Journal: Journal of Advanced Research

Article Title: The pro-cancer and immunosuppressive activity of macrophage-transformed cancer-associated fibroblasts in oral squamous cell carcinoma

doi: 10.1016/j.jare.2025.07.027

Figure Lengend Snippet: MMTs recruit a large number of MDSCs through CXCL10 pathway. A Significantly upregulated genes in MMTs versus non-MMT fibroblasts from OSCC database (GSA-Human: HRA007439). B Differences in CXCL10 expression between non-MMT fibroblasts and MMTs were demonstrated in HNSCC (figure S1). C CXCL10 expression and release were detected by PCR and ELISA. D-H Exogenous CXCL10 was added to BMDMs, while the CXCL10 inhibitor AMG487 was administered to MMTs, followed by co-culturing with MDSCs. The migration of MDSCs was evaluated using transwell chambers and crystal violet staining ( D ). Flow cytometry was used to detect cell apoptosis ( E ), macrophage markers (F4/80 + CD11b + ) ( F ), as well as CFSE for assessing T cell proliferation inhibition ( G ). PCR and ELISA were employed to measure the expression of immunosuppressive factors IL10 and iNOS ( H ). I, J Tumor tissue photographs ( I ) and the tumor growth curve were taken and subjected to statistical analysis regarding volume and weight ( J ). K-M Flow cytometry was used to detect the proportion of MDSCs in the spleen ( K ) and tumors ( L ) of each group, while immunofluorescence staining ( M ) revealed the densest field of MDSCs within the tumor tissues across all groups. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs experimental group. “ns” represents no statistical significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: According to the manufacturer's instructions, the cytokine concentrations of TGFβ1, iNOS, IL-10, and CXCL10 in the supernatant were determined using ELISA kits (Boster, California, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Migration, Staining, Flow Cytometry, Inhibition, Immunofluorescence

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