Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: SPP1 promotes colorectal cancer metastasis through a positive feedback loop mediated by CAF-secreted CXCL12. A, Mass spectrometry analyzed supernatants from SPP1-stimulated and unstimulated CAFs, showing fold changes in secreted proteins (SPP1/control). B, A bubble chart displays commonly secreted protein levels in fibroblasts. C and D, Uniform Manifold Approximation and Projection (UMAP) plots and quantitative analysis reveal CXCL12 expression in fibroblasts within OE-SPP1 and vector groups. E, ELISA measured CXCL12 in CAF supernatants with/without SPP1 (1 µg/mL), n = 3. F, A flowchart shows CAF-conditioned medium’s (CM) impact on colorectal cancer (CRC) cell migration and invasion. G and H, Transwell and wound healing assays evaluated the effects of CAF-conditioned media or CXCL12-neutralizing antibody (100 ng/mL) on colorectal cancer cell migration and invasion ( n = 3). I–K, Flowchart illustrating the effects of CXCL12 or neutralizing antibody treatment on the colorectal cancer cell migration and invasion, assessed via transwell and wound healing assays ( n = 3). L, The effect of CXCL12 (100 ng/mL) or a neutralizing antibody (100 ng/mL) on the epithelial–mesenchymal transition markers expression in the colorectal cancer cells was analyzed using Western blotting ( n = 3). M, Correlation analysis of CXCL12 with SPP1 and TGFB1 in the TCGA dataset. N and O, The effect of CXCL12 (100 ng/mL) or neutralizing antibody (100 ng/mL) on the SPP1 and TGFβ expression in the colorectal cancer cells was evaluated using Western blotting ( N ) or ELISA ( O ), n = 3. Results are presented as mean ± SEM. P values were calculated using a two-tailed unpaired Student t test ( E ), whereas one-way ANOVA was used for the other comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Mass Spectrometry, Control, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Migration, Western Blot, Two Tailed Test

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: SPP1 inhibits T-cell infiltration and cytotoxicity via CXCL12 secretion from CAFs. A, Schematic of the coculture system with PDOs, T cells, and CAFs. CRC, colorectal cancer; E:T, effector to target. B and C, Confocal microscopy assessing the effect of SPP1 overexpression on T-cell infiltration and cytotoxicity in PDOs with or without CAFs ( n = 3). D and E, Impact of rhSPP1 (1 µg/mL) or CXCL12-neutralizing antibody (100 ng/mL) on T-cell infiltration and cytotoxicity in PDOs ( n = 3). Results are presented as mean ± SEM. P values were determined using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant. PI, propidium iodide.

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Confocal Microscopy, Over Expression

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: SPP1 activates the β-catenin/HIF1α axis in the CAFs to drive CXCL12 secretion. A, Western blotting assessed key signaling pathway in CAFs after 24 hours of SPP1 protein stimulation. B–E, β-catenin and HIF1α expressions were analyzed following SPP1 or conditioned medium treatments, including from SPP1-overexpressing or -knockdown cells. F–H, HIF1α degradation was evaluated with MSAB or si-CTNNB1 transfection after cycloheximide (CHX) treatment, and HIF1α levels were measured after MSAB (1 µmol/L) or MG132 (20 µmol/L) pretreatment. I and J, Coimmunoprecipitation examined the HIF1α and β-catenin interaction. K and L, Immunofluorescence and nuclear–cytoplasmic fractionation assays assessed HIF1α and β-catenin localization ( n = 3). Scale bar, 25 μm. M–O, CXCL12 levels in conditioned media were measured after SPP1 (1 µg/mL) or MSAB treatments (24 hours). P, Correlation analysis of HIF1α and CXCL12 expression in 50 CAF samples using transcriptome data. Q, Dual-luciferase assays evaluated CXCL12 promoter activity ( n = 3). R and S, T-cell migration and infiltration were analyzed with or without SPP1 protein or MSAB treatment, n = 3. Scale bar, 50 μm. Western blotting ( A–J and L ) and ELISA ( M–O ) were repeated three times, with data representative of three independent experiments. Results are presented as mean ± SEM. P values were determined by one-way ANOVA ( M –O , R , and S ) and two-tailed unpaired Student t test ( F , G , and Q ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. R and S , Created with Figdraw.com .

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Western Blot, Knockdown, Transfection, Immunofluorescence, Fractionation, Expressing, Luciferase, Activity Assay, Migration, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cancer Research

Article Title: SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

doi: 10.1158/0008-5472.CAN-24-4916

Figure Lengend Snippet: Blocking the SPP1/CXCL12 axis alleviates immunosuppression in the liver microenvironment and augments the benefits of immunotherapy. A, Flowchart of the intrasplenic injection model of liver metastasis using OE-SPP1 MC38 cells ( i.s.v. , intrasplenic injection; i.p. , intraperitoneal injection). B–D, Representative tumor morphology, hematoxylin and eosin staining, liver weight, and tumor burden ( n = 5 mice/group). Scale bar, 1 mm. E and F, Flow cytometric analysis of IFNγ + CD8 + and GZMB + CD8 + T cells in liver metastases ( n = 5 mice/group). G, Flowchart of the cecal orthotopic injection model of liver metastasis in the NOG mice using HCT116-HM cells. H and I, Luciferase images and bioluminescence quantification of metastatic livers. J, Hematoxylin and eosin staining and the number of liver metastases ( n = 5 mice/group). K, ELISA analysis of IFNγ levels in liver metastases ( n = 5 mice/group). L–N, ELISA of SPP1 and CXCL12 in peripheral blood of responders ( n = 25) and nonresponders ( n = 12) in immunotherapy-treated colorectal cancer cohorts. O, Diagram of tumor-derived SPP1 activation of CAFs to promote immunotherapy resistance in CRLM. Data are presented as mean ± SEM. P values were determined using one-way ANOVA ( C–F , and I–K ) and two-tailed unpaired Student t test ( L and M ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. O, Created in BioRender. Liu, F. (2025) https://BioRender.com/k7tx8am .

