Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: Murine and Human CXCL9 Transgenes Engineered in Recombinant VSVs Do Not Alter the Growth Kinetics or Viral Killing Ability In Vitro Schematic depiction of the genomes of recombinant VSVs encoding murine (A) or human (B) CXCL9, CXCLi, and GFP. (C) Replication kinetics of VSVs encoding murine or human CXCL9 were performed in a multistep viral growth curve in Vero cells. Data are shown from duplicate experiments as average titer + standard deviation. (D) Viability of VSV-mCXCL9-, VSV-mCXCLi-, and VSV-GFP-infected Vero and murine LM2 tumor cells was assessed at 24 and 48 h postinfection at an MOI of 10. (E) Viability of VSV-M51R-hCXCL9- and VSV-M51R-hCXCLi-infected Vero and human FaDu-Luc tumor cells was measured at 24 and 72 h postinfection at an MOI of 10. Data are presented as duplicate experiments as average percent viability compared with mock-infected cells + standard deviation. Significance was determined by paired t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Recombinant, In Vitro, Standard Deviation, Infection

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: CXCL9 Expressed from VSV-mCXCL9 Is Biologically Active (A) Murine CXCL9 secretion was evaluated in vitro in the LM2 non-small cell lung cancer cell line. Supernatants of VSV-infected LM2 cells (MOI 0.1) were collected 24 h postinfection, and chemokine concentration was determined by ELISA. Concentrations are presented as average concentration + standard deviation. (B) Chemotactic activity of virally encoded mCXCL9 was assessed in an in vitro migration assay adapted from Campanella et al. Numbers of migrated cells are presented as average percent increase in migration compared with mock treated + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: In Vitro, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Activity Assay, Migration, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: Generation of a Tumor to Blood CXCL9 Chemokine Gradient after Intratumoral VSV-mCXCL9 Administration Intratumoral and serum mCXCL9 protein concentrations were determined after intratumoral injection of VSV-mCXCL9 or VSV-GFP in LM2 tumor-bearing mice by ELISA (n = 3 mice/group for each time point). Values are presented as average chemokine concentration in pg/mL + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: VSV-M51R-hCXCL9 Expresses hCXCL9 Chemokine In Vitro and In Vivo (A) Human CXCL9 expression from VSV-M51R-hCXCL9 was tested in vitro in FaDu-Luc cells. Supernatants of FaDu-Luc cells were collected 48 h postinfection with VSV-M51R-hCXCL9, VSV-M51R-hCXCLi, (MOI 0.01) or after mock infection, and were assayed by ELISA (first panel). Data are presented as average concentration + standard deviation. Intratumoral and plasma hCXCL9 concentrations at various time points following virus injection were determined in FaDu-Luc tumor-bearing mice by ELISA (n = 3 mice/group for each time point). Values are presented as average chemokine concentration in pg/mL + standard deviation (second and third panels). Significance was determined by t test (*p < 0.05, **p < 0.01). (B) Human T cells were activated, and CXCR3 expression was tested by flow cytometry. Fluorescence minus one-stained cells are shown in red, and CXCR3-stained cells are shown in blue. FaDu-Luc tumors were harvested and processed 2 and 4 days after intratumoral injection of VSV-M51R-hCXCL9, VSV-M51R-hCXCLi, or PBS treatment (corresponding, respectively, to 1 and 3 days post adoptive T cell transfer). Flow data were gated on live hCD45 + or hCD3 + cells. T cell numbers are presented as average number of immune cells ± standard deviation (n = 3 mice/group for 48 h; n = 4 for PBS treated, n = 3 for VSV-M51R-hCXCL9 treated, and n = 2 for VSV-M51R-hCXCLi at 96 h).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: In Vitro, In Vivo, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Injection, Flow Cytometry, Fluorescence, Staining

Journal: Journal for immunotherapy of cancer

Article Title: Endosialin-positive CAFs promote hepatocellular carcinoma progression by suppressing CD8 + T cell infiltration.

