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: bioRxiv

Article Title: Crosslinked CXCR4 Signals Decreased Motility and Increased Adhesion of T Cells

doi: 10.1101/2025.06.04.652236

Figure Lengend Snippet: The e%ect of crosslinking CXCR4 on T cell motility and adhesion to fibronectin. A. HSB2 T cells were treated with 50 nM CXCL12 monomer, the (CXCL12) 2 -Fc fusion protein, and the (CXCL12) 2 -Fc/protein A complex, respectively, and motility of single T cells on a fibronectin-coated surface was measured for 2 hours (n = 30). B. HSB2 T cells were treated with 150 nM of the (CXCL12) 2 -Fc fusion protein for 1 hour then added to fibronectin-coated plates for di%erent time intervals. The plates were lightly washed after which the number adherent T cells were counted (n = 3). The treatments of the HSB2 T cells in panel A were applied to the adhesion assay (n = 4). C. Shown are the results of stimulating primary human e%ector T cells with incremental concentrations of the (CXCL12) 2 -Fc fusion protein on spontaneous motility (n = 40) and adhesion to fibronectin (n = 3). D. HSB2 T cells were incubated with CDF21, a human anti-human CXCR4 antibody, washed, and incubated with an isotype control rabbit IgG or a rabbit anti-human IgG antibody. The motility (n = 30) and adhesion (n = 2) of the HSB2 T cells were measured. Mean ± SEM; ns, not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Article Snippet: Cells were treated with 150 nM (CXCL12) 2 -Fc (Sinobiological, 10118-H01H) or vehicle control for 1 hour at 37°C in chemotaxis buffer (RPMI-1640 supplemented with 0.5% BSA and 20 mM HEPES).

Techniques: Cell Adhesion Assay, Incubation, Control

Journal: bioRxiv

Article Title: Crosslinked CXCR4 Signals Decreased Motility and Increased Adhesion of T Cells

doi: 10.1101/2025.06.04.652236

Figure Lengend Snippet: Tyrosine phosphorylation of PTK2B and crosslinking of CXCR4. A. HSB2 T cells were incubated with the (CXCL12) 2 -Fc fusion protein for timed intervals, after which PTK2B was immunoprecipitated from cell lysates, subjected to SDS-PAGE, and the gel was immunoblotted with antibodies to PTK2B and phosphotyrosine, respectively. B. HSB2 T cells were incubated with the (CXCL12) 2 -Fc fusion protein and cell lysates were subjected to SDS-PAGE and immunoblotting with antibodies to pTyr579/580 of PTK2B and pTyr402, respectively. C. HSB2 T cells were incubated with the monomeric CXCL12, dimeric CXCL12 and tetrameric CXCL12, respectively, and the cell lysates were analyzed as in panel A. D. HSB2 T cells were pre-incubated with the PTK2B inhibitor, PF-4618433, followed by incubation with the (CXCL12) 2 -Fc fusion protein, after which cell motility (n = 30) and adhesion to fibronectin (n = 3) were assessed. E. HSB2 T cells were subjected to treatment with PTX or bu%er control, followed stimulation with the (CXCL12) 2 -Fc fusion protein. The cell lysates were analyzed for tyrosine phosphorylation of PTK2B. F. HSB2 T cells were treated with CDF21 human anti-CXCR4 antibody followed by anti-human IgG antibody, and cell lysates were analyzed for tyrosine phosphorylation of PTK2B. Mean ± SEM; ns, not significant, **P < 0.01, ****P < 0.0001, Student’s t test.

Article Snippet: Cells were treated with 150 nM (CXCL12) 2 -Fc (Sinobiological, 10118-H01H) or vehicle control for 1 hour at 37°C in chemotaxis buffer (RPMI-1640 supplemented with 0.5% BSA and 20 mM HEPES).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: bioRxiv

Article Title: Crosslinked CXCR4 Signals Decreased Motility and Increased Adhesion of T Cells

doi: 10.1101/2025.06.04.652236

Figure Lengend Snippet: The role of integrins in T cell immotility and adhesion. A. HSB2 T cells were pretreated with the anti-ITGA4 antibody followed by treatment with the (CXCL12) 2 -Fc fusion protein, after which ( A ) cell motility (n = 30) and ( B ) adhesion to fibronectin (n = 3) were measured, respectively. C. HSB2 T cells were pretreated with PTX followed by treatment with the (CXCL12) 2 -Fc fusion protein, after which adhesion to fibronectin was measured in duplicates. Mean ± SEM; ns, not significant, **P < 0.01, ****P < 0.0001, Student’s t test.

Article Snippet: Cells were treated with 150 nM (CXCL12) 2 -Fc (Sinobiological, 10118-H01H) or vehicle control for 1 hour at 37°C in chemotaxis buffer (RPMI-1640 supplemented with 0.5% BSA and 20 mM HEPES).

