spp1 protein  (R&D Systems)

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) Scheme of human cancer types included in the integrated analysis. (B) Numbers of samples in each normal tissue or cancer type. (C) Marker genes of the major cell types in the pan-cancer scRNA-seq atlas. (D) UMAPs showing LUM , MCAM , CSPG4 , MYH11 , ACTA2 , FAP , MMP11 , PIEZO2 , C1QA , C1QB , C1QC , CD34 expression in CAFs. (E) Signature genes of apCAF subclusters. (F) Expression of CD24 and CD37 in the two apCAF lineages. (G) Combined overall survival of the 14 types of cancer with SPP1 expression. (H) Regulon of SPI1 in each CAF subcluster revealed by SCENIC algorithm. (I) Regulon of POU5F1 in each CAF subcluster revealed by SCENIC algorithm. (J) ChIP-seq binding peaks of OCT4 at the SPP1 promoter in fibroblasts visualized using the WashU Epigenome Browser from the Cistrome project.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Marker, Expressing, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) All apCAFs marked by MHC II molecule expression are extracted from and re-clustered, revealing four apCAF subclusters. (B) UMAPs of signature genes of apCAF subclusters including CD74 , MSLN , UPK3B , KRT19 , PTPRC , CD52 (C) Pseudotime analysis reveals two distinct trajectories of apCAFs. Expression of CD74 , HLA-DRA , MSLN , PTPRC and SPP1 along the trajectories are shown. (D) Up-regulated genes in the F-apCAF lineage (subcluster 2 vs 1) are used to perform GSEA pathway analysis. Significant pathways are shown. (E) Up-regulated genes in the M-apCAF lineage (subcluster 3 vs 0) are used to perform GSEA pathway analysis. Significant pathways are shown. (F) Differentially expressed genes in apCAFs in cancer compared to normal tissues. Six most up-regulated and robustly expressed genes are identified: NDUFA4L2 , SPP1 , PLOD2 , EGLN3 , ANGPTL4 , HILPDA . (G) Expression of NDUFA4L2 , SPP1 , PLOD2 , EGLN3 , ANGPTL4 , HILPDA in each CAF subcluster. (H) Combined overall survival of the 14 types of cancer with the six-gene signature ( NDUFA4L2 , SPP1 , PLOD2 , EGLN3 , ANGPTL4 , HILPDA ). (I) Regulatory network of transcription factors in each CAF subcluster revealed by SCENIC algorithm. (J) Regulatory network of genes by SPI1 in F-apCAFs. (K) Regulatory network of genes by POU5F1 in M-apCAFs. (L) Abundance of F-apCAFs in different cancer types. (M) Abundance of M-apCAFs in different cancer types.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Expressing

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) IHC staining for pan-cytokeratin (PanCK) in human PM samples. Scale bars, 250 μm. (red arrow, normal mesothelium; blue arrow, cytokeratin + CAFs). (B) Multiplex IHC staining for PanCK, SPINK4 and DAPI in human PM samples. Scale bars, 10 μm. (C) Visualization of normal mesothelium adjacent to M-apCAF-enriched areas from Xenium assay. M-apCAFs, cancer cells and the expression of normal mesothelial cell genes MSLN and UPK3B are shown. (D) Ligand-receptor interaction analysis between M-apCAFs (ligands) and different populations of immune cells (receptors). (E) Ligand-receptor interaction analysis between M-apCAFs (ligands) and cancer cells (receptors). (F) SPP1 expression in the iCMS2 CAFs (patient 2, 3, 5, 8 (P2, P3, P5, P8)) and iCMS3 CAFs (patient 1, 4, 6, 7 (P1, P4, P6, P7)) from the GeoMx assay.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Immunohistochemistry, Multiplex Assay, Expressing

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) Robust cell type decomposition is performed in human PM sample with robust cytokeratin + CAF formation to deconvolve the Xenium data into cell types using our pan-cancer scRNA-seq atlas as reference. (B) Four spatial niches are identified. Percentages of cell types within each niche are shown. (C) Visualization of the spatial distribution of different cell types in four M-apCAF-enriched regions. (D) Expression of T cell immunosuppressive genes across four spatial niches. (E) RT-PCR (n=3/group) and western blots measuring the expression of SPP1 in OmMeso cells after tumor conditioned medium treatment. (F) Wound healing assays are performed to measure the migration capability of MC38 colon cancer cells in the presence of mouse recombinant protein or anti-SPP1 mAb. Representative pictures of cell migration at 0h, 24h, 48h are shown. n=3/group. (G) Matrigel transwell assays in the presence of mouse recombinant protein or anti-SPP1 mAb for 24 hours are performed. Representative pictures for each group are shown. n=3/group. (H) MC38 cancer cells are injected intraperitoneally into wildtype (WT) or Spp1 knockout (KO) mice on a C57BL/6 background (WT, n=8; KO, n=10). Mice are sacrificed 4 weeks after cancer cell injection. Peritoneal cancer index (PCI) scores and ascites formation are measured. (I) MC38 cancer cells are injected intraperitoneally into wildtype C57BL/6 mice. Mice are treated with control Ab or anti-SPP1 mAb (n=5/group) one week after cancer cell injection and maintained at two doses/week. Mice are sacrificed 4 weeks after cancer cell injection. PCI scores and ascites formation are measured.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Migration, Recombinant, Injection, Knock-Out, Control

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) UMAP of major cell types across the 6 human PDAC samples identified from the Xenium assays. (B) Marker genes of the major cell types in PDAC. (C) Proportions of the major cell types in the treatment naïve and chemoradiotherapy (chemo-RT)-treated PDAC samples. (D) Expression of SPP1 in cancer cell 1 and cancer cell 2 populations. (E) Robust cell type decomposition is performed in PDAC to deconvolve the Xenium data into cell types using our pan-cancer scRNA-seq atlas as reference. (F) Spp1 WT or KO tumors are digested into single cell suspension and subjected to scRNA-seq. Major cell types are identified in the merged data from Spp1 WT and KO groups. (G) Marker genes of major cell types in Spp1 WT and KO tumors. (H) IHC staining and quantification for CD3 and CD8 in Spp1 WT and KO tumors (n=3/group). (I) Expression of Spp1 , Ptprc , Cd24a and Msln in CAFs of Spp1 WT and KO tumors.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Marker, Expressing, Suspension, Immunohistochemistry

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) Spatial niches are identified in human PDAC sample with TLS formation. (B) Based on the spatial niches and expression of SPP1 in cancer cells, PDAC sample is classified into four areas: stroma, TLS, SPP1 - and SPP1 + cancer. Deconvolved cell types are shown in each area. (C) Expression of SPP1 is visualized in SPP1 - and SPP1 + cancer areas. (D) Proportions of F-apCAFs and M-apCAFs are quantified in stromal, TLS, SPP1 - and SPP1 + cancer areas (n=3 for each area). (E) Western blots measuring the expression of SPP1 in PanMeso cells after tumor conditioned medium treatment. (F) GFP + PanMeso cells are co-injected with a murine PDAC cell line (BMFA3: In Vivo 1 or CT1BA5: In Vivo 2) at a 1:1 ratio. Tumors are harvested 1 month after injection and digested into single-cell suspension. GFP + cells are collected by flow sorting and subjected to RNA-seq analysis in comparison to parental PanMeso cells to evaluate the Spp1 expression. (G) Syngeneic PDAC cancer cells (6620c1) are injected orthotopically into wildtype (WT) or Spp1 knockout (KO) C57BL/6 mice (n=6/group). Tumors are harvested 1 month after injection. (H) Spp1 WT or KO tumors are digested into single cell suspension and subjected to scRNA-seq (6 tumors/group, every two tumors are pooled together for library construction). Ratio of each cell type between WT and KO group is compared and quantified. (I) CAFs from both Spp1 WT or KO tumors are extracted from the scRNA-seq data. iCAF, myCAF and apCAF clusters are identified. (J) Signature genes of each CAF subtype. (K) Proportional changes of CAF subtypes between Spp1 WT and KO group. (L) UMAPs showing sslCAF marker Pi16 and Dpt expression between Spp1 WT and KO tumors. (M) CytoTRACE analysis determining the progenitor and differentiation status among iCAFs, myCAFs and apCAFs, with higher score indicating more stem-like and less differentiated status. (N) Quantification of the expression of T cell chemoattractant genes in CAFs between Spp1 WT and KO tumors. (O) Syngeneic PDAC cancer cells (6620c1) are injected orthotopically into wildtype C57BL/6 mice. Mice are treated with control Ab (n=5) or anti-SPP1 mAb (n=7) one week after cancer cell injection and maintained at two doses/week. Mice are sacrificed 4 weeks after cancer cell injection.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Expressing, Western Blot, Injection, In Vivo, Suspension, RNA Sequencing Assay, Comparison, Knock-Out, Marker, Control

Journal: Nature

Article Title: Signalling by senescent melanocytes hyperactivates hair growth.

doi: 10.1038/s41586-023-06172-8

Figure Lengend Snippet: Fig. 3 | Secretome of nevus melanocytes contains SPP1 that promotes hair growth. a,d, On cytometry, SPP1 was increased in P56 Tyr-NrasQ61K (a) and P69 Tyr-CreERT2;Braf V600E (d) melanocytes. In a, for the permeabilized condition, n = 3 in WT and n = 5 in Tyr-NrasQ61K (P = 0.000000115); for the surface-bound condition, n = 3 in WT and n = 5 in Tyr-NrasQ61K (P = 0.0257). In d, for the permeabilized condition, n = 3 (P = 0.001397); for the surface-bound condition, n = 3 (P = 0.2888). See Extended Data Fig. 4. c,f, On western blot, SPP1 levels were increased in P56 Tyr-NrasQ61K (c) and P69 Tyr-CreERT2;Braf V600E (f) melanocytes. In c, n = 3; P = 0.00784. In f, n = 3; P = 0.0109. Uncropped gels are shown in Supplementary Fig. 1. b,e, On ELISA, SPP1 levels increased in day 5 cultures of P56 Tyr-NrasQ61K (b) and P69 Tyr-CreERT2;Braf V600E (e) melanocytes. In b, n = 3 in WT and n = 4 in Tyr-NrasQ61K; P = 0.00072. In e, n = 4; P = 0.00224. See Extended Data Fig. 4e. g, Unlike WT, Tyr-NrasQ61K skin contained Trp2+Spp1+ melanocytes adjacent to HF bulges. h, Anagen HF quantification in Tyr-NrasQ61K;Spp1−/− versus Tyr-NrasQ61K;Spp1+/− control mice. At P44, n = 12 in control and n = 14 in Tyr-NrasQ61K;Spp1−/− (P = 0.0000000191); at P56, n = 12 in control and n = 15 in Tyr-NrasQ61K;Spp1−/− (P = 0.0000195). i, Tyr-CreERT2;Braf V600E;Spp1fl/fl mice showed

Article Snippet: In brief, recombinant SPP1 protein (441-OP, R&D) was reconstituted in 0.1% BSA to a final concentration of 1.3 mg ml−1.

Techniques: Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature

Article Title: Signalling by senescent melanocytes hyperactivates hair growth.

doi: 10.1038/s41586-023-06172-8

Figure Lengend Snippet: Fig. 4 | Effect of SPP1 on hair growth depends on CD44. a,b, Epithelial HF cells in both WT control (a) and Tyr-NrasQ61K (b) mice strongly expressed CD44. Samples were also stained for the epithelial keratin marker KRT14. c,d, Co-staining for SPP1 and CD44 in Tyr-NrasQ61K (c) and Tyr-CreERT2;Braf V600E (d) skin revealed SPP1high clusters of dermal cells adjacent to CD44+ bulge cells with weaker colocalizing SPP1 signal (yellow arrows). e, Cd44−/− mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00938. f, Cd44−/− mice showed reduced wound-induced hair growth compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 6 in WT and n = 5 in Cd44−/−; P = 0.0494. g,h, Tyr-NrasQ61K;CD44−/− mice lacking Cd44 showed rescue of hair cycle quiescence. At P44, Tyr-NrasQ61K;Cd44−/− HFs were in coordinated telogen (g).

Article Snippet: In brief, recombinant SPP1 protein (441-OP, R&D) was reconstituted in 0.1% BSA to a final concentration of 1.3 mg ml−1.

Techniques: Control, Staining, Marker, Activation Assay

Journal: Nature

Article Title: Signalling by senescent melanocytes hyperactivates hair growth.

doi: 10.1038/s41586-023-06172-8

Figure Lengend Snippet: Fig. 5 | Human nevi feature secretome enriched for SPP1. a, Bulk RNA-seq reveals prominent differences between hairy nevi and adjacent normal facial skin in humans. A principal component analysis plot is shown. See Extended Data Fig. 9. b, Selected upregulated (by 2× or more; green) and downregulated (by 2× or more; red) differentially expressed genes in nevus versus normal human skin. Bold and underlined genes were validated by qRT-PCR. c, qRT–PCR of selected differentially expressed genes from bulk RNA-seq data. n = 3. d,e, SPP1 and TRP2 co-staining. In normal skin, TRP2+ melanocytes did not express SPP1 (d), whereas in nevus skin, clusters of TRP2+SPP1+ cells were seen next to HF bulge regions (e). f, SPP1 and SOX10 co-staining. Nevus skin

Article Snippet: In brief, recombinant SPP1 protein (441-OP, R&D) was reconstituted in 0.1% BSA to a final concentration of 1.3 mg ml−1.

Techniques: RNA Sequencing, Quantitative RT-PCR, Staining