thbs2 heterozygote mice  (Jackson Laboratory)

Journal: FASEB BioAdvances

Article Title: Age and Sex Impact the Role of Thrombospondin‐2 and Thrombospondin‐5 in Response to Hindlimb Ischemia

doi: 10.1096/fba.2025-00258

Figure Lengend Snippet: (A, B) Thrombospondin effect on angiogenesis within respective age group. Young (14–16 weeks) and old (105–110 weeks) male and female WT, THBS2 −/− and THBS5 −/− mice underwent femoral artery ligation. Angiogenesis was assessed 14 days postoperatively. (A) THBS2 −/− young male and female mice had increased angiogenesis in young age. THBS5 −/− did not affect angiogenesis compared to respective WT controls. (B) THBS2 −/− or THBS5 −/− did not affect angiogenesis in old mice. Values are reported as means ± SEM. * p < 0.05, gene knockout compared to WT. WT, wild‐type.

Article Snippet: THBS2 + heterozygote mice were obtained from Jackson Laboratory (Bar Harbor, ME), bred to homozygote THBS2 −/− at Loyola University Chicago Comparative Medicine Facility and genotyped at Loyola University Chicago.

Techniques: Ligation, Gene Knockout

Journal: FASEB BioAdvances

Article Title: Age and Sex Impact the Role of Thrombospondin‐2 and Thrombospondin‐5 in Response to Hindlimb Ischemia

doi: 10.1096/fba.2025-00258

Figure Lengend Snippet: (A, B) Thrombospondin effect on arteriogenesis within respective age group. Young (14–16 weeks) and old (105–110 weeks) male and female WT, THBS2 −/− and THBS5 −/− mice underwent femoral artery ligation. Arteriogenesis was assessed 14 days postoperatively. (A) THBS5 −/− young males had reduced arteriogenesis compared to WT, THBS5 −/− young females had increased arteriogenesis than WT. (B) THBS2 −/− and THBS5 −/− in old male and female mice increased arteriogenesis compared to their respective WT controls. 5hpf signifies arterioles counted at 20× magnification of five random fields using ImageJ 1.54 g. Values are reported as means ± SEM. * p < 0.05, gene knockout compared to wild‐type (WT).

Article Snippet: THBS2 + heterozygote mice were obtained from Jackson Laboratory (Bar Harbor, ME), bred to homozygote THBS2 −/− at Loyola University Chicago Comparative Medicine Facility and genotyped at Loyola University Chicago.

Techniques: Ligation, Gene Knockout

Journal: FASEB BioAdvances

Article Title: Age and Sex Impact the Role of Thrombospondin‐2 and Thrombospondin‐5 in Response to Hindlimb Ischemia

doi: 10.1096/fba.2025-00258

Figure Lengend Snippet: (A–C) The effect of sex or age among the thrombospondins. Young and old male and female mice and ovariectomized young females underwent femoral artery ligation. Angiogenesis and arteriogenesis were assessed on postoperative day 14. The figure depicts differences in angiogenesis and arteriogenesis in (A) wild‐type mice, (B) THBS2 −/− mice, (C) THBS5 −/− mice. 5hpf signifies arterioles counted at 20× magnification of five random fields using ImageJ 1.54 g. Values are depicted as means ± SEM. § p < 0.05 females compared to males, brackets determine whether comparison is between non‐OVX or OVX females, * p < 0.05, OVX females compared to non‐OVX females, # p < 0.05, old age compared to young in respective group.

Article Snippet: THBS2 + heterozygote mice were obtained from Jackson Laboratory (Bar Harbor, ME), bred to homozygote THBS2 −/− at Loyola University Chicago Comparative Medicine Facility and genotyped at Loyola University Chicago.

