Journal: Journal for Immunotherapy of Cancer

Article Title: An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade

doi: 10.1136/jitc-2021-002843

Figure Lengend Snippet: Intratumoral injection of VV-scFv-TIGIT enhanced infiltration of immune cells in the CT26 tumor model. (A) Treatment scheme of CT26 subcutaneous (S.C.) tumor model. (B) Mean tumor volume of mice. (C) Individual tumor growth curve of mice. (D) Body weight of mice. (E) Representative diagram of flow cytometric analysis of immune cell infiltration in tumor tissues. (F) Flow cytometric analysis of the proportion of lymphocytes and their subpopulations in tumor tissues. (G) Flow cytometric analysis of the expression of immune checkpoints on CD8 + T cells. (H) Flow cytometric analysis of the expression of immune checkpoints on CD4 + T cells. (I) Flow cytometric analysis of the expression of CD107A on CD8 + T cells. (J) Flow cytometric analysis of the proportion of lymphocytes subsets in splenocytes. (K) The virus titers in tumor tissues or sera of mice were quantified by the TCID50 method. ns, no significant differences; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. PBS, phosphate-buffered saline; scFv, single-chain variable fragment; TIGIT, T-cell immunoglobulin and ITIM domain; VV, vaccinia virus.

Article Snippet: For depletion of CD8 + T or NK cells, each mouse was injected intraperitoneally with 500 μg of anti-CD8α antibody (Clone YTS 169.4, Cat# BP0117, BioXCell, USA) or anti-NK1.1 antibody (Clone PK136, Cat# BP0036, BioXCell).

Techniques: Injection, Expressing, Virus, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade

doi: 10.1136/jitc-2021-002843

Figure Lengend Snippet: Intraperitoneal injection of VV-scFv-TIGIT enhanced antitumor efficacy in ascites tumor models. (A) Treatment scheme of H22 ascites model. (B) Flow cytometric analysis of the proportion of tumor cells, lymphocytes, and their subpopulations in tumor tissues. (C) Flow cytometric analysis of the expression of immune checkpoint molecules on the surface of CD8 + T cells. (D) Surface and intracellular staining of CD8 + T cells for T cell activation markers and analysis by flow cytometry. (E) The levels of cytokines in the ascites were detected by ELISA. (F) Kaplan-Meier survival curves of tumor-bearing mice. ns, no significant differences; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. PBS, phosphate-buffered saline; scFv, single-chain variable fragment; TIGIT, T-cell immunoglobulin and ITIM domain; VV, vaccinia virus.

Article Snippet: For depletion of CD8 + T or NK cells, each mouse was injected intraperitoneally with 500 μg of anti-CD8α antibody (Clone YTS 169.4, Cat# BP0117, BioXCell, USA) or anti-NK1.1 antibody (Clone PK136, Cat# BP0036, BioXCell).

Techniques: Injection, Expressing, Staining, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Saline, Virus

Journal: Journal for Immunotherapy of Cancer

Article Title: An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade

doi: 10.1136/jitc-2021-002843

Figure Lengend Snippet: CD8 + T cells mediated the antitumor immunity of VV-scFv-TIGIT. (A) Treatment scheme of H22 ascites model. (B, C) The depletion of CD8 + T cells and NK cells in the blood (B) or ascites (C) of mice was analyzed by flow cytometry. (D) Representative diagram of flow cytometric analysis of tumor cells and lymphocytes in the ascites. (E) Flow cytometric analysis of the proportion of tumor cells, lymphocytes, and their subpopulations in the ascites. (F) The levels of cytokines in the ascites were detected by ELISA. (G) Kaplan-Meier survival curves of tumor-bearing mice. ns, no significant differences; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. PBS, phosphate-buffered saline; scFv, single-chain variable fragment; TIGIT, T-cell immunoglobulin and ITIM domain; VV, vaccinia virus.

