Figures and data
Keratinocytes in the melanoma microenvironment undergo EMT-like changes.
(A) Generation of transparent zebrafish with GFP-labeling of keratinocytes. Casper Triples (mitfa-/-;mitfa:BRAFV600E;tp53-/-;mpv17-/-) were injected with the Tol2Kit 394 vector containing a krt4:eGFP cassette and tol2 mRNA. Brightfield shows a transparent zebrafish while fluorescence imaging shows eGFP-labeling of keratinocytes. (Scale bar = 5mm)
(B) Schematic of TEAZ (Transgene Electroporation in Adult Zebrafish). Plasmid mix containing miniCoopR:tdTomato, mitfa:Cas9, zU6:sgptena, zU6:sgptenb, and the tol2 plasmid was injected superficially in the flank of the zebrafish. Electroporation of the injection site results in rescue of melanocyte precursors and the generation of a localized melanoma that could be analyzed by microscopy and FACS.
(C) Brightfield and immunofluorescence of zebrafish 8-weeks post-TEAZ with localized and fluorescently labeled melanoma. (Scale bar = 5mm)
(D) Immunofluorescence imaging of TEAZ region after 8-weeks, comparing empty vector control vs. miniCoopR:tdT conditions, with yellow dotted circles indicating general area of dissection for FACS. (Scale bar = 1mm)
(E) Confocal imaging of zebrafish epidermis. Normal epidermis of Tg(krt4:eGFP) Casper Triple post-TEAZ with empty vector control shows eGFP-labeled, polygonal shaped keratinocytes regularly connected while epidermis with melanoma generated with miniCoopR-drived melanocyte rescue shows disrupted epidermis and irregularly shaped keratinocytes. (Scale bar = 50um)
(F) qPCR of FACS sorted zebrafish epidermis with or without melanoma. tdTomato-labeled melanoma cells, eGFP-labeled keratinocytes and non-fluorescently labeled TME cells were isolated by dissection (as indicated in G) and FACS. Comparison of mitfa and krt4 expression of samples normalized to non-fluorescent cells, either ‘TME-Other’ in tumor samples or ‘Other’ in non-tumor samples, shows enrichment of mitfa in melanoma sample and krt4 in keratinocyte sample. ns is non-significant, * is p ≤ 0.05, *** is p ≤ 0.001, **** is p ≤ 0.0001 by Tukey’s multiple comparisons test.
(G) Comparison of the EMT-markers vim and cdh2 shows enrichment in TME keratinocytes vs. keratinocytes from epidermis without melanoma. * is p ≤ 0.05 by Welch’s t-test.
(H) Schematic of keratinocyte-melanoma co-culture experiment. HaCaTs were cultured in monoculture or co-culture with A375 melanoma cells in triplicates for 21 days, followed by FACS isolation of keratinocytes for RNA-sequencing comparing co-culture vs. monoculture keratinocytes.
(I) Top 5 enriched Hallmark pathways in HaCaTs co-cultured with A375 melanoma cells compared with HaCaTs in monoculture.
(J) Normalized counts of EMT biomarkers vimentin (VIM) and N-cadherin (CDH2).
Schematics in panels A, B and H created with bioRender (BioRender.com/l71i763; BioRender.com/l01p550; BioRender.com/j99d044).
Zebrafish scRNA-sequencing shows upregulation of EMT-TFs in tumor-associated keratinocytes.
(A) Schematic of scRNA-sequencing experiment. Embryo injection with miniCoopR:eGFP plasmid and tol2 mRNA in Casper Triples (mitfa-/-;mitfa:BRAFV600E;tp53-/-;mpv17-/-) results in melanocyte rescue and subsequent melanoma formation. Melanoma was dissected and dissociated to single cell suspension for FACS isolation of eGFP+ melanoma cells and non-fluorescent TME cells for single cell RNA-sequencing.
(B) Dimensionality reduction and subsequent analysis with zebrafish keratinocyte gene module scoring highlights two keratinocyte clusters.
(C) Top 6 GSEA Hallmark pathways enriched in comparison between keratinocyte clusters.
(D) Hallmark EMT pathway enrichment in keratinocyte clusters.
(E) Expression of EMT-transcription factors Snail (snai1a), Slug (snai2), and Twist1 (twist1a) in TAK vs. NKC, Melanoma, and Other TME cells. ns is no significance, * is p ≤ 0.05, **** is p ≤ 0.0001.
Schematic in panel A created with bioRender (BioRender.com/m35x698).
Overexpression of twist1a/b results in improved survival of fish with melanoma.
(A) Schematic of zebrafish melanoma model with labeling and perturbation of keratinocytes. Twist1a and twist1b are overexpressed under the keratinocyte-specific krt4 promoter in the TWIST condition and an empty vector control was used in the CTRL condition. Plasmid mix containing miniCoopR-tdTomato, krt4:eGFP, either empty vector or krt4:twist1a/b, with tol2 mRNA were injected into Casper Triples (mitfa-/-;mitfa:BRAFV600E;tp53-/-;mpv17-/-). Fish were sorted at 5 days for eGFP and tdTomato positivity as marker of successful keratinocyte labeling and melanocyte rescue.
