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: Bone Research

Article Title: Neonatal bone marrow interstitial fluid supports expansion and osteogenic ability of human bone marrow mesenchymal stromal cells

doi: 10.1038/s41413-025-00496-z

Figure Lengend Snippet: NBIF promotes CXCR4 expression and enhances hBMSC homing to bone marrow. a, b Quantitative RT-PCR analysis of CXCR4 expression in hBMSCs after 7-day culture ( a ) and in mouse primary mBMSCs after 2 passages (7 days) ( b ). Data [and also in ( d )] were expressed as mean ± standard error of the mean (SEM) of the fold change across three replicates for each group. P -values were obtained from an unpaired t -test; ** P ≤ 0.01, **** P ≤ 0.000 1. c, d Representative images ( c ) and quantification data ( d ) of migratory hBMSCs in the Transwell culture (see the Materials and Methods section for details). rhCXCL12, recombinant human CXCL12 protein; AMD3100, the CXCR4 antagonist. Scale bar, 100 μm. e Scheme of the experimental design for mouse transplantation and analysis of GFP-labeled hBMSCs. f Quantification of the proportion of GFP + -hBMSCs in the whole bone marrow of host mice. Data were expressed as mean ± standard error of the mean (SEM) across indicated replicates for each group. 5 mice for the FBS-fed hBMSCs group and 5 mice for NBIF-fed hBMSCs group at each time point. P -values were obtained from an unpaired t -test; * P < 0.05, ** P < 0.01. g Representative immunofluorescent images of markers at 14 days post-transplantation. Scale bar, 10 μm. h–j Quantification of GFP + - ( h ), LEPR + - ( i ) or LEPR + ; GFP + - cells ( j ) in FBS-fed hBMSCs group ( n = 5 mice) or NBIF-fed hBMSC group ( n = 10 mice) 14 days post transplantation. Data were expressed as mean ± standard error of the mean (SEM) for each group. P -values were obtained from an unpaired t -test; * P < 0.05, ** P < 0.01

Article Snippet: To assess the effect of CXCL12, recombinant human CXCL12 was added to the lower chamber at a concentration of 100 ng/mL, with or without 50 nmol/L CXCR4 inhibitor AMD3100 (Medchemexpress) for 24 hours.

Techniques: Expressing, Quantitative RT-PCR, Recombinant, Transplantation Assay, Labeling

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