Article Snippet: For depletion of CD8 + T or NK cells, each mouse was injected intraperitoneally with 500 μg of anti-CD8α antibody (Clone YTS 169.4, Cat# BP0117, BioXCell, USA) or anti-NK1.1 antibody (Clone PK136, Cat# BP0036, BioXCell).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Saline, Virus

Journal: Journal for Immunotherapy of Cancer

Article Title: An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade

doi: 10.1136/jitc-2021-002843

Figure Lengend Snippet: Treatment of mice with VV-scFv-TIGIT established long-term tumor-specific immunological memory. (A) Rechallenge scheme. (B) Kaplan-Meier survival curves of the treatment-naïve mice or the mice cured of H22 by VV-scFv-TIGIT. (C) Tumor volumes of previously cured mice rechallenged with H22 or MC38 cells subcutaneously. (D) Rechallenge scheme and representative diagram of flow cytometric analysis of memory T cells. (E) Flow cytometric analysis of the proportion of CD8 + T cells, naïve (CD62L + CD44 − ), effector memory (CD62L - CD44 + ) and central memory (CD62L + CD44 + ) CD8 + T cells. (F) Co-culture of H22 cells with splenocytes from the H22 rechallenged mice. The proportion of lymphocytes and tumor cells in the co-culture system was detected by flow cytometry. The levels of cytokines in the co-culture system were detected by ELISA. ns, no significant differences; *p<0.05; ***p<0.001; ****p<0.0001. scFv, single-chain variable fragment; TIGIT, T-cell immunoglobulin and ITIM domain; VV, vaccinia virus.

Article Snippet: For depletion of CD8 + T or NK cells, each mouse was injected intraperitoneally with 500 μg of anti-CD8α antibody (Clone YTS 169.4, Cat# BP0117, BioXCell, USA) or anti-NK1.1 antibody (Clone PK136, Cat# BP0036, BioXCell).

Techniques: Co-Culture Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Virus

Journal: bioRxiv

Article Title: An AhR-Ovol1-Id1 regulatory axis in keratinocytes promotes skin homeostasis against atopic dermatitis

doi: 10.1101/2024.01.29.577821

Figure Lengend Snippet: (A) RT-qPCR results of Ovol1 and Il1a expression in epidermal cells of the HDM/SEB-treated mice at day 7. n = 3 mice per group. (B) Experimental design for IL-1R Ab treatment in (C-K). Samples were harvested at day 11 after treatment for downstream analysis. IgG was used as a control. (C-D) Flow cytometry analysis of dermal γδT cells in the back skin of IgG- or IL-1R Ab-treated SSKO mice. See legends for additional details. n = 4 mice per group. (E) Representative photographs of IgG- or IL-1R Ab-treated SSKO mice. (F) Clinical scores of IgG- or IL-1R Ab-treated SSKO mice. n = 5 mice per group. (G) Representative skin histology (H/E staining). Scale bar = 100 μm. (H) Quantification of epidermal, dermal and total skin thickness in IgG- or IL-1R Ab-treated SSKO mice. n = 4 mice per group. (I) TEWL values of IgG- or IL-1R Ab-treated SSKO mice. n = 5 mice per group. (J-K) Flow cytometry analysis of the indicated cell types in the back skin of IgG- or IL-1R Ab-treated SSKO mice. See legends for additional details. n = 4 mice per group. (L) RT-qPCR results of Il4 , Il17a and Il33 expression in whole skin. Data are mean + SEM. n = 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. p values were calculated using 2-tailed unpaired Student t test or Mann-Whitney U test.

Article Snippet: For IL-1R blocking, mice were i.p injected with 200 μg IL-1R antibody (BioXcell, Cat# BE0256, Clone JAMA-147) or IgG (BioXcell, Cat# BE0091) at days −1, 0, 3, 7 and 10 of HDM/SEB treatment.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: An AhR-Ovol1-Id1 regulatory axis in keratinocytes promotes skin homeostasis against atopic dermatitis