Article Snippet: Human recombinant SPP1 (HY- P70499 ) and CXCL12 (HY- P70469 ) proteins were obtained from MedChemExpress.

Techniques: Blocking Assay, Injection, Staining, Luciferase, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: CXCR4-engineered fibroblast membrane nanovesicles for Photothermal-enhanced ferroptotic therapy through chemokine-navigated tumor homing

doi: 10.1186/s12951-025-03951-5

Figure Lengend Snippet: Characterization of CXCR4-OE fibroblasts. ( A ) RT-qPCR analysis of CXCR4 expression in murine dermal fibroblasts. ( B ) Western blotting analysis of CXCR4 in fibroblasts and fibroblast membranes. C and D. Representative fluorescence images of fibroblasts stained with FITC-labeled CXCR4 antibody ( C ), and the relative FITC intensity in cells ( D ) ( n = 3). E and F. Representative transwell invasion images of fibroblasts towards different treatments (Control, CXCL12, CXCL12 + LIT-927, CXCL12 + CTR-FbM@IR NPs and CXCL12 + FbM@IR NPs) ( E ), and the number of invaded cells per field ( F ). Data are displayed as mean ± SEM. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). CXCR4-OE, CXCR4-overexpressed fibroblasts; CTR, control fibroblasts

Article Snippet: One hour prior to NPs injection, mice received an intraperitoneal injection of the CXCR4 antagonist Plerixafor (3.1 mg/kg, MedChemExpress, USA) or the CXCL12 inhibitor LIT-927 (3.3 mg/kg).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Fluorescence, Staining, Labeling, Control

Journal: Journal of Nanobiotechnology

Article Title: CXCR4-engineered fibroblast membrane nanovesicles for Photothermal-enhanced ferroptotic therapy through chemokine-navigated tumor homing

doi: 10.1186/s12951-025-03951-5

Figure Lengend Snippet: Cellular internalization and CXCR4-CXCL12 axis-mediated targeting of FbM@IR NPs in tumor spheroids. A and B. Representative CLSM images showing the cellular internalization of FITC-labeled CTR-FbM@IR NPs and FbM@IR NPs into KYSE-30 cells ( A ) and MDA-MB-468 cells ( B ) after 3-hour and 9-hour incubation. C and D. The relative FITC intensity in KYSE-30 ( C ) and MDA-MB-468 cells ( D ). E. ELISA of CXCL12 levels in different cell culture supernatants. F. 3D-merged CLSM images showing the uptake of FITC-labeled FbM@IR NPs, FbM@IR NPs + plerixafor, and CTR-FbM@IR NPs in KYSE-30 spheroids after 24 h of incubation. Data are indicated as mean ± SEM. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***)

Article Snippet: One hour prior to NPs injection, mice received an intraperitoneal injection of the CXCR4 antagonist Plerixafor (3.1 mg/kg, MedChemExpress, USA) or the CXCL12 inhibitor LIT-927 (3.3 mg/kg).

Techniques: Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Journal of Nanobiotechnology

Article Title: CXCR4-engineered fibroblast membrane nanovesicles for Photothermal-enhanced ferroptotic therapy through chemokine-navigated tumor homing

doi: 10.1186/s12951-025-03951-5

Figure Lengend Snippet: Enhanced in vivo tumor targeting of FbM@IR NPs via CXCR4-CXCL12 interaction. A and B. In vivo IR780 distribution in MDA-MB-468 ( A ) and KYSE-30 ( B ) tumor-bearing mice at different time points after i.v. injection of FbM@IR NPs, FbM@IR NPs + Plerixafor, FbM@IR NPs + LIT-927 and CTR-FbM@IR NPs. (Green dashed circles indicate tumor sites). C and D. Ex vivo IR780 distribution in tissues (heart, liver, spleen, lung, kidneys, tumor and lymph nodes) harvested from mice bearing MDA-MB-468 ( C ) and KYSE-30 ( D ) tumor at 48 h after injection of FbM@IR NPs, FbM@IR NPs + Plerixafor, FbM@IR NPs + LIT-927 and CTR-FbM@IR NPs. E and F. Ex vivo IR780 signals of tumors from MDA-MB-468 tumor-bearing mice ( E ) and KYSE-30 tumor-bearing mice (F) ( n = 3). Data are presented as mean ± SEM. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***)

Article Snippet: One hour prior to NPs injection, mice received an intraperitoneal injection of the CXCR4 antagonist Plerixafor (3.1 mg/kg, MedChemExpress, USA) or the CXCL12 inhibitor LIT-927 (3.3 mg/kg).

Techniques: In Vivo, Injection, Ex Vivo