Fig 2: Establishment of CAFs. (A) Rosa26-mT mouse treated with DEN for 17 weeks, and waiting 30 weeks for tumor formation. Then mouse CAFs were cultured according to our protocol. (B) Representative immunohistochemistry staining of a-SMA in mouse and human primary tissue (magnification, 400×). (C) Representative image of established human and mouse CAFs (magnification, 100×). (D) Representative immunofluorescence staining of a-SMA, FAP, EpCAM, AFP, CD45, and CD31 in mouse and human CAFs (magnification, 400×). DAPI, 4',6-diamidino-2-phenylindole; hCAF, human cancer associated fibroblast; mCAF, mouse cancer associated fibroblast; mT, membrane tomato; RFP, red fluorescent protein.

Fig 3: Patient-derived multicellular spheroids and melanoma patient-derived organoids (MPDOs). (A) Time course of patient-derived multicellular spheroid growth. The adherent cultured cells from disassociated fresh patient melanoma tissues were digested and seeded on the agarose for 48 h to form multicellular spheroids. Images were taken by microscopy. Scale bar, 250 µm. (B) PD-L1 expression in patient-derived multicellular spheroid (patient 1 and patient 2) with or without IFN-? stimulation (50 ng/ml) was measured by FACS after spheroids were disassociated into single cells. (C) Immunofluorescent staining of Phalloidine, ICAM-1 and a-SMA in patient-derived multicellular spheroid. Scale bar, 200 µm. (D) Representative images of MPDOs grew on the matrigel over time. Images were taken at days 2, 5, and 7. Single cells derived from fresh patient melanoma tissues were seeded onto the matrigel and cultured. Scale bar, 150 µm. (E) Cellular components of CD45+, CD8+, CD4+ and CD11b+ cells in the MPDOs were measured by flow cytometry. P1–P6, patient 1 to patient 6. (F) Immunofluorescent staining of HMB-45, a-SMA, ICAM-1, Vimentin, CD45+ and CD3+ T cells in MPDOs. Scale bar, 100 µm

Fig 4: MPDOs preserve diverse immune contexture (a). FACS analysis of CD45+, CD4+, CD8+, CD11b+, CD14+, CD15+ and CD68+ immune cells in MPDOs cultured in Matrigel at different time points over 1 month period. MPDOs were passaged every 5–7 days. (b). Immunofluorescent staining of CD45+ leukocytes, CD3+ T cells, CD8+ T cells, CD14+ monocytic cells and CD11b+ myeloid cells in MPDOs of 3 other different patients cultured in collagen gel and Matrigel, respectively. Scale bar, 100 µm.

Fig 5: Small melanoma tissue fragment-derived organoids (a). Representative phase contrast images of small melanoma tissue fragments from different locations of a melanoma cultured for 14 days. Scale bar, 500 µm. (b). H & E staining of tissue fragment-derived organoids from different location of a melanoma. Right panels are the enlarged view of the areas in the black frame on the left panels. Scale bar, 200 µm. (c). Immunofluorescent staining of a-SMA, vimentin, CD45+, CD3+, CD8+, CD14+ and CD11b+ in tissue fragment-derived organoids from different locations of a melanoma. Scale bar, 100 µm.