doi: 10.1101/2024.01.29.577821

Figure Lengend Snippet: Related to . (A-B) Percentages of different immune cell populations out of total skin cells (A) or skin CD45 + cells (B) in IgG- or IL-1R Ab-treated SSKO mice at day 11. n = 4 mice per group. (C) Clinical scores of skin eruption, scaling, bleeding and redness at day 11. n = 5 mice per group. (D) Quantification of dermal and total skin thickness. n = 4 mice per group. (E) Quantification of the indicated immune cell types per gram of skin tissue at day 11. n = 4 mice per group. (F) RT-qPCR analysis of the indicated genes in whole skin at day 11. n = 5 mice per group. (G) Quantification of the indicated immune cell types per gram of skin tissue in DMSO- or AGX51-treated SSKO mice at day 11. n = 4 mice per group. (H-I) Percentages of different immune cell populations out of total skin cells (H) or skin CD45 + cells (I) at day 11. n = 4-5 mice per group. (J) RT-qPCR results of the indicated genes in the whole skin at day 11. Data are mean + SEM. n = 4 mice per group. * p < 0.05, ** p < 0.01. p values were calculated using 2-tailed unpaired Student t test.

Article Snippet: For IL-1R blocking, mice were i.p injected with 200 μg IL-1R antibody (BioXcell, Cat# BE0256, Clone JAMA-147) or IgG (BioXcell, Cat# BE0091) at days −1, 0, 3, 7 and 10 of HDM/SEB treatment.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A, Western blot demonstrates high expression of SPP1 during acquired CDDP resistance in HN30-R8 HNSCC cells compared to HN30 CDDP sensitive parental cells. B, Restoration of KEAP1 or knockdown of NRF2 modulates SPP1 expression in CDDP resistant HNSCC cells. C, Western blot verifies downregulation efficiency of SPP1 in stable CDDP resistant cells (HN30-R8). D, Bar graph depicting levels of SPP1 determined by ELISA in CDDP sensitive (HN30), resistant (HN30- R8) and their SPP1 knockdown derivatives. E and F, Representative images of clonogenic survival and curves demonstrating increased sensitivity to CDDP in HN30-R8 cells stably expressing shRNA SPP1 after treatment with the indicated doses of cisplatin. G and H, Annexin V-APC/7-AAD–positive staining (percentage of dead cells) confirming induction of low levels of apoptosis in SPP1 knockdown HN30-R8 cells treated with CDDP (8 µmol/L) for 48 hr. I, Fluorescence level of intracellular oxidized C11-BODIPY (581/591) measured by Flow cytometry. J, Bar graph showing increased lipid peroxidation levels calculated from the fluorescent integrated density of the oxidized BODIPY indicates ferroptosis in HN30-R8 cells expressing SPP1 shRNA following treatment with CDDP (8 mol/L) for 48 hr compared to untreated ShCtrl control cells. K, Western blot demonstrates decreased protein level of anti- ferroptotic markers, ACSL4 and GPX4 and absence of apoptotic marker, PARP cleavage. Data shown are representative of three independent experiments. Bar graphs are mean ± SEM, unpaired student t-test and two-way ANOVA respectively. **P < 0.001, ***P< 0.0001, *P=0.027, **P=0.0027, ***P=0.0015.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Western Blot, Expressing, Knockdown, Enzyme-linked Immunosorbent Assay, Stable Transfection, shRNA, Staining, Fluorescence, Flow Cytometry, Control, Marker

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A, Western blot demonstrates high expression of SPP1 during acquired CDDP resistance in PCI13-wtp53 HNSCC cells compared to PCI13-wtp53 CDDP sensitive parental cells. B , Western blot verifies downregulation efficiency of SPP1 in stable CDDP resistant cells (PCI13-wtp53-resistant). C and D, Representative images of clonogenic survival and curves demonstrating increased sensitivity to CDDP in PCI13-wtp53 cells stably expressing shRNA SPP1 after treatment with the indicated doses of cisplatin.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Western Blot, Expressing, Stable Transfection, shRNA

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: The HN30-R8 and their shRNA SPP1 derivatives were cultured in presence and absence of CDDP (8 mol/L) for 48 hr and subjected to colony growth on soft agar and Matrigel invasion assays as described in Methods . A and B, Knock-down of SPP1 alone or with CDDP treatment decreases the colony growth on soft agar. C and D, The invasion ability of HN30-R8 cells expressing shRNA SPP1 is significantly decreased compared to the shCtrl control cells. E and F, Cell invasion is inhibited following neutralization of the SPP1 with the anti-SPP1(osteopontin) antibody and in the presence of CDDP in HN30-R8 CDDP resistant cells. Data shown are representative of three independent experiments. Error bars are mean ± SEM, unpaired student t-test and two-way ANOVA respectively. *P<0.05, *P<0.01, ***P<0.001, ****P < 0.0001. The scale bar is 100 µm. Images were taken using Leica DMLA microscope.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: shRNA, Cell Culture, Knockdown, Expressing, Control, Neutralization, Microscopy