doi: 10.1136/jitc-2024-009111

Figure Lengend Snippet: Figure 4 Knockdown of endosialin promotes the expression and secretion of CXCL9/10 in cancer-associated fibroblasts (CAFs) by activating INF-γ-STAT1 pathway. (A) Chemokine array to show the levels of different chemokines in IFN-γ-stimulated endosialin knockdown or control HFL1 cells. (B) RT-qPCR to show the mRNA level of CXCL9/10 in endosialin knockdown or control HFL1 cells. (C) ELISA to show the protein level of CXCL9/10 in the culture medium of endosialin knockdown or control HFL1 cells. (D) RT-qPCR to show the mRNA level of CXCL9/10 in endosialin overexpressing or control LX-2 cells. (E) ELISA to show the protein level of CXCL9/10 in the culture medium of endosialin overexpressing or control LX-2 cells. (F) ELISA to show the level of CXCL9/10 in subcutaneous Hepa1-6 tumor tissues of WT and ENKO mice (n=3). (G) Fluorescent imaging to show the migrated 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled T cells after co-culture with endosialin knockdown or control HFL1 cells in the presence of CXCL10 neutralizing antibody or control antibody (left) and quantification of the migrated T cells (right). Scale bar, 100 µm. (H) Western blot to detect the level of pSTAT1 in endosialin knockdown or control HFL1 cells. (I) IF staining to show the localization of STAT1 in endosialin knockdown or control HFL1 cells. Scale bar, 20 µm. (J) Western blot to detect the level of pSTAT1 in endosialin overexpressing or control LX-2 cells. (K) IF staining to show the localization of STAT1 in endosialin overexpressing or control LX-2 cells. Scale bar, 20 µm. Representative images are shown. Data are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: To examine CXCL9/10 level in the tumor tissues of ENKO or WT mice, tumor tissues were isolated and prepared into a tissue homogenate by tissue fragmentation, and then examined using mouse CXCL9 (CUSABIO, #CSB- EL006252MO) and CXCL10 (CUSABIO, #CSBE08183m) ELISA kits according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Imaging, Labeling, Co-Culture Assay, Western Blot, Staining

Journal: Journal for immunotherapy of cancer

Article Title: Endosialin-positive CAFs promote hepatocellular carcinoma progression by suppressing CD8 + T cell infiltration.

doi: 10.1136/jitc-2024-009111

Figure Lengend Snippet: Figure 5 High endosialin expression is correlated with low CD8+ T infiltration in clinical hepatocellular carcinoma (HCC) tissues. (A) Immunohistochemistry (IHC) staining of endosialin in HCC tissues and adjacent normal tissues (n=5). Scale bar, 100 µm (top panel), 20 µm (bottom panel). (B) Dual immunofluorescent (IF) staining of HCC tissues to show the correlation between endosialin expression and CD8+ T cell infiltration (n=4). Scale bar, 50 µm. (C) RT-qPCR to show the mRNA level of CXCL9/10 in endosialin knockdown or control primary cancer-associated fibroblasts (CAFs. (D) ELISA to show the protein level of CXCL9/10 in the culture medium of endosialin knockdown or control primary CAFs. (E) Fluorescent imaging to show the migrated 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled T cells after co-culture with endosialin knockdown or control primary CAFs at the presence of CXCL10 neutralizing antibody or control antibody (left) and quantification of the migrated T cells (right). Scale bar, 100 µm. (F) Western blot to detect the level of pSTAT1 in endosialin knockdown or control primary CAFs. (G) IF staining to show the localization of STAT1 in endosialin knockdown or control primary CAFs. Scale bar, 20 µm. Representative images are shown. Data are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: To examine CXCL9/10 level in the tumor tissues of ENKO or WT mice, tumor tissues were isolated and prepared into a tissue homogenate by tissue fragmentation, and then examined using mouse CXCL9 (CUSABIO, #CSB- EL006252MO) and CXCL10 (CUSABIO, #CSBE08183m) ELISA kits according to the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry, Staining, Quantitative RT-PCR, Knockdown, Control, Enzyme-linked Immunosorbent Assay, Imaging, Labeling, Co-Culture Assay, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Endosialin-positive CAFs promote hepatocellular carcinoma progression by suppressing CD8 + T cell infiltration.

doi: 10.1136/jitc-2024-009111

Figure Lengend Snippet: Figure 6 Combination therapy with endosialin antibody and PD-1 antibody shows synergistic antitumor effect. (A) Schematic image to show the process of combination therapy. (B) Picture of isolated tumors (left) and tumor weight (right) after mice were sacrificed after treatment (n=8). (C) Growth curve of the tumors in different groups after treatment. (D) Flow cytometry to show the percentage of CD4+ T cells and CD8+ T cells in the tumor tissues of different groups after treatment (left panel) and quantification of the flow cytometry data (right panel) (n=6). (E) Graphic diagram to describe the function of endosialin, which can inhibit the secretion of CXCL9/10 by inhibiting IFN-γ/JAK/STAT1 pathway in CAFs, thus inhibit the infiltration of CD8+ T cells into tumor tissues; and endosialin antibody can enhance the anti-tumor effect of PD1 antibody by increasing the infiltration of CD8+ T cells. Representative images are shown. Data are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. CAFs, cancer-associated fibroblasts.