Techniques: Ligation, Comparison

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a, b THBS2 expression in each subtype of CMS ( a , n: CMS1 = 85, CMS2 = 132, CMS3 = 78, CMS4 = 184) and IMF ( b n: MSI = 82, iCMS2_MSS_NF = 117, iCMS3_MSS_NF = 73, iCMS2_MSS_F = 101, iCMS3_MSS_F = 51) in TCGA-COADREAD dataset. c THBS2 expression in iCMS2 or iCMS3 in TCGA (n: iCMS2 = 298, iCMS3 = 232). d Kaplan-Meier curves for overall survival of iCMS2 or iCMS3 patients according to THBS2 expression in TCGA dataset (n: iCMS2, THBS2 high = 139, THBS2 low = 143; iCMS3, THBS2 high = 118, THBS2 low = 106). e–m scRNA-seq analyses of Colorectal Atlas dataset (n = 192). Uniform manifold approximation and projection (UMAP) plot of all cells colored by tissue origin ( e ) and cell types ( f ). UMAP feature plot colored by THBS2 expression ( g ). Violin plots for indicated gene expression in fibroblast subsets ( h ). UMAP plot of tumor fibroblast subsets colored by cell types ( i ). Dot plots of indicated gene expression across CAF subtypes ( j ). Violin plots for indicated gene expression ( k ) and gene signatures ( l ) in tumor fibroblast subsets. Scatter plots showing the proportions of THBS2-positive CAFs and mCAF-positive CAFs in individual patients ( m ). n Co-immunostaining for THBS2 (RNAscope) and CD8 in CMS-annotated human CRC. T: tumor, NT: non-tumor, F: tumor front, I: tumor interior. White lines denote tumor borders. o–t Xenium 5K panel analyses of tumor–non-tumor interfaces in CMS1/MSI and CMS4/MSS CRCs. T tumor, NT non-tumor, F tumor front. Spatial maps ( o,q,r ) and corresponding pie charts ( p ) showing annotated cell types. Black dashed lines denote tumor borders. Three-dimensional density maps ( s ) and quantification of CD8⁺ T-cell density (n = 6, respectively; technical replicates) ( t ). Dunnett’s test, two-sided ( a, b ), unpaired Student’s t-test, two-sided ( c ), Log-rank test ( d ), Wilcoxon rank sum test, two-sided ( k, l ), Pearson correlation coefficient, two-sided ( m ), and Mann-Whitney test, two-sided ( t ). Mean ± SEM. Scale bars, 200 μm ( n, o, q, r ). Adjustments for multiple comparisons were made in ( a, b ), and not in ( k, l ). Box and whiskers graphs indicate the median and the 25th and 75th percentiles, with minimum and maximum values at the extremes of the whiskers. Source data are provided as a file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: Expressing, Gene Expression, Immunostaining, RNAscope, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a Schematic representation of orthotopic implantation of MTO to WT mice. b Immunofluorescence (left) for CD8 and THBS2 in orthotopic MTO tumors, and quantification of CD8 + cells (right, n = 3; biological replicates). Scale bars, 500 μm (top), 100 μm (bottom). White lines denote tumor borders. c Schematic representation of orthotopic implantation of MTO to WT or Thbs2 -/- mice. d Macroscopic images (top) and H&E staining (bottom) of orthotopic implantation in WT or Thbs2 -/- mice. Scale bars, 1 mm. e, f Volume ( e , n: WT = 15, Thbs2 -/- = 16) and the change in diameter ( f , n: WT = 12, Thbs2 -/- = 15) of orthotopic tumors in WT or Thbs2 -/- mice. g, h Incidence ( g , left), macroscopic numbers ( g , right), and H&E staining ( h ) of liver metastasis (met, arrows) in WT or Thbs2 -/- mice (n: WT = 15, Thbs2 -/- = 16). Scale bars, 1 mm. i Kaplan-Meier curves after MTO implantation (n: WT = 17, Thbs2 -/- = 19). j , Immunofluorescence for CD8, THBS2, and αSMA in orthotopic tumors in WT or Thbs2 -/- mice 5-weeks post-injection of MTOs. Scale bars, 500 μm. k , Volcano plot of upregulated genes in Thbs2 -/- mice versus WT mice after MTO implantation (n = 3 biological replicates). l–n Anti-CD8 antibody (αCD8 ab) treatment on MTO-bearing WT or Thbs2 -/- mice (n: WT control = 6, WT αCD8 ab = 6, Thbs2 -/- control = 7, Thbs2 -/- αCD8 ab = 8). Schematic representation ( l ), macroscopic images ( m ), and volumes and weights ( n ) of tumors. o , p Immunohistochemistry for indicated proteins ( o ) and quantification ( p ) in orthotopic tumors of WT or Thbs2 -/- mice (n = 3). Red dashed lines denote tumors ( d, m ). Scale bars, 50 μm. Tukey’s test, two-sided ( b ), Unpaired Student’s t-test, two-sided ( e, g ), two-way ANOVA ( f ), Log-rank test ( i ), Fisher’s exact test, two-sided ( g ), Fisher’s LSD test, two-sided ( n, p ). Mean ± SEM. Adjustments for multiple comparisons were made in ( b ). Source data are provided as a Source Data file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: Immunofluorescence, Staining, Injection, Control, Immunohistochemistry

