Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: Myofibroblast activation in ISG15-deficient cells. (A) Spatial expression and intensity levels of two myofibroblast markers ( ACTA2 and COL5A1 ). (B) Relative expression of various myofibroblast markers for clusters 1 and 5 separated by sample biopsy. (C) Percentage of total spots that express TGFβ in the entire biopsy, within CD68 + spots only, and within myofibroblast cluster 5. (D) Significance of pathway enrichment for clusters 0, 1, 5, 3, 7, and 9 generated by MSigDB Hallmark 2020 and BioPlanet 2019 analysis from Enrichr. Adjusted P values are shown. (E) Relative RNA levels of myofibroblast markers ( PDGFA , ITGB6 , FGF2 ) following a 72-h treatment with 1,000 IU/ml IFNα2b, 10 ng/ml TGFβ, or both of WT and ISG15 KO lung epithelial cells (A549). (F) Percentage of cells undergoing EMT upon TGFβ/IFNα stimulation. (G) Relative RNA levels of myofibroblast markers ( PDGFA , ITGB6 , FGF2 ) following a 5-day treatment with 1,000 IU/ml IFNα2b, 10 ng/ml TGFβ, or both of WT and ISG15 KO fibroblasts. (H) ACTA2 (myofibroblasts) IHC for lesion 1 and lesion 3. P values were calculated with two-tailed t test. *P < 0.05.

Article Snippet: The probes used were the following: TGFB1 Hs07289533_m1, ISG15 Hs01921425_s1, IFIT1 Hs03027069_s1, IFI27 Hs01086373_g1, SIGLEC1 Hs00988063_m1, MX1 Hs00895608_m1, FGF-2 Hs00266645_m1, ITGB6 Hs00168458_m1, PDGFA Hs00234994_m1.

Techniques: Activation Assay, Expressing, Generated, Two Tailed Test

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: ITGB6 shows specificity and upregulation in human cancers. (A) Human Protein Atlas analysis of RNA expression of ITGB6 across various cancer types. Normalized RNA expression levels are given in fragments per kilobase million (FPKM). Data are acquired from TCGA. Graphs show the mean and SD. (B) Differential gene expression analysis of ITGB6 across tumor and normal tissue. Data are obtained from a standardized concatenated data set from GEO, GTex, TCGA, and TARGET databases. RNA-seq tumor and normal tissue samples are obtained from the same patients and from adjacent sites. Graphs were generated using the TNMplot tool and were normalized using DESeq2. Fold-change (FC) of medians and Mann-Whitney test p -values are shown.

Article Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay.

Techniques: RNA Expression, Gene Expression, RNA Sequencing, Generated, MANN-WHITNEY

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay. Statistical analysis was performed using an unpaired t-test. Randomly chosen fields of view from the co-culture of FaDu (F), CAL-27 (G), or Capan-2 (H) cells (green) and TALL-104 T-cells (blue) (n = 10). Cells were pretreated for 8 hrs with latent TGFβ. Quantification of cancer cell counts for FaDu (I), CAL27 (M), and Capan-2 (Q). Quantification of the percentage of cancer cells that are dead for FaDu (J), CAL27 (N), and Capan-2 (R). Quantification of TALL-104 T-cell counts for the FaDu cohort (K), the CAL27 cohort (O), and the Capan-2 cohort (S). Quantification of the percentage of T cells that are dead for the FaDu cohort (L), the CAL27 cohort (P), and the Capan-2 cohort (T). One-way ANOVA: P < 0.0001 (****), P < 0.01 (**).

Article Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay.