Article Snippet: To examine CXCL9/10 level in the tumor tissues of ENKO or WT mice, tumor tissues were isolated and prepared into a tissue homogenate by tissue fragmentation, and then examined using mouse CXCL9 (CUSABIO, #CSB- EL006252MO) and CXCL10 (CUSABIO, #CSBE08183m) ELISA kits according to the manufacturer’s instructions.

Techniques: Isolation, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Opening of the blood-brain barrier using low-intensity pulsed ultrasound enhances responses to immunotherapy in preclinical glioma models

doi: 10.1158/1078-0432.CCR-20-3760

Figure Lengend Snippet: A. Schema of the lentivirus gene transfer plasmid structure for murine CXCL9 or CXCL10 B. and gene transfer plasmid of control methionine-deficient green fluorescent protein alone. C. Flow cytometry analysis of the percentage of CD3+ T cells within the total CD45+ immune cell infiltration within intracerebral gliomas in the different treatment groups (n=5-7/group). Statistics: PBS versus CXCL10 with ultrasound, p=0.0167; CXCL10 versus CXCL10 with ultrasound, p=0.0412; PBS with ultrasound versus CXCL10 with ultrasound, p=0.0004. D. Flow cytometry analysis of CD4+ T cells within the total CD45+ immune cell infiltration within intracerebral gliomas. Statistics: PBS versus CXCL10 with ultrasound, p=0.0098; CXCL10 versus CXCL10 with ultrasound, p=0.0371; PBS with ultrasound versus CXCL10 with ultrasound, p=0.0003. E. Flow cytometry analysis of CD8+ T cells within the total CD45+ immune cell infiltration within intracerebral gliomas. Statistics: PBS with ultrasound versus CXCL10 with ultrasound, p=0.0023; PBS versus CXCL10 with ultrasound, p=0.153; CXCL10 versus CXCL10 with ultrasound, p=0.2154, as assessed by the two-sided unpaired t test. F. Treatment schema for C57BL/6 mice bearing GL261 tumors treated with CXCL9 or CXCL10 APCs. G. Kaplan-Meier survival analysis of mice treated with PBS, i.c. CXCL10 APCs, i.v. CXCL10 APCs, or i.v. CXCL10 APCs with ultrasound. PBS with sonication control for the GL261 model is demonstrated in Supplement 1A. The MS durations in the treatment groups were as follows: PBS (3 mice), 24 days; i.c. CXCL10 APCs (4 mice), 28 days; i.v. CXCL10 APCs (4 mice), 24 days; i.v. CXCL10 APCs with ultrasound (3 mice), 34 days. Statistics: PBS versus i.c. CXCL10 APCs, p=0.0476; PBS versus i.v. CXCL10 APCs, p=0.6041; PBS versus i.v. CXCL10 APCs with ultrasound, p=0.0246; i.c. CXCL10 APCs versus i.v. CXCL10 APCs, p=0.6349; i.c. CXCL10 APCs versus i.v. CXCL10 APCs with ultrasound, p=0.0213; i.v. CXCL10 APCs versus i.v. CXCL10 APCs with ultrasound, p=0.0415.

Article Snippet: CXCL9 and CXCL10 gene transduction into F4/80+CD11c+ antigen presenting cells To transduce the CXCL9 and CXCL10 immune chemokine genes into the APCs, we prepared lentiviruses that encoded the cDNA of murine CXCL9 or CXCL10 gene transfer plasmids (cat. # MR200667L3 and cat. # MR200291L4, respectively; Origene, Rockville, MD).