Techniques:

Journal: bioRxiv

Article Title: Crosslinked CXCR4 Signals Decreased Motility and Increased Adhesion of T Cells

doi: 10.1101/2025.06.04.652236

Figure Lengend Snippet: TNFα and TNFRSF1B signaling and T cell motility and adhesion. A. HSB2 T cells were pretreated with a neutralizing antibody to TNF followed by stimulation with the (CXCL12) 2 -Fc fusion protein. T cell motility (n = 40) and adhesion to fibronectin (n = 3) were then measured. B. Jurkat T cells were lentiviral vector expressing TNFRSF1B and empty vector control, respectively. Combinations of the (CXCL12) 2 -Fc fusion protein and TNFα were added to the Jurkat T cells and they were assessed for motility (n = 30) ( C ). D. Jurkat T cells that had been transduced with a lentiviral vector expressing TNFRSF1B or an empty vector control were incubated with increasing concentrations of TNFα, and adhesion of the T cells to fibronectin was measured (n = 3). Mean ± SEM; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Article Snippet: Cells were treated with 150 nM (CXCL12) 2 -Fc (Sinobiological, 10118-H01H) or vehicle control for 1 hour at 37°C in chemotaxis buffer (RPMI-1640 supplemented with 0.5% BSA and 20 mM HEPES).

Techniques: Plasmid Preparation, Expressing, Control, Transduction, Incubation

Journal: Cell Death Discovery

Article Title: CXCL12 alone is enough to Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts

doi: 10.1038/s41420-025-02420-0

Figure Lengend Snippet: A Schematic diagram showing the generation of NFs treated with CAFs-CM. B Phase-contrast microscopy revealed the typical spindle-like features of fibroblasts in CAFs and NFs isolated from lung cancer tissues, Scale bar = 100 μm. C Western blot analysis showed the expression of the fibroblast markers (FAP and Vimentin), the epithelial markers (CK-19) and the endothelial marker (CD31) in CAFs and NFs. D The impact of NFs and CAFs on the migratory capabilities of H1299 and A549 was assessed using transwell assay. The results include representative images of cell migration counts (left) and statistical data (right). Data are presented as the mean ± SD of three biological replicates.*, p < 0.05; ** , p < 0.005. Scale bar = 200 μm. E Western blot analysis showed the expression of CXCL12, CXCR4, pSTAT3/STAT3, Vimentin, and α-SMA in NFs treated with DMEM or CAFs CM.

Article Snippet: Recombinant human CXCL12 protein was purchased from Sino Biological (Beijing, China).

Techniques: Microscopy, Isolation, Western Blot, Expressing, Marker, Transwell Assay, Migration

Journal: Cell Death Discovery

Article Title: CXCL12 alone is enough to Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts

doi: 10.1038/s41420-025-02420-0

Figure Lengend Snippet: A . Western blot analysis of the expression of CXCL12, p-STAT3/STAT3, Vimentin, and α-SMA in p53S-CAFs treated with DMEM and NFs treated with DMEM, p53 -/- fibroblast CM, and p53S-CAFs CM. B Immunofluorescence analysis of the expression of Vimentin and α-SMA in NFs treated with DMEM, p53 -/- fibroblast CM, and p53S-CAFs CM. C Calculation of the percentage of cells that incorporated EdU or the number of migration cells for Transwell migration assay in NFs alone and treated with p53S-CAFs-CM. *, p < 0.05. **, p < 0.005. D Transwell migration assay of lung cancer cells H1299 and A549 cultured with NFs CM or CEFs CM. Average migration ±SEM from three independent experiments performed( n = 3). **, p < 0.005. ***, p < 0.001.

Article Snippet: Recombinant human CXCL12 protein was purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Migration, Transwell Migration Assay, Cell Culture

Journal: Cell Death Discovery

Article Title: CXCL12 alone is enough to Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts

doi: 10.1038/s41420-025-02420-0

Figure Lengend Snippet: A Western blot analysis of the expression of CXCL12 in NFs and CAFs. B Correlation of CXCL12 expression with CAFs markers α-SMA and Vimentin ( P < 0.001, R > 0).