Techniques: Knock-Out, Flow Cytometry, Western Blot, Biomarker Discovery, CRISPR, Colony Assay, Control, Co-Culture Assay

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: Suppressed growth of ITGB6 -knockout tumors in immunocompetent mice. (A) Flow cytometry analysis of the percentage of mouse cancer cells that are ITGB6+ compared to unstained controls. (B) Western blot validation of CRISPR knockdown of ITGB6 in MOC1 and KPCY cells. (C) Colony formation assay of CRISPR control (CTRL) cells and ITGB6-knockout (KO-1, KO-2) cells for MOC1 and KPCY cells. (D) Quantification of colony formation assay. Statistical analysis was performed using one-way ANOVA: P < 0.05 (*). (E) Experimental timeline for C57BL/6 mice injected with syngeneic tumor cells. Tumor volume of mice starting upon tumor formation at day 5 for MOC1 (F) and KPCY (H) cohorts (n = 10). Mass of MOC1 (G) and KPCY (I) tumors harvested at day 25. Statistical comparison between CTRL and KO-1 cohorts was performed using an unpaired t-test: P < 0.001 (***), P < 0.05 (*). (J) Heat map of Luminex panel showing fold change of cytokines of tumor interstitial fluid harvested from MOC1 and KPCY tumors (n = 5). Red indicates upregulation and green downregulation of cytokines in ITGB6 KO tumors compared to control tumors. (K) Concentrations of select tumor interstitial fluid cytokines from MOC1 and KPCY (L) as measured by the Luminex panel. Statistical comparison between CTRL and KO-1 cohorts was performed using an unpaired t-test. Representative 10× images of MOC1 (M) and KPCY (O) from scanned IHC slides showing CD8 T-cell infiltration across control and ITGB6-deficient cells. Quantification of CD8-stained IHC slides for MOC1 (N) (control: n = 5, KO: n = 10) and KPCY (P) (control: n = 7, KO: n = 8) cohorts, showing p -values calculated using an unpaired t-test.

Article Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay.

Techniques: Knock-Out, Flow Cytometry, Western Blot, Biomarker Discovery, CRISPR, Knockdown, Colony Assay, Control, Injection, Comparison, Luminex, Staining

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: ITGB6 decreases overall survival in patients. Curves show the overall survival of patients with various cancer types stratified based on ITGB6 expression. Patients with high ITGB6 have expression levels above +0.25 σ of the mean, and ITGB6 low patients have expression below -0.25 σ. The graphs were generated using TCGA data through the cBioPortal. Statistical analysis of Kaplan-Meier curves and corresponding hazard ratios (HR) was performed using the Log-rank test.

Article Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay.

Techniques: Expressing, Generated

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: Immune checkpoint blockade is modulated by ITGB6 expression. (A) Pan-cancer analysis of ITGB6 RNA expression level normalized using DESeq2 of patients who are either responsive or unresponsive to immune checkpoint blockade. RNA data are extracted from a TCGA data set of multiple cancer types of patients who underwent immune checkpoint blockade therapy. Graphs were generated using the ROCplot tool, and the results of an unpaired t-test are shown. (B) Kaplan-Meier curves of overall survival of patients undergoing immune checkpoint blockade therapy stratified into low and high ITGB6 expression about the mean. Statistical analysis was performed using the Log-rank test.

Article Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay.

Techniques: Expressing, RNA Expression, Generated

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: ITGB6 shows specificity and upregulation in human cancers. (A) Human Protein Atlas analysis of RNA expression of ITGB6 across various cancer types. Normalized RNA expression levels are given in fragments per kilobase million (FPKM). Data are acquired from TCGA. Graphs show the mean and SD. (B) Differential gene expression analysis of ITGB6 across tumor and normal tissue. Data are obtained from a standardized concatenated data set from GEO, GTex, TCGA, and TARGET databases. RNA-seq tumor and normal tissue samples are obtained from the same patients and from adjacent sites. Graphs were generated using the TNMplot tool and were normalized using DESeq2. Fold-change (FC) of medians and Mann-Whitney test p -values are shown.

Article Snippet: The human cell lines HCT116WT, Capan-2, FaDu, and CAL27 were screened via flow cytometry using an APC-conjugated human ITGB6 antibody (R&D Systems, # FAB4155A).