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a Gene targeting strategy for Thbs2 . b Schematic representation of orthotopic implantation of MTO to Thbs2 f/f or fibroblast-specific Thbs2 knockout mice ( Thbs2 ΔFibro). c Thbs2 expression in MTO-derived orthotopic tumors from Thbs2 f/f or Thbs2 ΔFibro mice analyzed by qRT-PCR (n = 3). d Macroscopic images of orthotopic tumors in Thbs2 f/f or Thbs2 ΔFibro mice. e Tumor diameter in Thbs2 f/f or Thbs2 ΔFibro mice (n = 11). f Immunofluorescence for CD8 and THBS2 in orthotopic MTO tumors. F: tumor front, I: tumor interior. Magnified views (middle and right) correspond to the yellow and magenta boxed regions in the left panels. g Immunohistochemistry for cleaved-caspase 3 (C-Cas3) in orthotopic MTO tumors. h Quantification of CD8 + cells in ( f ) and C-Cas3 + cells in ( g ; n = 3; biological replicates). i , Schematic representation of orthotopic co-implantation of MTO and WT CAF or Thbs2 -/- CAF into WT or Thbs2 -/- mice. Comparisons were made between tumors in WT mice implanted with WT CAF, tumors in Thbs2 -/- mice implanted with Thbs2 -/- CAF, and tumors in Thbs2 -/- mice implanted with WT CAF. j, k Macroscopic images ( j ) and volumes ( k ) of orthotopic tumors in ( i )(n: MTO & WT CAF → WT = 12, MTO & Thbs2 -/- CAF → Thbs2 -/- = 10, MTO & WT CAF→ Thbs2 -/- = 10). l–o Immunofluorescence ( l ) and immunohistochemistry ( m ) for indicated proteins in orthotopic tumors from ( i ), with quantifications ( n, o ) (n = 3; biological replicates). F: tumor front, I: tumor interior. Magnified views (middle and bottom) correspond to the yellow and magenta boxed regions in the top panels ( l ). Scale bars: 1 mm ( d, j ), 200 μm ( f, l ), 50 μm ( g, m ). Unpaired Student’s t-test, two-sided ( c, e, h ), Šídák’s multiple comparison test, two-sided ( k ), Tukey’s test, two-sided ( n, o ). Mean ± SEM. Adjustments for multiple comparisons were made in ( n, o, k ). Source data are provided as a file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: Knock-Out, Expressing, Derivative Assay, Quantitative RT-PCR, Immunofluorescence, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a, b UMAP of single-cell RNA sequencing (scRNA-seq) data of orthotopic MTO tumors in WT or Thbs2 -/- mice. all tumor cells colored by host mouse genotypes ( a ) and major cellular components ( b ). c Pie charts showing the proportions of indicated cell types in each genotype. d, e UMAP of immune cells colored by cell types ( d ) and proportion of each cell type ( e ). f UMAP of T and natural killer cells (T/NK cells) colored by cell types. g Proportion of T/NK cell types. h, i UMAP of CD8 + T cells colored by cell subtypes ( h ) and cell counts of each subtype ( i ). j, k Violin plots of CD8 + T cells ( j ) and monocytes/macrophages (Mono/Macs) ( k ) for indicated gene signatures. l Immunofluorescence staining of indicated proteins in orthotopic tumors. Scale bars, 100 μm. m Violin plots of Mono/Mac for indicated gene signatures. n UMAP of Mono/Mac colored by cell subtypes. o–q Proportion ( o ) and violin plots for indicated gene signatures and gene expression ( p, q ) of Mono/Mac subtypes. r–t UMAP of DCs colored by DC subtypes ( r ). UMAP feature plots of DCs in WT and Thbs2 -/- mice for indicated gene expression ( s ). t Violin plots of cDCs for indicated genes signatures. u, v UMAP of all tumor cells ( u ) and immune cells ( v ) colored by cell types (left) and feature plots for Cxcl10 (right). w UMAP of T/NK cells colored by cell types (left) and feature plots for Cxcr3 (right). x Schematic representation of models for CD8 + T cell recruitment regulated by THBS2 + CAF. Wilcoxon rank sum test, two-sided ( j,k,m,p,q,t ). Source data are provided as a file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: RNA Sequencing, Immunofluorescence, Staining, Gene Expression

