage due to ultraviolet radiation. Photoageing along with chronological ageing are therefore critically important factors in melanoma biology. The tissue microenvironment is also heavily affected by induced senescence. There is growing evidence that senescent dermal fibroblasts can consequently promote tumour progression. We focused on the analysis of microenvironmental factors represented in melanoma heterogeneous spheroids by either photodamaged or normal dermal fibroblasts. Our effort was primarily focused on the determination of the functional diversity of fibroblasts in heterogeneous spheroids. Therefore, we analysed these 3D models by single-cell RNA sequencing and advanced bioinformatic analysis. We aimed to map the fibroblast diversity resulting from previously acquired damage caused by exposure to extrinsic and intrinsic stimuli. Using this robust methodology, we highlighted molecules that could become important for the control of melanoma cell–cancer-associated fibroblast interaction as an essential part of the tumour microenvironment.
Heterogeneous spheroids have recently acquired a prominent position in melanoma research because they incorporate microenvironmental cues relevant for melanoma. In this study, we focused on the analysis of microenvironmental factors introduced in melanoma heterogeneous spheroids by different dermal fibroblasts. We aimed to map the fibroblast diversity resulting from previously acquired damage caused by exposure to extrinsic and intrinsic stimuli. To construct heterogeneous melanoma spheroids, we used normal dermal fibroblasts from the sun-protected skin of a juvenile donor. We compared them to the fibroblasts from the sun-exposed photodamaged skin of an adult donor. Further, we analysed the spheroids by single-cell RNA sequencing. To validate transcriptional data, we also compared the immunohistochemical analysis of heterogeneous spheroids to melanoma biopsies. We have distinguished three functional clusters in primary human fibroblasts from melanoma spheroids. These clusters differed in the expression of (a) extracellular matrix-related genes, (b) pro-inflammatory factors, and (c) TGFβ signalling superfamily. We observed a broader deregulation of gene transcription in previously photodamaged cells. We have confirmed that pro-inflammatory cytokine IL-6 significantly enhances melanoma invasion to the extracellular matrix in our model. This supports the opinion that the aspects of ageing are essential for reliable melanoma 3D modelling in vitro. View Full-Text
Keywords: melanoma; spheroids; fibroblasts; subpopulation; heterogeneity; Interleukin-6; cytokine; senescence-associated secretory phenotype; extracellular matrix; single-cell sequencing melanoma; spheroids; fibroblasts; subpopulation; heterogeneity; Interleukin-6; cytokine; senescence-associated secretory phenotype; extracellular matrix; single-cell sequencing
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MDPI and ACS Style

Novotný, J.; Strnadová, K.; Dvořánková, B.; Kocourková, Š.; Jakša, R.; Dundr, P.; Pačes, V.; Smetana, K., Jr.; Kolář, M.; Lacina, L. Single-Cell RNA Sequencing Unravels Heterogeneity of the Stromal Niche in Cutaneous Melanoma Heterogeneous Spheroids. Cancers 2020, 12, 3324. https://doi.org/10.3390/cancers12113324

AMA Style

Novotný J, Strnadová K, Dvořánková B, Kocourková Š, Jakša R, Dundr P, Pačes V, Smetana K Jr., Kolář M, Lacina L. Single-Cell RNA Sequencing Unravels Heterogeneity of the Stromal Niche in Cutaneous Melanoma Heterogeneous Spheroids. Cancers. 2020; 12(11):3324. https://doi.org/10.3390/cancers12113324

Chicago/Turabian Style

Novotný, Jiří, Karolína Strnadová, Barbora Dvořánková, Šárka Kocourková, Radek Jakša, Pavel Dundr, Václav Pačes, Karel Smetana Jr., Michal Kolář, and Lukáš Lacina. 2020. "Single-Cell RNA Sequencing Unravels Heterogeneity of the Stromal Niche in Cutaneous Melanoma Heterogeneous Spheroids" Cancers 12, no. 11: 3324. https://doi.org/10.3390/cancers12113324

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