Article Snippet: Recombinant human CXCL12 protein was purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Expressing

Journal: Cell Death Discovery

Article Title: CXCL12 alone is enough to Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts

doi: 10.1038/s41420-025-02420-0

Figure Lengend Snippet: A Western blotting analysis was conducted on NFs that were either untreated or treated with CXCL12, p53S-CAFs-CM, or a combination of p53S-CAFs-CM and AMD3100. B EdU incorporation assay of NFs treated with DMEM, CXCL12, CXCL12 and AMD3100, p53S-CAFs CM, p53S-CAFs CM and AMD3100. Representative images (left) and statistics (right) of the percentage of cells that incorporated EdU were shown. **, p < 0.005. C Transwell assay of NFs treated with DMEM, CXCL12, CXCL12 and AMD3100, p53S-CAFs CM, p53S-CAFs CM and AMD3100. Representative images (left) and statistics (right) of migration assay were shown. **, p < 0.005. ***, p < 0.001. D Transwell co-culture assay was employed to evaluate the pro-migratory capacity of normal fibroblasts, CEFs-12 (normal fibroblasts treated with CXCL12), CEFs (normal fibroblasts treated with p53S-CAFs conditioned medium), and CEFs+AMD3100 towards lung cancer cells H1299 and A549. The results include representative images of cell migration (left) and statistical data (right). **, p < 0.005, ***, p < 0.0005. Scale bar = 200 μm. E The flowchart for the experimental design of xenograft tumor models. F Representative images show the xenograft tumors in the backs of nude mice formed by mixed subcutaneous injection of CEFs-12 (or NFs) and A549 cells. G Tumor growth graphs indicated the tumor volumes at different time course ( n = 3 or 4 mice per group). ** p < 0.01. H Precise weighing of the tumors was performed upon dissection, ** p < 0.01.

Article Snippet: Recombinant human CXCL12 protein was purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transwell Assay, Migration, Co-culture Assay, Injection, Dissection

Journal: Cell Death Discovery

Article Title: CXCL12 alone is enough to Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts

doi: 10.1038/s41420-025-02420-0

Figure Lengend Snippet: A p53S-CAFs-derived CXCL12 activated STAT3 pathway. NFs were treated with CXCL12 (20 ng/ml, 24 hours). The protein levels of CXCL12, CXCR4, IL6, p-JAK2, JAK2, p-STAT3, STAT3, SHP2, α-SMA, vimentin, p-ERK, ERK, p-AKT and AKT were detected by Western blot. B Western blot analysis of CXCL12, p-STAT3, STAT3, α-SMA and Vimentin from NFs alone and treated with CXCL12, or CXCL12 and Stattic. C Proliferative ability of NFs treated with CXCL12, CXCL12 and Stattic (upper), or p53S-CAFs CM, p53S-CAFs CM and Stattic(down) were measured by EdU incorporation assay. **, p < 0.005. D Migratory ability of NFs treated with CXCL12, CXCL12 and Stattic (upper), or p53S-CAFs CM, p53S-CAFs CM and Stattic(down) were measured by transwell migration assay. **, p < 0.005. ***, p < 0.001. Scale bar = 200 μm. E NFs were treated with CXCL12 and then transfected with STAT3 siRNA or control siRNA. STAT3 and p-STAT3 expression was measured by Western blot. *, p < 0.05. **, p < 0.005. F Proliferative and Migratory ability were measured by EdU incorporation assay and transwell assay. **, p < 0.005. ***, p < 0.001. G CEFs (NFs were treated with CXCL12) were transfected with STAT3 siRNA or control siRNA. Transwell assays detected the migration ability of H1299 and A549 co-cultured with CEFs or CEFs-SiSTAT3. **, p < 0.005. ***, p < 0.001. Scale bar = 200 μm.

Article Snippet: Recombinant human CXCL12 protein was purchased from Sino Biological (Beijing, China).

Techniques: Derivative Assay, Western Blot, Transwell Migration Assay, Transfection, Control, Expressing, Transwell Assay, Migration, Cell Culture

Journal: Cell Death Discovery

Article Title: CXCL12 alone is enough to Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts

doi: 10.1038/s41420-025-02420-0

Figure Lengend Snippet: A Western blot analysis of the knockdown efficiency following the transfection of CXCL12 siRNA into p53S-CAFs. B Western blot assessment of the impact on the expression of CAFs marker proteins in normal fibroblasts by conditioned medium after CXCL12 knockdown in p53S-CAFs. C Immunofluorescence analysis was conducted on the expression of CAFs markers α-SMA and Vimentin in p53 +/+ fibroblasts treated with either p53S-CAFs+NC CM or p53S-CAFs+siCXCL12 CM, in addition to the percentage of EdU-incorporated positive population. * p < 0.05, ** p < 0.005. D The impact of CEFs and CEFs+siCXCL12 on the migratory capabilities of H1299 and A549 was assessed using transwell assay. The results include representative images of cell migration counts (left) and statistical data (right). * p < 0.05; ** p < 0.005. Scale bar = 200 μm.

Article Snippet: Recombinant human CXCL12 protein was purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Knockdown, Transfection, Expressing, Marker, Immunofluorescence, Transwell Assay, Migration