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: HN30-R8 cells stably expressing shRNA SPP1 #4 and shCtrl constructs were orthotopically injected into the tongues of male athymic nu/nu mice and treated intravenously via tail injection with 4 mg/kg of CDDP for 4 weeks as indicated. Each treatment group contains 9-12 mice. Tumor growth was measured and reported as tumor volume means ± SEM. A, Tumor growth curves calculated after 4 weeks of injection and treatment. Statistical analyses were performed by a two-way ANOVA test. ****P < 0.0001 CDDP treated HN30-R8 shRNA SPP1 #4 tumor bearing mice versus all other treatment groups. B and C, Kaplan–Meier survival curve and percent of body weight loss of mice from A. The death of animals occurred when tumor compromised the animal welfare. D and E, Incidence of lymph node and lung tumor metastases in mice assessed microscopically by hematoxylin and eosin staining. F, Bar graph depicting the impact of SPP1 knockdown on the number of lymph node (N score) and lung tumor metastases present concurrently in mice. G, Table summarizes the number of mice with lung metastatic nodules, where No (indicates no nodule present). H, Kaplan–Meier survival curve of mice injected with HN30-R8 (shCtrl) and HN30-R8 (shRNA SPP1 #2 construct) cells through the tail vein. I, Macroscopic nodules can be seen in the representative images from the lungs of mice injected with the tumor cells via tail vein. J and K, respective microscopic images and Table depicting the number of the lung metastatic nodules, and their sizes appearing in mice following tail vein injection with the tumor cells. All in vivo data were expressed as ± SEM and Log-rank, *P< 0.05 was considered significant for mice survival. The Fisher exact test statistic value, *P<0.05 was used to detect significant difference in lymph node and lung metastasis in mice.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Stable Transfection, Expressing, shRNA, Construct, Injection, Staining, Knockdown, In Vivo

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A) Schematic procedure of the in vivo mouse xenograft experiment and treatment schedule. Mice were injected with CDDP resistant HN30-R8 cells and after tumor formed, mice were treated with CDDP, anti-OPN, and in combination as indicated. B, Tumor growth curves calculated after 4 weeks of injection and treatment with drugs. C, Kaplan–Meier survival curve demonstrating improved survival of mice following combination of anti-OPN and CDDP. D, Percent body weight loss among the treatment groups. E, SPP1 levels determined by ELISA-based assay in plasma prepared from mice blood samples collected following treatment with drugs. The plasma samples were diluted 100 times in the ELISA dilution buffer. F, Table showing microscopic evaluation of incidence and percentage of tumor metastatic nodules in mice lymph nodes and lungs sections stained with H&E as indicated. All in vivo data were expressed as ± SEM and two-way ANOVA and Log-rank tests, *P< 0.05 were considered significant for tumor growth reduction and mice survival, respectively.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: In Vivo, Injection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Staining