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a Imaging mass cytometry (IMC) images of orthotopic tumors in WT or Thbs2 -/- mice at two weeks post-injection of MTO. White lines denote tumor borders. Scale bars, 100 μm. T: tumor, NT: non-tumor. b–d Neighborhood composition analyses using CytoMAP. Region prevalence ( b ) and dimensionality reduction UMAP plots ( c, d ) of neighborhoods involving CD8 + T cells, F4/80 + TAMs, and CD11c + DCs in tumors of WT or Thbs2 -/- mice. Regions are color-coded based on types defined in Supplementary Fig. . e–g CD8 + T cell infiltration analysis. Representative images showing infiltrating CD8⁺ T cells; inset images highlight infiltration bands at the tumor–non-tumor interface ( e ). Green lines indicate tumor borders. Histograms ( f ) and scatter plots ( g ) show distances of CD8 + T cells from borders (n: WT = 261, Thbs2 -/- = 2459). h–m Proximity analyses between CD8 + T cells, F4/80 + macrophages, and CD11c + DCs. Green lines indicate tumor borders. Representative images ( h ) histograms ( i ) and scatter plots ( j ) show the nearest distances between CD8 + T cells and CD11c + DCs (n: WT = 706, Thbs2 -/- = 880). Representative images ( k ) histograms ( l ) and scatter plots ( m ) show the nearest distances between CD8 + T cells and F4/80 + macrophages (n: WT = 960, Thbs2 -/- = 1022). Green lines denote tumor borders. Mann-Whitney U-test, two-sided ( g, j, m ). Mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: Imaging, Mass Cytometry, Injection, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a–e Combined blockage of CXCL9 (αCXCL9 ab) and CXCL10 (αCXCL10 ab) in MTO-bearing WT or Thbs2 -/- mice (n: WT control = 8, WT αCXCL9 ab/CXCL10 ab = 9, Thbs2 -/- control = 6, Thbs2 -/- αCXCL9 ab/CXCL10 ab = 9). Schematic representation ( a ), macroscopic images ( b ), and volumes and weights ( c ) of tumors. Immunofluorescence for CD8 ( d ) and its quantification ( e ). Bottom images show magnified views of the yellow dashed boxes in the top panels ( d ). f–o Anti-CXCR3 antibody (αCXCR3 ab) treatment in MTO-bearing WT or Thbs2 -/- mice (n: WT control = 10, WT αCXCR3 ab = 10, Thbs2 -/- control = 7, Thbs2 -/- αCXCR3 ab = 8). Schematic representation ( f ) macroscopic images ( g ) and volumes and weights ( h ) of tumors. i Representative IMC images of orthotopic tumors. T: tumor, NT: non-tumor. j, k CD8 + T cell infiltration analysis from ( f ). Histograms ( j ) and scatter plots ( k ) show distances of CD8 + T cells from tumor borders. l–o , Proximity analyses between CD8 + T cells and F4/80 + macrophages or CD11c + DCs in ( f ). Histograms ( l ) and scatter plots ( m ) show the nearest distances between CD8 + T cells and CD11c + DCs. Histograms ( n ) and scatter plots ( o ) show the nearest distances between CD8 + T cells and F4/80 + macrophages. Red dashed lines indicate tumor regions in ( b,g ). White lines denote tumor borders in ( i ). Scale bars, 1 mm ( b, g ), 500 μm ( d , top), 50 μm ( d , bottom), 100 μm ( i ). Fisher’s LSD test ( c,e,h ), Mann-Whitney U-test, two-sided ( k,m,o ). Mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: Control, Immunofluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a–f Immunofluorescence for CD8 and PD-1 in MTO-derived orthotopic tumors from WT or Thbs2 -/- mice at 2-weeks ( a ), 5-weeks ( c ), and 9-weeks ( e ) post-injection of MTO. White lines denote the tumor borders. Scale bars, 100 μm. Quantification of PD-1 + and PD-1 - CD8 + T cells at 2-weeks ( b ), 5-weeks ( d ), and 9-weeks ( f ) post-injection of MTO. F tumor front, I tumor interior. Magnified views (right panels for WT and Thbs2 -/- ) correspond to the yellow and magenta boxed regions in the left panels. g Violin plots of T cells for indicated gene signatures in tumors from WT and THBS2 -/- mice. h, i Box plots of GSVA values for indicated gene signatures in each CMS (n: CMS1 = 85, CMS2 = 132, CMS3 = 78, CMS4 = 184) subtype of CRC in TCGA-COADREAD dataset. j GSEA for Thbs2 KO signatures comparing CMS1 CRCs vs. CMS4 CRCs in TCGA. k–n Anti-PD-1 antibody (αPD-1 ab) or anti-CTLA-4 antibody (αCTLA-4 ab) treatment in MTO-bearing WT or Thbs2 -/- mice (n: WT control = 12, WT αCTLA-4 ab = 11, WT αPD-1 ab = 12, Thbs2 -/- control = 11, Thbs2 -/- αCTLA-4 ab = 11, Thbs2 -/- αPD-1 ab = 13). Schematic representation ( k ) changes in tumor diameter ( l ) tumor weights and volumes ( m ) and H&E images of tumors ( n ). Scale bars, 1 mm ( n ). Wilcoxon rank sum test, two-sided ( g ) Dunnett’s test, two-sided ( h, i ) Two-way ANOVA ( l ) Unpaired t-test, two-sided ( m ). Mean ± SEM. Adjustments for multiple comparisons were made in ( h, i ) and not in ( m ). Box and whiskers graphs indicate the median and the 25 and 75th percentiles, with minimum and maximum values at the extremes of the whiskers. Source data are provided as a file.

Article Snippet: The concentration of THBS2 in the serum was measured using the Human Thrombospondin-2 Quantikine ELISA Kit (R&D systems; DTSP20).