Techniques: RNA Expression, Gene Expression, RNA Sequencing, Generated, MANN-WHITNEY

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: ITGB6 knockout induces T-cell killing of HNSCC and PAAD cells. (A) ITGB6 RNA of selected human cancer cell lines from the Human Protein Atlas. (B) Flow cytometry analysis of the percentage of cancer cells that are ITGB6+ compared to unstained controls. (C) Western blot validation of the CRISPR knockout of ITGB6 in FaDu, CAL27, and Capan-2 cells. (D) Colony formation assay of CRISPR control (CTRL) cells and ITGB6 knockouts (KO-1) for FaDu and CAL27 cells (n = 3). (E) Quantification of colony formation assay. Statistical analysis was performed using an unpaired t-test. Randomly chosen fields of view from the co-culture of FaDu (F), CAL-27 (G), or Capan-2 (H) cells (green) and TALL-104 T-cells (blue) (n = 10). Cells were pretreated for 8 hrs with latent TGFβ. Quantification of cancer cell counts for FaDu (I), CAL27 (M), and Capan-2 (Q). Quantification of the percentage of cancer cells that are dead for FaDu (J), CAL27 (N), and Capan-2 (R). Quantification of TALL-104 T-cell counts for the FaDu cohort (K), the CAL27 cohort (O), and the Capan-2 cohort (S). Quantification of the percentage of T cells that are dead for the FaDu cohort (L), the CAL27 cohort (P), and the Capan-2 cohort (T). One-way ANOVA: P < 0.0001 (****), P < 0.01 (**).

Article Snippet: The human cell lines HCT116WT, Capan-2, FaDu, and CAL27 were screened via flow cytometry using an APC-conjugated human ITGB6 antibody (R&D Systems, # FAB4155A).

Techniques: Knock-Out, Flow Cytometry, Western Blot, Biomarker Discovery, CRISPR, Colony Assay, Control, Co-Culture Assay

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: Suppressed growth of ITGB6 -knockout tumors in immunocompetent mice. (A) Flow cytometry analysis of the percentage of mouse cancer cells that are ITGB6+ compared to unstained controls. (B) Western blot validation of CRISPR knockdown of ITGB6 in MOC1 and KPCY cells. (C) Colony formation assay of CRISPR control (CTRL) cells and ITGB6-knockout (KO-1, KO-2) cells for MOC1 and KPCY cells. (D) Quantification of colony formation assay. Statistical analysis was performed using one-way ANOVA: P < 0.05 (*). (E) Experimental timeline for C57BL/6 mice injected with syngeneic tumor cells. Tumor volume of mice starting upon tumor formation at day 5 for MOC1 (F) and KPCY (H) cohorts (n = 10). Mass of MOC1 (G) and KPCY (I) tumors harvested at day 25. Statistical comparison between CTRL and KO-1 cohorts was performed using an unpaired t-test: P < 0.001 (***), P < 0.05 (*). (J) Heat map of Luminex panel showing fold change of cytokines of tumor interstitial fluid harvested from MOC1 and KPCY tumors (n = 5). Red indicates upregulation and green downregulation of cytokines in ITGB6 KO tumors compared to control tumors. (K) Concentrations of select tumor interstitial fluid cytokines from MOC1 and KPCY (L) as measured by the Luminex panel. Statistical comparison between CTRL and KO-1 cohorts was performed using an unpaired t-test. Representative 10× images of MOC1 (M) and KPCY (O) from scanned IHC slides showing CD8 T-cell infiltration across control and ITGB6-deficient cells. Quantification of CD8-stained IHC slides for MOC1 (N) (control: n = 5, KO: n = 10) and KPCY (P) (control: n = 7, KO: n = 8) cohorts, showing p -values calculated using an unpaired t-test.

Article Snippet: The human cell lines HCT116WT, Capan-2, FaDu, and CAL27 were screened via flow cytometry using an APC-conjugated human ITGB6 antibody (R&D Systems, # FAB4155A).

Techniques: Knock-Out, Flow Cytometry, Western Blot, Biomarker Discovery, CRISPR, Knockdown, Colony Assay, Control, Injection, Comparison, Luminex, Staining

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: ITGB6 decreases overall survival in patients. Curves show the overall survival of patients with various cancer types stratified based on ITGB6 expression. Patients with high ITGB6 have expression levels above +0.25 σ of the mean, and ITGB6 low patients have expression below -0.25 σ. The graphs were generated using TCGA data through the cBioPortal. Statistical analysis of Kaplan-Meier curves and corresponding hazard ratios (HR) was performed using the Log-rank test.

Article Snippet: The human cell lines HCT116WT, Capan-2, FaDu, and CAL27 were screened via flow cytometry using an APC-conjugated human ITGB6 antibody (R&D Systems, # FAB4155A).

Techniques: Expressing, Generated

Journal: American Journal of Cancer Research

Article Title: Inhibition of ITGB6 stimulates potent anti-tumor responses in immunocompetent mouse models of head and neck squamous cell carcinoma and pancreatic adenocarcinoma

doi: 10.62347/MEAD1055

Figure Lengend Snippet: Immune checkpoint blockade is modulated by ITGB6 expression. (A) Pan-cancer analysis of ITGB6 RNA expression level normalized using DESeq2 of patients who are either responsive or unresponsive to immune checkpoint blockade. RNA data are extracted from a TCGA data set of multiple cancer types of patients who underwent immune checkpoint blockade therapy. Graphs were generated using the ROCplot tool, and the results of an unpaired t-test are shown. (B) Kaplan-Meier curves of overall survival of patients undergoing immune checkpoint blockade therapy stratified into low and high ITGB6 expression about the mean. Statistical analysis was performed using the Log-rank test.

Article Snippet: The human cell lines HCT116WT, Capan-2, FaDu, and CAL27 were screened via flow cytometry using an APC-conjugated human ITGB6 antibody (R&D Systems, # FAB4155A).

Techniques: Expressing, RNA Expression, Generated

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: (a) Experimental design for the pull-down of Siglec-10 ligands on PDAC cells. Recombinant Siglec-10 Fc (as well as a no-protein control or a Siglec-5 control) was allowed to bind its physiological ligands on the surface of PDAC cells, followed by an HRP-conjugated anti-Fc secondary antibody. In the presence of H₂O₂, HRP generated short-lived radicals that facilitated the transfer of biotin to proximal Siglec-10 ligands. Biotinylated Siglec-10 ligands were pulled down using streptavidin beads and identified by mass spectrometry. Created in BioRender. Saini, P. (2025) https://BioRender.com/lhnibjl . (b) A total of 4,044 proteins were identified, with enriched binding compared to a control using the anti-Fc antibody only without Siglec-10 protein. Of these, 110 proteins showed enrichment relative to a Siglec-5 control. Six proteins, CD47, CD59, CD73, ITGB6, ITGA3, and ITGB1, were significantly overexpressed in PAAD tissues compared to normal tissues in the TCGA dataset. (c) Response curves showing interactions between Siglec-10 and the six glycoproteins measured by surface plasmon resonance (SPR). Two concentrations (1000 nM, green; 100 nM, red) were tested for all glycoproteins, while ITGA3 was also tested at 300 nM (green) and 30 nM (red). (d) Binding of the SNA lectin (specific for sialic acid) to ITGA3 and ITGB1 recombinant glycoproteins was measured by a lectin array. Sialidase-treated glycoproteins (blue bars) showed significantly reduced binding compared to untreated glycoproteins (red bars). Unpaired t-tests. Means with SEM are shown. (e) SPR response curves comparing the binding of intact (sialylated) ITGA3 and desialylated ITGA3 to immobilized Siglec-10. (f) SPR response curves comparing the binding of intact (sialylated) ITGB1 and desialylated ITGB1 to immobilized Siglec-10.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Recombinant, Control, Generated, Mass Spectrometry, Binding Assay, SPR Assay