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A, Levels of SPP1 mRNA are significantly higher in oral cavity (OC) squamous cell carcinoma (OCSCC) and laryngeal / hypopharyngeal (LH) squamous cell carcinoma tumors compared to normal samples from the TCGA cohort, regardless of HPV association. B, SPP1 expression is elevated in tumors harboring mutations in either NRF2 (orange symbols) or KEAP1 (blue symbols) genes. C, OCSCC tumors with elevated SPP1 mRNA expression (cohort cutoff determined by recursive portioning) have significantly lower median overall survival time. D, LHSCC tumors with elevated SPP1 mRNA expression (cohort cutoff determined by recursive portioning) have significantly lower median overall survival time. E, OCSCC tumors with elevated SPP1 mRNA expression have significantly lower median disease-free survival (DFS) times. F, LHSCC tumors with elevated SPP1 mRNA expression have significantly lower median DFS times. G, OSCC patients with pathological lymph node stage >N1 have significantly higher SPP1 mRNA expression compared to node negative (N0) patients. H, OCSCC patients with more advanced T stages (T3 & T4) have significantly elevated SPP1 mRNA expression. I, Primary tumors with higher levels of SPP1 mRNA had a greater incidence of gross extracapsular lymph node extension. J, Primary OCSCC tumors associated with microscopic (micro) or gross extracapsular lymph node spread had a trend towards elevated SPP1 mRNA expression. K, Elevated expression of SPP1 mRNA was associated with less differentiated OCSCC tumors. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.001. L, Hierarchical clustering of the cross-correlation coefficients featuring modules highly co-correlated among a subset of leukocytes including eosinophils, neutrophils, monocytes, mast cells, and macrophages which all moderately correlated with SPP1 expression levels. M, Regression model revealing that NRF2 is the strongest predictor of SPP1 (****p < 0.00001), followed by macrophages. (****p < 0.00001), and CAF (****p < 0.00001).

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Expressing

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A, Volcano plot showing negative log10 adjusted P values (y-axis) verses differences in RPPA values (x-axis) measured for analytes derived from HN30R8 after infection with either control shRNA (shCtrl) or shRNA SPP1 knockdown (SPP1 KD). Positive delta values indicate analytes that decrease after SPP1 KD compared to control. Analytes in the AKT / mTOR pathway are represented with blue symbols, while analytes from the FAK pathway are represented with red symbols. A doted horizontal line at Y =1 corresponds to a minimum false discovery rate of 0.1. B, Heatmap of select analytes measured by RPPA after one-way unsupervised hierarchical clustering, showing that samples clustered by the status of SPP1 KD. Gene Ontology (GO) enrichment was performed on all analytes found significantly different after SPP1 KD and analytes from select GO pathways found to be significantly enriched were included in the cluster analysis and are annotated by GO process with black squares above the heatmap. C, Western blot analyses confirming differential expression of most of these proteins, including AKT/ mTOR and FAK among the control and SPP1 KD cell lines.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Derivative Assay, Infection, Control, shRNA, Knockdown, Western Blot, Quantitative Proteomics

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A, Hematoxylin and eosin staining and UMAP plots showing the histological annotation of the H&E -stained Visium slides and cell types in patient primary and metastatic lung tumors. B, Near super-pixel resolution analysis using iSTAR and visualization of differential expression of SPP1, NFE2L2 (NRF2), KEAP1 and KEAP1 in primary and metastatic lung tumors. C, Near super-pixel resolution analysis using iSTAR and visualization of the differential expression of SPP1 receptors in primary and metastatic lung tumors. D, COMMOT clustering analysis and similar communication intensity signals demonstrating possible interaction between SPP1 and its receptors, CD44 and integrins in primary and metastatic lung tumors in HNSCC patient. E, Western blot analysis showing positive interaction of SPP1 with CD44 and several integrin receptors in vitro in CDDP-resistant HN30-R8 cell lines.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Staining, Quantitative Proteomics, Western Blot, In Vitro

Journal: bioRxiv

Article Title: Targeted suppression of SPP1 inhibits tumor invasion and metastasis in NRF2 hyperactivated cisplatin resistant HNSCC

