Journal: Frontiers in Immunology
Article Title: SLFN11 expression correlates with immune microenvironment and predicts prognosis in melanoma
doi: 10.3389/fimmu.2025.1607056
Figure Lengend Snippet: SLFN11 Overexpression Reprograms the Tumor Immune Microenvironment in Melanoma. (a, b) Validation of SLFN11-overexpressing (SLFN11-OE) and negative control (NC) SK-Mel-246 stable cell lines by PCR (A) and Western blot (B) . GAPDH served as loading control. (C) CCK-8 proliferation assay showing no significant difference in viability between SLFN11-OE and NC cells over 7 days (n = 3). (D) Schematic of co-culture system: SLFN11-OE or NC melanoma cells were co-cultured with PMA-induced THP-1 M0 macrophages. (E) qPCR analysis of macrophage polarization markers after co-culture. Macrophages exposed to SLFN11-OE cells exhibited upregulated M1 markers (NOS2, CXCL10, TNFα) and downregulated M2 markers (CD163, CD206, ARG1) compared to NC (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t-test). (F) ELISA quantification of CXCL10 in supernatants from macrophages isolated post-co-culture and maintained for 48 hours (n = 4). (G) PD-L1 mRNA levels in SLFN11-OE versus NC cells (n = 3). (H) Representative crystal violet staining images showing migration of CFSE-labeled THP-1-derived M0 macrophages to the lower surface of the upper transwell chamber after 48 hours of co-culture with SLFN11-overexpressing (SLFN11-OE) or control tumor cells (SK-Mel-246 and A375) in the lower chamber. Quantification indicates significantly more migrated macrophages in the SLFN11-OE group. (I) Flow cytometry analysis of CFSE + macrophages in the lower chamber, confirming a higher proportion of migrated macrophages in the SLFN11-OE group compared to controls, consistent with enhanced chemotaxis. (J) Flow cytometry analysis of CFSE-labeled CD8 + T cells migrating to the lower transwell chamber after co-culture with SLFN11-OE or control tumor cells. (K) Percentage of CD8 + IFNG + cells after co-culture with SLFN11-overexpressing vs. control (NC) melanoma cells. (L) Quantification of CD8+IFNG+ T cell frequency, presented as box and bar plots. Error bars represent the mean ± SEM (n = 3). Statistical significance was determined using an unpaired Student’s t-test.
Article Snippet: Supernatants were collected, and CXCL10 secretion was quantified using a Human CXCL10 ELISA Kit (Proteintech) following the manufacturer’s protocol.
Techniques: Over Expression, Biomarker Discovery, Negative Control, Stable Transfection, Western Blot, Control, CCK-8 Assay, Proliferation Assay, Co-Culture Assay, Cell Culture, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Migration, Labeling, Derivative Assay, Flow Cytometry, Chemotaxis Assay
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