(B) Tumor-free survival and overall survival of A. ns is no significance, ** is p ≤ 0.01 by Log-rank (Mantel-Cox) test.
(C) Sample images of zebrafish with melanoma at 26 weeks post-injection. Melanomas are pigmented in brightfield images. Keratinocytes are labeled by eGFP and melanoma are labeled by tdTomato in fluorescence images. Scale bar = 5mm.
(D) H&E and IHC of cross-sections through zebrafish body and melanoma. Dotted yellow line demarcates border of body. Percent of tumor in body is calculated as tumor area within body border divided by total tumor area. Scale bar = 500um.
Schematic in panel A created with bioRender (BioRender.com/u52i228).
Zebrafish findings are recapitulated in human cell lines.
(A) Schematic of coverslip cell infiltration assay. Melanoma cells are plated on a coverslip and allowed to attach overnight. The coverslip is then transferred into a well of keratinocytes to assess melanoma infiltration into keratinocytes.
(B) Generation of a HaCaT cell line overexpressing TWIST1. HaCaT-BFP was infected with lentivirus containing cassette with nuclear localized GFP and CMV driven TWIST1 or no ORF. Infected cell lines were allowed to grow for a week before sorting for nuclear GFP as a marker of successful integration.
(C) Western blot for Twist expression in HaCaT-CTRL and HaCaT-TWIST.
(D) Immunofluorescence imaging for Twist localization in HaCaT-CTRL and HaCaT-TWIST. TWIST staining is pseudo-colored in magenta, DAPI in blue, phalloidin in white. Scale bar = 50um.
(E) Immunofluorescence imaging of coverslip cell infiltration assay after 20 hours with HS294T-tdT (orange) melanoma cells in co-culture with either HaCaT-CTRL or HaCaT-TWIST (cyan). Scale bar = 500um.
(F) Quantification of E. Infiltrating HS294T melanoma cells from each image were counted and averaged across four images per well. Resulting cell counts were normalized to average cell counts of HaCaT-CTRL from each set. N = 9, 3 sets, 3 replicates/wells per set. * is p ≤ 0.05 by t-test.
(G) Immunofluorescence imaging of coverslip cell infiltration assay after 20 hours with SKMEL2-tdT (orange) melanoma cells in co-culture with either HaCaT-CTRL or HaCaT-TWIST (cyan). Scale bar = 500um.
(H) Quantification of G. Infiltrating SKMEL2 melanoma cells from each image were counted and averaged across four images per well. Resulting cell counts were normalized to average cell counts of HaCaT-CTRL from each set. N = 9, 3 sets, 3 replicates/wells per set. ** is p ≤ 0.01 by t-test.
Schematics in panels A and B created with bioRender (BioRender.com/a03i794; BioRender.com/w80w034).
scRNA-sequencing shows unique keratinocyte-melanoma communication with twist1a/b overexpression in keratinocytes.
(A) Schematic of scRNA-sequencing protocol. Melanoma and surrounding tissue were dissected from 26-weeks old zebrafish from either CTRL or TWIST conditions as shown in Figure 3. Samples were dissociated to single cell suspensions for FACS isolation of keratinocytes (GFP) and melanoma (tdTomato). Keratinocytes and melanoma were recombined per condition at a ratio of 7:3 for enrichment of keratinocytes for scRNA-sequencing.
(B) UMAP dimensional reduction and feature plots of scRNA-sequencing dataset. CTRL and TWIST samples were sequenced, and cell types were identified using eGFP+ for keratinocytes and tdTomato+ for melanoma, after which the datasets were integrated.
(C) UMAP showing cell type assignments of each cluster within either CTRL or TWIST conditions. Arrow indicates a TWIST-melanoma population clustering separately from other melanoma clusters.
(D) UMAP highlighting keratinocyte clusters, with a Tumor-Associated Keratinocyte (TAK) cluster, a Normal Keratinocyte Cluster (NKC), and a Twist-High cluster unique to the TWIST condition.
(E) Melanoma cell state analysis of the melanoma cluster unique to the Twist condition indicated by arrow in Figure 5C.
(F) Schematic overview of CellChat analysis. In CTRL condition, we analyzed Ligand-Receptor pairs with NKC as sender and CTRL-Melanoma as receiver. In TWIST condition, we analyzed L-R pairs with both NKC and Twist-High as sender and TWIST-Melanoma as receiver.
(G) CellChat analysis results. L-R pairs shown at p<0.01, with color scale indicating communication probability of L-R pair.
Schematics in panels A and F created with bioRender (BioRender.com/w59i021; BioRender.com/h74w538).
