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: High SPP1 levels are linked to CRLM and immunotherapy resistance. A, Schematic diagram shows the construction of a CRLM cell line. B, Volcano plot of differentially expressed genes in the RNA-seq analysis of the LoVo and LoVo-HM cells. C, The GSE41568 , GSE14297 , GSE128213 , and LoVo-HM datasets were combined, identifying upregulated genes with a fold change >1.5 and P < 0.05. D, Venn diagram revealing SPP1 as the only gene upregulated in all datasets. E and F, Western blot analysis of SPP1 expression in primary tumors (P) and liver metastases (L) from five patients with colorectal cancer is shown as a heatmap ( E ) and in PDOs from three patients with colorectal cancer ( F ). G, ELISA measures SPP1 in the blood of 45 patients with colorectal cancer without metastasis (M0) and 46 with metastasis (M1). H, Imaging assessment before and after immunotherapy in a patient with colorectal cancer #1 with liver metastasis are shown, with tumor diameters analyzed ( n = 3 patients). I, Confocal microscopy to evaluate T-cell infiltration into the PDOs from patients with colorectal cancer ( n = 3 patients). Scale bar, 50 μm. J, Confocal microscopy shows T-cell cytotoxicity in PDOs of patients with colorectal cancer with propidium iodide (PI) labeling for dead cells, n = 3. Scale bar, 50 μm. K–M, Spatial transcriptomics revealing tumor regression and residual tumor regions ( n = 1). Spatial visualization of the cell types ( L ) and hierarchical clustering of the localized spots using Uniform Manifold Approximation and Projection (UMAP; M ). N and O, UMAP plots illustrating SPP1 expression in different cell clusters. P, mIHC examines SPP1 and TME in primary and liver metastases from five patients with colorectal cancer, with representative images. n = 5. Scale bar, 200 μm. Data are presented as mean ± SEM. Statistical analysis: two-tailed unpaired Student t test ( F , G , I , and J ) and paired-samples Student t test ( H and P ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. CRC, colorectal cancer; LM, liver metastasis; mIHC, multiplex IHC; PT, primary tumor.

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

Techniques: RNA Sequencing, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Imaging, Confocal Microscopy, Labeling, Two Tailed Test, Multiplex Assay

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 the occurrence of colorectal cancer metastasis and immunotherapy resistance. A–C, The impact of SPP1 overexpression or knockdown on LoVo and LoVo-HM cell migration and invasion was assessed using transwell and wound healing assays. Scale bar, 100 μm. n = 3. D–F, Liver metastasis in C57BL/6J mice injected with MC38 cells was evaluated through tumor burden quantification and hematoxylin and eosin (H&E) staining ( n = 5 mice/group). Scale bar, 50 μm. G, Schematic representation of the in vivo experiment. CRC, colorectal cancer; LM, liver metastasis; PT, primary tumor. H, Flow cytometry confirmed human CD45 + (hCD45 + ) cell engraftment at 7 days after implantation ( n = 3 mice/group). I and J, Tumor morphology, weight, volume, and IFNγ expression were analyzed ( n = 3 mice/group). K–M, ELISA measured IFNγ ( K ), granzyme B ( L ), and SPP1 ( M ) levels in the tumor tissues ( n = 3 mice/group). N, Western blot analysis of SPP1 in the tumor tissues ( n = 3 mice/group). O, Hematoxylin and eosin analysis of the tumor tissues. P and Q, Masson trichrome staining and IHC were used to evaluate collagen content and αSMA expression. R and S, TUNEL and Ki-67 staining were performed on PDOX tumor tissues. Data are presented as mean ± SEM. P values were determined by two-tailed unpaired Student t test ( A , C , E , H–N , and P–S ) and one-way ANOVA ( B and C ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant. IOD, integrated optical density.

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

Techniques: Over Expression, Knockdown, Migration, Injection, Staining, In Vivo, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, TUNEL 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: SPP1 enhances CAF infiltration into the TME and promotes their malignant phenotypes. A, Correlation analysis of SPP1 with key microenvironmental cells in TCGA datasets. DC, dendritic cell; MDSC, myeloid-derived suppressor cell; Treg, regulatory T cell. B–E, Subcutaneous tumor models in the MC38 cells overexpressing SPP1 or a vector control were used to examine cell subpopulations via single-cell RNA-seq. UMAP, Uniform Manifold Approximation and Projection. F, Brightfield images of the CAFs and representative IHC staining images of CDX2, CK20, β-catenin, and Ki-67 in PDOs. H&E, hematoxylin and eosin. G–I, Transwell and wound healing assay of the CAFs with SPP1 overexpression or treatment ( n = 3). J, EdU assay for proliferation in CAFs with SPP1 overexpression or treatment. ( n = 3). K, Schematic representation of the collagen contraction assays. L, Collagen contraction assay with CAFs treated with or without SPP1 protein (1 µg/mL), n = 3. Data are presented as mean ± SEM. P values were determined using a two-tailed unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. CM, conditioned medium.