doi: 10.1101/2025.05.28.656679

Figure Lengend Snippet: A, All spots filtered by pathology annotation from the Visium slides analyzed by Seurat showing 14 cell clusters (0-13) in primary and metastatic lung tumors. B, Box plot showing scaled expression levels of SPP1 in each cell cluster (*p=0.001687, t-test). C, Volcano plot depicting the log fold change (logFC>1.5 and FDR <0.01) of the differentially expressed genes in primary and metastatic lung tumors. D, Heatmap showing HALLMARK enrichment analyses for 50 MSigDB ALLMARK gene sets profile obtained from the differentially expressed gene list of each cluster using the AUCell package. Red horizontal bar represents primary tumor with clusters (0, 1, 2, 7, 9, and 10) and blue horizontal bar represents metastatic lung tumor with clusters (3 and 6). E, Bar graph showing top five enriched gene signatures selected from the 50 MSigDB HALLMARK gene sets in primary and metastatic lung tumors. F, Proposed signaling model summarizing the oncogenic role of SPP1 in HNSCC. We hypothesized that under NRF2-stressed conditions, hyperactivated SPP1 interacts with specific class of α/β integrins and CD44 in the tumors and activate downstream signaling effectors including AKT/mTOR, MAPK and FAK proteins. Consequently, this interaction between SPP1 and its receptors in cisplatin-resistant HNSCC enhances oncogenic cellular responses and inhibits ferroptosis, ultimately contributing to therapy resistance, tumor progression, and metastasis.

Article Snippet: For the in vivo drug combination experiment, the HN30-R8 CDDP-resistant cells were injected into the mice oral tongues as described above, and treated with either CDDP alone, the SPP1 inhibitor (osteopontin monoclonal antibody, BioXCell #Cat #BE0382, 10 mg/kg, 3 times a week, given intraperitoneally, I.P.) alone, and their combination or PBS vehicle (given I.P.) for 4 weeks.

Techniques: Expressing

Journal: bioRxiv

Article Title: Induction of immortal-like and functional CAR-T cells by defined factors

doi: 10.1101/2023.11.06.565785

Figure Lengend Snippet: a, The composition of anti-mCD19 CAR. b , Vector design of pMSCV- sgRNA-CAR19. Thy1.1 is co-expressed with CAR at protein level via P2A, serving as a marker of CAR expression. c , Experimental design. CD8 + Cas9 + T cells were activated by anti-CD3/anti-CD28 (1 μg/ml), infected with retrovirus expressing CAR19 with sgRNA targeting indicated genes. One million of CD8 + Thy1.1 + CAR19T cells were transferred into B6 mice. CAR19T cells and B cells from peripheral blood were monitored by flow cytometry. d-f , Representative plots ( d ) and statistical analysis ( e,f ) of Thy1.1 + CAR19T cells and CD19 + B cells among single live cells from peripheral blood 7 days and 28 days post-transfer are shown (n = 4 mice for each group). g , Experimental design for genome-wide dual sgRNA screening for gene whose loss of function confers persistence to ZC3H12A-deficient CAR19T cells. Indicated sgRNA library was used to infect activated CD8 + T cells, and 150 million CAR19T cells were transferred into 50 B6 mice (3 million per mouse). Seven days and 3 months later, Thy1.1 + CAR19T cells were sorted from spleen for sgRNA analysis. h , Screening result. i , Experimental design for validation of the screening hit BCOR. j,k , Representative plots ( j ) and statistical analysis ( k ) of Thy1.1 + CAR19T cells and CD19 + B cells among single live cells from peripheral blood of mice transferred with CAR19TIF cells expressing indicated sgRNAs are shown (n = 3 mice in PBS group, n = 6 mice in sgNT group, n = 5 mice in sg Zc3h12a group, n = 5 mice in sg Bcor / Zc3h12a group). l , Percentages of CD19 + B cells from spleen of mice 6 months after transferring CAR19TIF cells expressing indicated sgRNAs (n = 4 mice in sgNT group, n = 5 mice in sg Bcor / Zc3h12a group). e, f, k and l , data represent mean ± SEM from one of three independent experiments. * P < 0.05, **** P < 0.0001, ns, not significant, one-way ANOVA multiple-comparisons test in ( e,f ), two-way ANOVA multiple-comparisons test in ( k ), two-tailed unpaired Student’s t test in ( l ).

Article Snippet: CD8 + T cells were purified by a negative selection Kit (BioLegend, Cat# 480035), and purified CD8 + T cells were activated by 1 μg/ml anti-CD3 (BioXCell, Cat# BP0001-1, RRID:AB 1107634) and 1 μg/ml anti-CD28 (BioXCell, Cat# BP0015-1, RRID:AB 1107624) overnight.

Techniques: Plasmid Preparation, Marker, Expressing, Infection, Flow Cytometry, Genome Wide, Transferring, Two Tailed Test