Techniques: Immunofluorescence, Derivative Assay, Injection, Control

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a, b THBS2 expression in each subtype of CMS ( a , n: CMS1 = 85, CMS2 = 132, CMS3 = 78, CMS4 = 184) and IMF ( b n: MSI = 82, iCMS2_MSS_NF = 117, iCMS3_MSS_NF = 73, iCMS2_MSS_F = 101, iCMS3_MSS_F = 51) in TCGA-COADREAD dataset. c THBS2 expression in iCMS2 or iCMS3 in TCGA (n: iCMS2 = 298, iCMS3 = 232). d Kaplan-Meier curves for overall survival of iCMS2 or iCMS3 patients according to THBS2 expression in TCGA dataset (n: iCMS2, THBS2 high = 139, THBS2 low = 143; iCMS3, THBS2 high = 118, THBS2 low = 106). e–m scRNA-seq analyses of Colorectal Atlas dataset (n = 192). Uniform manifold approximation and projection (UMAP) plot of all cells colored by tissue origin ( e ) and cell types ( f ). UMAP feature plot colored by THBS2 expression ( g ). Violin plots for indicated gene expression in fibroblast subsets ( h ). UMAP plot of tumor fibroblast subsets colored by cell types ( i ). Dot plots of indicated gene expression across CAF subtypes ( j ). Violin plots for indicated gene expression ( k ) and gene signatures ( l ) in tumor fibroblast subsets. Scatter plots showing the proportions of THBS2-positive CAFs and mCAF-positive CAFs in individual patients ( m ). n Co-immunostaining for THBS2 (RNAscope) and CD8 in CMS-annotated human CRC. T: tumor, NT: non-tumor, F: tumor front, I: tumor interior. White lines denote tumor borders. o–t Xenium 5K panel analyses of tumor–non-tumor interfaces in CMS1/MSI and CMS4/MSS CRCs. T tumor, NT non-tumor, F tumor front. Spatial maps ( o,q,r ) and corresponding pie charts ( p ) showing annotated cell types. Black dashed lines denote tumor borders. Three-dimensional density maps ( s ) and quantification of CD8⁺ T-cell density (n = 6, respectively; technical replicates) ( t ). Dunnett’s test, two-sided ( a, b ), unpaired Student’s t-test, two-sided ( c ), Log-rank test ( d ), Wilcoxon rank sum test, two-sided ( k, l ), Pearson correlation coefficient, two-sided ( m ), and Mann-Whitney test, two-sided ( t ). Mean ± SEM. Scale bars, 200 μm ( n, o, q, r ). Adjustments for multiple comparisons were made in ( a, b ), and not in ( k, l ). Box and whiskers graphs indicate the median and the 25th and 75th percentiles, with minimum and maximum values at the extremes of the whiskers. Source data are provided as a file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: Expressing, Gene Expression, Immunostaining, RNAscope, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a Schematic representation of orthotopic implantation of MTO to WT mice. b Immunofluorescence (left) for CD8 and THBS2 in orthotopic MTO tumors, and quantification of CD8 + cells (right, n = 3; biological replicates). Scale bars, 500 μm (top), 100 μm (bottom). White lines denote tumor borders. c Schematic representation of orthotopic implantation of MTO to WT or Thbs2 -/- mice. d Macroscopic images (top) and H&E staining (bottom) of orthotopic implantation in WT or Thbs2 -/- mice. Scale bars, 1 mm. e, f Volume ( e , n: WT = 15, Thbs2 -/- = 16) and the change in diameter ( f , n: WT = 12, Thbs2 -/- = 15) of orthotopic tumors in WT or Thbs2 -/- mice. g, h Incidence ( g , left), macroscopic numbers ( g , right), and H&E staining ( h ) of liver metastasis (met, arrows) in WT or Thbs2 -/- mice (n: WT = 15, Thbs2 -/- = 16). Scale bars, 1 mm. i Kaplan-Meier curves after MTO implantation (n: WT = 17, Thbs2 -/- = 19). j , Immunofluorescence for CD8, THBS2, and αSMA in orthotopic tumors in WT or Thbs2 -/- mice 5-weeks post-injection of MTOs. Scale bars, 500 μm. k , Volcano plot of upregulated genes in Thbs2 -/- mice versus WT mice after MTO implantation (n = 3 biological replicates). l–n Anti-CD8 antibody (αCD8 ab) treatment on MTO-bearing WT or Thbs2 -/- mice (n: WT control = 6, WT αCD8 ab = 6, Thbs2 -/- control = 7, Thbs2 -/- αCD8 ab = 8). Schematic representation ( l ), macroscopic images ( m ), and volumes and weights ( n ) of tumors. o , p Immunohistochemistry for indicated proteins ( o ) and quantification ( p ) in orthotopic tumors of WT or Thbs2 -/- mice (n = 3). Red dashed lines denote tumors ( d, m ). Scale bars, 50 μm. Tukey’s test, two-sided ( b ), Unpaired Student’s t-test, two-sided ( e, g ), two-way ANOVA ( f ), Log-rank test ( i ), Fisher’s exact test, two-sided ( g ), Fisher’s LSD test, two-sided ( n, p ). Mean ± SEM. Adjustments for multiple comparisons were made in ( b ). Source data are provided as a Source Data file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: Immunofluorescence, Staining, Injection, Control, Immunohistochemistry