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

Techniques: Derivative Assay, Plasmid Preparation, Control, RNA Sequencing, Immunohistochemistry, Wound Healing Assay, Over Expression, EdU Assay, Contraction 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: 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: Talabostat mesylate reverses CRLM progression and synergizes with PD-1/PD-L1 blockade therapy. A, Schematic of the subcutaneous mouse model established with MC38 cells. B–D, Representative tumor morphology, weight, and volume ( n = 5 mice/group). E, ELISA measured the IFNγ, granzyme B, and TGFβ levels in the tumor tissues. F, Schematic representation of the colorectal cancer (CRC) PDX model in PBMC-reconstituted NOG mice. G–I, Tumor morphology, weight, and volume data ( n = 5 mice/group). J, ELISA measured the SPP1, IFNγ, granzyme B, and TGFβ levels in the tumor tissues. K and L, Hematoxylin and eosin staining of metastatic livers from C57BL/6J mice implanted with MC38-GFP or MC38-SPP1 cells, treated with talabostat mesylate, showed liver weight and tumor burden ( n = 5 mice/group). Scale bar, 1 mm. M and N, Flow cytometric analysis of the IFNγ + CD8 + and GZMB + CD8 + T-cell populations in liver metastases ( n = 5 mice/group). Results are presented as mean ± SEM. P values were determined via one-way ANOVA ( D , E , H , J , and L–N ). *, P < 0.05; **, P < 0.01; ***, P < 0.001. aP, anti–PD-1 antibody; aP + i, anti–PD-1 antibody + talabostat mesylate.

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

Techniques: Enzyme-linked Immunosorbent Assay, Staining

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: Cell Regeneration

Article Title: Integrative analysis and experimental validation identify the role of CD44 and Nucleolin in regulating gliogenesis following spinal cord injury

doi: 10.1186/s13619-025-00253-x

Figure Lengend Snippet: Dynamic cell portion changes and intercellular communication analysis revealed SPP1 signaling pathway was critical in microglia after SCI. A , B Stacked bar plots depicting changes in the relative abundance of major cell types in spinal cord ( A ) and peripheral immune cell populations ( B ) across various time points. Astrocytes, microglia, OPCs, and MDMs show marked shifts, particularly in the acute (1~3 dpi) and subacute phases of SCI, the absence of 42 dpi stems directly from the source data ( GSE172167 ), where immune cell clusters were not identified or annotated at this specific time point in the original study. C Intercellular communication networks illustrate increased signaling complexity at 1 and 3 dpi compared to the sham condition. D Quantitative result of the total number of interactions in sham, 1 dpi, and 3 dpi samples, showing a significant increase in cell–cell interactions post-injury. E Heatmaps displaying the changes in signaling patterns for key cell types. SPP1 became prominent at 1 dpi. F Information flow of microglia indicated the SPP1 signal was significant

Article Snippet: After adhesion, recombinant mouse SPP1 protein (MCE, Cat No. HY- P71786 ) and recombinant mouse PTN protein (MCE, Cat No. HY- P71213 ) were separately administered to the microglia and astrocytes at concentrations of 0, 0.1, 0.5, and 1 μg/mL for a duration of 24 h. Subsequent to the stimulation period, the culture medium was carefully removed, and the cells were gently washed twice with PBS.

Techniques:

Journal: Cell Regeneration

Article Title: Integrative analysis and experimental validation identify the role of CD44 and Nucleolin in regulating gliogenesis following spinal cord injury

doi: 10.1186/s13619-025-00253-x

Figure Lengend Snippet: SPP1-CD44 signaling promotes microglial activation and inflammatory response. A SPP1 signaling pathway network showing interactions between microglia and other cell types. B A circular plot illustrating the interaction network of microglia with other cells in various ligand -receptor pairs, including Spp1 - Cd44 . C , D Violin plots showing expression levels of Spp1 and Cd44 across different cell types in sham (blue) and 1 dpi (red). Both genes show elevated expression in microglia following injury. E Violin plot of Cd44 expression in microglia subclusters, showing the upregulation in the wound healing and inflammatory response2 cluster at 1 dpi. F The microglia were sorted by flow cytometry and ( G ) Cd44 gene expression was detected by qRCR. H Flow cytometry analysis of CD44 positive microglia after SCI, showing a marked increase in CD44 + microglia during 7 dpi. I Immunofluorescence images of spinal cord lesion site stained for Iba1, CD44, SPP1, and merged with DAPI. White arrows indicate the co-stained CD44 + and SPP1 + signals in Iba1 positive microglia ( J ) Quantification results of CD44 + and SPP1 + in Iba1 positive microglia cells. K Using PLA to detect specific SPP1-CD44 interactions of spinal cord lesion site in situ. L Quantification of PLA results, the PLA signal is quantified and plotted as the area of PLA signal per Iba1 positive cell. M , N qRT-PCR showing dose- and time-dependent increases of Cd44 expression in BV2 microglia after recombinant SPP1 stimulation. O Representative images of PLA assay specific SPP1-CD44 interactions of BV2 microglia cells in vitro. P PLA signal was quantified and plotted as the area of PLA signal per cell. Q ELISA quantification of IL-6 levels in cell supernatant after SPP1 stimulation. R Western blot showing the time course of CD44 and p-NF-κB p65 protein expression in BV2 cells after SPP1 treatment. ( S – T ) Quantification of CD44 and p-NF-κB p65 protein levels. Data are presented as mean ± SEM. ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: After adhesion, recombinant mouse SPP1 protein (MCE, Cat No. HY- P71786 ) and recombinant mouse PTN protein (MCE, Cat No. HY- P71213 ) were separately administered to the microglia and astrocytes at concentrations of 0, 0.1, 0.5, and 1 μg/mL for a duration of 24 h. Subsequent to the stimulation period, the culture medium was carefully removed, and the cells were gently washed twice with PBS.

Techniques: Activation Assay, Expressing, Flow Cytometry, Gene Expression, Immunofluorescence, Staining, In Situ, Quantitative RT-PCR, Recombinant, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot