A Unique Signature for Cancer-Associated Fibroblasts in Melanoma Metastases

Abstract

Cancer-associated fibroblasts (CAFs) represent a central cell population of the tumor microenvironment (TME). Recently, single-cell RNA-sequencing (scRNA-seq) analyses of primary tumors of different cancer entities yielded different classifications of CAF subsets underscoring the heterogeneity of CAFs within the TME. Here, we analyzed the transcriptional signatures of approximately 8400 CAFs and normal fibroblasts by scRNA-seq and compared genetic profiles of CAFs from murine melanoma primary tumors to CAFs from corresponding melanoma lung metastases. This revealed distinct subsets for primary tumor and metastasis-specific CAF populations, respectively. Combined with the spatial characterization of metastasis CAFs at the RNA and protein level, scRNA analyses indicate tumor-dependent crosstalk between neutrophils and CAFs, mediated via SAA3 and IL1b-related signaling pathways, which can be recapitulated in vitro. Analyzing tissue sections of human patient samples, this interaction was found to be present in human melanoma metastasis. Taken together, our data highlight unique characteristics of metastasis CAFs with potential therapeutic impact for melanoma metastasis.

Keywords: CAF; SAA; melanoma; metastasis; scRNA sequencing.

FIGURE 1

scRNA data mirror the complex cellular composition of primary tumors and metastases. (a) Schematics of workflow. (b) Cell clusters based on 10× Genomics scRNA‐sequencing analysis visualized by uniform manifold approximation and projection (UMAP). Cells are color coded according to cell types. (c) Fibroblast clusters extracted from (b). (d) The violin plot shows the expression of Saa3 for fibroblast clusters. (e) IF staining of collagen I, and SAA3 on lung metastasis and primary tumor. Time point of metastasis = 6 weeks after primary tumor resection. Dashed line: tumor/TME margin. (f) RNAscopeTM in situ hybridization of Saa3 and IF staining of collagen I on lung metastasis. Upper line: Leading edge, lower line: Tumor core. Time point of metastasis = 2 months after primary tumor resection. Scale bar = 20 μm.

FIGURE 2

Il1b signaling as a putative activator of Saa3 expression in metastasis CAFs. (a) Expression of putatively Saa3‐inducing receptors on fibroblasts and (b) expression of corresponding ligands in all cells. The dashed rectangle in (a) highlights the metastasis CAF cluster. (c) Immunofluorescence staining of MPO and CitH3 on a lung metastasis developed in a mT/mG mouse. Time point of metastasis occurrence = 2 months after resection of the primary tumor. Scale bar = 20 μm. (d) mRNA expression of Saa3 after 6 h of IL1β treatment. Bar charts show mRNA levels as fold change to the corresponding PBS control condition of three biological replicates, respectively, with each of three technical replicates. Error bars show ± SD.

FIGURE 3

SAA1 levels in human CAFs increase upon Il1b treatment. (a) Immunofluorescence staining of aSMA and SAA1 on human lung and liver melanoma metastasis. Images represent the staining of one of three individual melanoma patient samples yielding very similar results. Scale bar = 20 μm. (b) mRNA expression of SAA1 in GMO1604A after coculture with murine lung metastasis melanoma cells. Bar charts show mRNA levels as fold change to the corresponding GMO1604A monoculture control condition of two biological replicates, respectively, with each of two technical replicates. Error bars show ± SD. (c) mRNA expression of SAA1 after 6 h of IL1β treatment. Bar charts show mRNA levels as fold change to the corresponding PBS control condition of three biological replicates, respectively, with each of two to three technical replicates. Error bars show ± SD.