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a Gene targeting strategy for Thbs2 . b Schematic representation of orthotopic implantation of MTO to Thbs2 f/f or fibroblast-specific Thbs2 knockout mice ( Thbs2 ΔFibro). c Thbs2 expression in MTO-derived orthotopic tumors from Thbs2 f/f or Thbs2 ΔFibro mice analyzed by qRT-PCR (n = 3). d Macroscopic images of orthotopic tumors in Thbs2 f/f or Thbs2 ΔFibro mice. e Tumor diameter in Thbs2 f/f or Thbs2 ΔFibro mice (n = 11). f Immunofluorescence for CD8 and THBS2 in orthotopic MTO tumors. F: tumor front, I: tumor interior. Magnified views (middle and right) correspond to the yellow and magenta boxed regions in the left panels. g Immunohistochemistry for cleaved-caspase 3 (C-Cas3) in orthotopic MTO tumors. h Quantification of CD8 + cells in ( f ) and C-Cas3 + cells in ( g ; n = 3; biological replicates). i , Schematic representation of orthotopic co-implantation of MTO and WT CAF or Thbs2 -/- CAF into WT or Thbs2 -/- mice. Comparisons were made between tumors in WT mice implanted with WT CAF, tumors in Thbs2 -/- mice implanted with Thbs2 -/- CAF, and tumors in Thbs2 -/- mice implanted with WT CAF. j, k Macroscopic images ( j ) and volumes ( k ) of orthotopic tumors in ( i )(n: MTO & WT CAF → WT = 12, MTO & Thbs2 -/- CAF → Thbs2 -/- = 10, MTO & WT CAF→ Thbs2 -/- = 10). l–o Immunofluorescence ( l ) and immunohistochemistry ( m ) for indicated proteins in orthotopic tumors from ( i ), with quantifications ( n, o ) (n = 3; biological replicates). F: tumor front, I: tumor interior. Magnified views (middle and bottom) correspond to the yellow and magenta boxed regions in the top panels ( l ). Scale bars: 1 mm ( d, j ), 200 μm ( f, l ), 50 μm ( g, m ). Unpaired Student’s t-test, two-sided ( c, e, h ), Šídák’s multiple comparison test, two-sided ( k ), Tukey’s test, two-sided ( n, o ). Mean ± SEM. Adjustments for multiple comparisons were made in ( n, o, k ). Source data are provided as a file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: Knock-Out, Expressing, Derivative Assay, Quantitative RT-PCR, Immunofluorescence, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a, b UMAP of single-cell RNA sequencing (scRNA-seq) data of orthotopic MTO tumors in WT or Thbs2 -/- mice. all tumor cells colored by host mouse genotypes ( a ) and major cellular components ( b ). c Pie charts showing the proportions of indicated cell types in each genotype. d, e UMAP of immune cells colored by cell types ( d ) and proportion of each cell type ( e ). f UMAP of T and natural killer cells (T/NK cells) colored by cell types. g Proportion of T/NK cell types. h, i UMAP of CD8 + T cells colored by cell subtypes ( h ) and cell counts of each subtype ( i ). j, k Violin plots of CD8 + T cells ( j ) and monocytes/macrophages (Mono/Macs) ( k ) for indicated gene signatures. l Immunofluorescence staining of indicated proteins in orthotopic tumors. Scale bars, 100 μm. m Violin plots of Mono/Mac for indicated gene signatures. n UMAP of Mono/Mac colored by cell subtypes. o–q Proportion ( o ) and violin plots for indicated gene signatures and gene expression ( p, q ) of Mono/Mac subtypes. r–t UMAP of DCs colored by DC subtypes ( r ). UMAP feature plots of DCs in WT and Thbs2 -/- mice for indicated gene expression ( s ). t Violin plots of cDCs for indicated genes signatures. u, v UMAP of all tumor cells ( u ) and immune cells ( v ) colored by cell types (left) and feature plots for Cxcl10 (right). w UMAP of T/NK cells colored by cell types (left) and feature plots for Cxcr3 (right). x Schematic representation of models for CD8 + T cell recruitment regulated by THBS2 + CAF. Wilcoxon rank sum test, two-sided ( j,k,m,p,q,t ). Source data are provided as a file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: RNA Sequencing, Immunofluorescence, Staining, Gene Expression

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a Imaging mass cytometry (IMC) images of orthotopic tumors in WT or Thbs2 -/- mice at two weeks post-injection of MTO. White lines denote tumor borders. Scale bars, 100 μm. T: tumor, NT: non-tumor. b–d Neighborhood composition analyses using CytoMAP. Region prevalence ( b ) and dimensionality reduction UMAP plots ( c, d ) of neighborhoods involving CD8 + T cells, F4/80 + TAMs, and CD11c + DCs in tumors of WT or Thbs2 -/- mice. Regions are color-coded based on types defined in Supplementary Fig. . e–g CD8 + T cell infiltration analysis. Representative images showing infiltrating CD8⁺ T cells; inset images highlight infiltration bands at the tumor–non-tumor interface ( e ). Green lines indicate tumor borders. Histograms ( f ) and scatter plots ( g ) show distances of CD8 + T cells from borders (n: WT = 261, Thbs2 -/- = 2459). h–m Proximity analyses between CD8 + T cells, F4/80 + macrophages, and CD11c + DCs. Green lines indicate tumor borders. Representative images ( h ) histograms ( i ) and scatter plots ( j ) show the nearest distances between CD8 + T cells and CD11c + DCs (n: WT = 706, Thbs2 -/- = 880). Representative images ( k ) histograms ( l ) and scatter plots ( m ) show the nearest distances between CD8 + T cells and F4/80 + macrophages (n: WT = 960, Thbs2 -/- = 1022). Green lines denote tumor borders. Mann-Whitney U-test, two-sided ( g, j, m ). Mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: Imaging, Mass Cytometry, Injection, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a–e Combined blockage of CXCL9 (αCXCL9 ab) and CXCL10 (αCXCL10 ab) in MTO-bearing WT or Thbs2 -/- mice (n: WT control = 8, WT αCXCL9 ab/CXCL10 ab = 9, Thbs2 -/- control = 6, Thbs2 -/- αCXCL9 ab/CXCL10 ab = 9). Schematic representation ( a ), macroscopic images ( b ), and volumes and weights ( c ) of tumors. Immunofluorescence for CD8 ( d ) and its quantification ( e ). Bottom images show magnified views of the yellow dashed boxes in the top panels ( d ). f–o Anti-CXCR3 antibody (αCXCR3 ab) treatment in MTO-bearing WT or Thbs2 -/- mice (n: WT control = 10, WT αCXCR3 ab = 10, Thbs2 -/- control = 7, Thbs2 -/- αCXCR3 ab = 8). Schematic representation ( f ) macroscopic images ( g ) and volumes and weights ( h ) of tumors. i Representative IMC images of orthotopic tumors. T: tumor, NT: non-tumor. j, k CD8 + T cell infiltration analysis from ( f ). Histograms ( j ) and scatter plots ( k ) show distances of CD8 + T cells from tumor borders. l–o , Proximity analyses between CD8 + T cells and F4/80 + macrophages or CD11c + DCs in ( f ). Histograms ( l ) and scatter plots ( m ) show the nearest distances between CD8 + T cells and CD11c + DCs. Histograms ( n ) and scatter plots ( o ) show the nearest distances between CD8 + T cells and F4/80 + macrophages. Red dashed lines indicate tumor regions in ( b,g ). White lines denote tumor borders in ( i ). Scale bars, 1 mm ( b, g ), 500 μm ( d , top), 50 μm ( d , bottom), 100 μm ( i ). Fisher’s LSD test ( c,e,h ), Mann-Whitney U-test, two-sided ( k,m,o ). Mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: Control, Immunofluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting fibroblast derived thrombospondin 2 disrupts an immune-exclusionary environment at the tumor front in colorectal cancer

doi: 10.1038/s41467-025-66485-2

Figure Lengend Snippet: a–f Immunofluorescence for CD8 and PD-1 in MTO-derived orthotopic tumors from WT or Thbs2 -/- mice at 2-weeks ( a ), 5-weeks ( c ), and 9-weeks ( e ) post-injection of MTO. White lines denote the tumor borders. Scale bars, 100 μm. Quantification of PD-1 + and PD-1 - CD8 + T cells at 2-weeks ( b ), 5-weeks ( d ), and 9-weeks ( f ) post-injection of MTO. F tumor front, I tumor interior. Magnified views (right panels for WT and Thbs2 -/- ) correspond to the yellow and magenta boxed regions in the left panels. g Violin plots of T cells for indicated gene signatures in tumors from WT and THBS2 -/- mice. h, i Box plots of GSVA values for indicated gene signatures in each CMS (n: CMS1 = 85, CMS2 = 132, CMS3 = 78, CMS4 = 184) subtype of CRC in TCGA-COADREAD dataset. j GSEA for Thbs2 KO signatures comparing CMS1 CRCs vs. CMS4 CRCs in TCGA. k–n Anti-PD-1 antibody (αPD-1 ab) or anti-CTLA-4 antibody (αCTLA-4 ab) treatment in MTO-bearing WT or Thbs2 -/- mice (n: WT control = 12, WT αCTLA-4 ab = 11, WT αPD-1 ab = 12, Thbs2 -/- control = 11, Thbs2 -/- αCTLA-4 ab = 11, Thbs2 -/- αPD-1 ab = 13). Schematic representation ( k ) changes in tumor diameter ( l ) tumor weights and volumes ( m ) and H&E images of tumors ( n ). Scale bars, 1 mm ( n ). Wilcoxon rank sum test, two-sided ( g ) Dunnett’s test, two-sided ( h, i ) Two-way ANOVA ( l ) Unpaired t-test, two-sided ( m ). Mean ± SEM. Adjustments for multiple comparisons were made in ( h, i ) and not in ( m ). Box and whiskers graphs indicate the median and the 25 and 75th percentiles, with minimum and maximum values at the extremes of the whiskers. Source data are provided as a file.

Article Snippet: Thbs2 floxed ( Thbs2 f/f ) mice were generated through outsourced services at Macrogen, Inc. (Seoul, Korea) using the CRISPR/Cas9 system.

Techniques: Immunofluorescence, Derivative Assay, Injection, Control

Journal: Melanoma Research

Article Title: Thrombospondin 2 drives liver metastasis in skin cutaneous melanoma via regulation of angiogenesis and extracellular matrix remodeling

doi: 10.1097/CMR.0000000000001055

Figure Lengend Snippet: THBS2 expression profiles in melanoma cell lines and genetic manipulation validation. (a) qRT-PCR analysis of THBS2 mRNA levels in normal melanocytes (HEMa) and melanoma cell lines (RPMI-7951, SK-MEL-28, G-361, MeWo, and A375). (b and c) Western blotting images and quantification of THBS2 protein expression normalized to β-actin. Data are shown as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001 versus HEMa cells. (d) Analysis of THBS2 expression data from the TIMER database shows significantly higher levels in metastatic melanomas ( n = 368) compared with primary melanomas ( n = 103), indicating a correlation with metastasis. (e–g) THBS2 knockdown in A375 cells confirmed by qRT-PCR (e) and western blotting analysis (f and g); **** P < 0.001 versus A375-mock control. (h–j) THBS2 overexpression in G-361 cells validated by qRT-PCR (h) and western blot analysis (i and j); **** P < 0.001 versus G-361-mock control. Data represent mean ± SD from three independent experiments. HEMa, human epidermal melanocyte; qRT-PCR, quantitative real-time PCR; RPMI, Roswell Park Memorial Institute; THBS2, thrombospondin 2; TIMER, Tumor Immune Estimation Resource.

Article Snippet: THBS2 overexpression was performed using THBS2 lentiviral activation particles (sc-401207-LAC; Santa Cruz Biotechnology), with corresponding control lentiviral activation particles (control vector, sc-437282).

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Knockdown, Control, Over Expression, Real-time Polymerase Chain Reaction

Journal: Melanoma Research

Article Title: Thrombospondin 2 drives liver metastasis in skin cutaneous melanoma via regulation of angiogenesis and extracellular matrix remodeling

doi: 10.1097/CMR.0000000000001055

Figure Lengend Snippet: THBS2 enhanced angiogenesis in melanoma through upregulation of angiogenesis-related factors. (a and b) Correlation analysis of THBS2 with angiogenesis-associated genes VEGFA , FGF2 , PECAM1, and FLT1 in primary melanoma (a) and metastatic melanoma (b) using the TIMER database. (c–f) Relative mRNA and protein expression of VEGFA, FGF2, PECAM1, and FLT1 in A375 cells with THBS2 knockdown (c and d) and G-361 cells with THBS2 overexpression (e and f), assessed by qRT-PCR and western blotting. (g and h) Representative images (g) and quantification (h) of tube formation in HUVECs cultured in conditioned media derived from melanoma cells with altered THBS2 expression. Data represent mean ± SD from three independent experiments. FGF2 , fibroblast growth factor 2; FLT1 , Fms-related tyrosine kinase 1; HUVECs, human umbilical vein endothelial cells; PECAM1 , platelet and endothelial cell adhesion molecule 1; qRT-PCR, quantitative real-time PCR; THBS2, thrombospondin 2; TIMER, Tumor Immune Estimation Resource; VEGFA , vascular endothelial growth factor A.

Article Snippet: THBS2 overexpression was performed using THBS2 lentiviral activation particles (sc-401207-LAC; Santa Cruz Biotechnology), with corresponding control lentiviral activation particles (control vector, sc-437282).

Techniques: Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Western Blot, Cell Culture, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Melanoma Research

Article Title: Thrombospondin 2 drives liver metastasis in skin cutaneous melanoma via regulation of angiogenesis and extracellular matrix remodeling

doi: 10.1097/CMR.0000000000001055

Figure Lengend Snippet: THBS2 regulated melanoma cell invasion and migration in-vitro. (a–c) Representative images of Transwell invasion assays (a) and wound healing assays at 0 and 24 h (b and c) in A375 cells with THBS2 knockdown and G-361 cells with THBS2 overexpression. (d) Fold change in invasive cell number relative to the mock group. (e) Quantification of wound closure percentage at 24 h. Data represent mean ± SD of three independent experiments. THBS2, thrombospondin 2.

Article Snippet: THBS2 overexpression was performed using THBS2 lentiviral activation particles (sc-401207-LAC; Santa Cruz Biotechnology), with corresponding control lentiviral activation particles (control vector, sc-437282).

Techniques: Migration, In Vitro, Knockdown, Over Expression

Journal: Melanoma Research

Article Title: Thrombospondin 2 drives liver metastasis in skin cutaneous melanoma via regulation of angiogenesis and extracellular matrix remodeling

doi: 10.1097/CMR.0000000000001055

Figure Lengend Snippet: THBS2 was associated with ECM-related gene expression and MMP activity in melanoma. (a and b) Correlation analysis between THBS2 and ECM- or MMP-related genes in primary (a) and metastatic (b) SKCM samples from the TIMER database. (c) qRT-PCR analysis of ECM-related genes and MMPs in A375 cells with THBS2 knockdown (c) and G-361 cells with THBS2 overexpression (d). (e) Western blotting analysis of ECM- and MMP-related protein expressions in A375 and G-361 cells following THBS2 knockdown or overexpression, respectively. Data are presented as mean ± SD from three independent experiments. COL1A1, collagen type I alpha 1 chain; COL4A1, collagen type IV alpha 1 chain; ECM, extracellular matrix; FN1, fibronectin 1; LAMA4, laminin subunit alpha-4; MMP2, matrix metallopeptidase 2; MMP9, matrix metallopeptidase 9; qRT-PCR, quantitative real-time PCR; SKCM, skin cutaneous melanoma; THBS2, thrombospondin 2; TIMER, Tumor Immune Estimation Resource.

Article Snippet: THBS2 overexpression was performed using THBS2 lentiviral activation particles (sc-401207-LAC; Santa Cruz Biotechnology), with corresponding control lentiviral activation particles (control vector, sc-437282).

Techniques: Gene Expression, Activity Assay, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Real-time Polymerase Chain Reaction

Journal: Melanoma Research

Article Title: Thrombospondin 2 drives liver metastasis in skin cutaneous melanoma via regulation of angiogenesis and extracellular matrix remodeling

doi: 10.1097/CMR.0000000000001055

Figure Lengend Snippet: THBS2 deficiency suppressed hepatic melanoma metastasis and downregulated angiogenesis- and ECM-related markers in peritumoral liver tissue. (a) Macroscopic images of representative metastatic livers from Thbs2 WT and KO mice 14 days after intrasplenic injection of B16-F10 cells. (b) Quantification of visible hepatic metastatic nodules. (c) Percentage of liver area occupied by metastases. (d) Relative mRNA expression levels of Lama4 , Pecam1 , Vegfa , Mmp2 , and Mmp9 in peritumoral liver tissues, measured by qRT-PCR. (e and f) Western blotting analysis and quantification of corresponding protein expression in peritumoral liver tissues. Data are shown as mean ± SD; n = 8 per group. ECM, extracellular matrix; KO, knockout; qRT-PCR, quantitative real-time PCR; THBS2, thrombospondin 2; WT, wild-type.

Article Snippet: THBS2 overexpression was performed using THBS2 lentiviral activation particles (sc-401207-LAC; Santa Cruz Biotechnology), with corresponding control lentiviral activation particles (control vector, sc-437282).

Techniques: Injection, Expressing, Quantitative RT-PCR, Western Blot, Knock-Out, Real-time Polymerase Chain Reaction