Techniques: Plasmid Preparation, Flow Cytometry, Sonication

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Opening of the blood-brain barrier using low-intensity pulsed ultrasound enhances responses to immunotherapy in preclinical glioma models

doi: 10.1158/1078-0432.CCR-20-3760

Figure Lengend Snippet: A. Anti-PD-1 delivered through an open BBB blocks immune exhaustion of effector T cells, thereby enabling T cells to exert an effector response through perforin and/or granzyme B. B. Compared with CAR T cells administered alone, ultrasound-administered CAR T cells can infiltrate the tumor microenvironment more diffusely and with longer persistence, thereby triggering tumor cytotoxicity through the CARs. C. Ultrasound BBB opening allows CXCL10 APCs and T cells to infiltrate the tumor microenvironment. Secondary to the APC presenting antigens to the T cell, the T cell becomes activated and thus can mediate direct tumor killing because the T cells have not been chronically stimulated.

Article Snippet: CXCL9 and CXCL10 gene transduction into F4/80+CD11c+ antigen presenting cells To transduce the CXCL9 and CXCL10 immune chemokine genes into the APCs, we prepared lentiviruses that encoded the cDNA of murine CXCL9 or CXCL10 gene transfer plasmids (cat. # MR200667L3 and cat. # MR200291L4, respectively; Origene, Rockville, MD).

Techniques:

Journal: Frontiers in Oncology

Article Title: Antitumor Effect and Immune Response of Nanosecond Pulsed Electric Fields in Pancreatic Cancer

doi: 10.3389/fonc.2020.621092

Figure Lengend Snippet: The concentrations of immune cytokines and chemokines before and after nsPEFs treatment. (A) Expression levels of TNF-α in the blood. (B) Expression levels of IL-1β in the blood. (C) Expression levels of IL-6 in the blood. (D) Expression levels of CCL2 in the tumor. (E) Expression levels of CXCL9 in the tumor. Data are presented as the mean ± SD. P-values were calculated based on a Student’s t-test (n>3 independent experiments). * p < 0.05, *** p < 0.001.

Article Snippet: The level of CCL2, CXCL9 in the tumor samples were analyzed using CCL2 ELISA kit (EK287/2-48, MULTI SCIENCES), CXCL9 ELISA kit (EK2143/2-96, MULTI SCIENCES) according to manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: Decomposition of the inflammatory score was conducted by estimating the most variable jacobians (first order partial derivative of inflammatory age) ( A ). Both positive and negative contributors to inflammatory age are observed. The top 15 most variable jacobians are CXCL9, EOTAXIN, Mip-1α, LEPTIN, IL-1β, IL-5, IFN-α and IL-4 (positive contributors) and TRAIL, IFN-β, CXCL1, IL-2, TGF-α, PAI-1 and LIF (negative contributors) (A). In a validation study, 97 healthy adults (age 25-90) well matched for cardiovascular risk factors, were selected form a total of 151 recruited subjects. Immune protein analysis was conducted in samples from these subjects. CXCL9, HGF, CXCL1 and LIF were found to change in the same direction in both the Stanford 1KIP and the validation cohort ( A and B ). Cardiovascular age was estimated using 3 parameters: (1) aortic pulse wave velocity, a measure of vascular stiffness; (2) relative wall thickness (RWT), a measure of ventricular remodeling, and (3) early diastolic mitral annular velocities (e’), a measure of ventricular relaxation. After adjusting for age, sex, BMI, heart rate, systolic blood pressure, fasting glucose and total cholesterol to HDL ratio, positive correlations were obtained between CXCL9 and PWV ( R = 0.22) and RWT ( R = 0.3) ( P < 0.05), and negative correlations were observed between LIF and PWV ( R = −0.27), and RWT ( R = −0.22) ( C and D ). No variable included in the models had high co-linearity as suggested by variance inflation factors (VIF) < 3 for each factor. Induced pluripotent stem cells (hiPSCs) were obtained from isolated fibroblasts ( N = 5, in duplicates) using the Yamanaka factors and differentiated them into endothelial cells (hiPSC-ECs) under well-defined conditions as previously described . Expression levels of CXCL9 and SIRT3 were measured by RT-PCR as described under Methods. A significant age-dependent increase in CXCL9 mRNA expression levels is observed ( P < 0.01), which reaches a plateau after the sixth cell passage ( E ). Concomitant with the increase in CXCL9, down-regulation in SIRT3 mRNA can be observed after the second cell passage ( P < 0.01) (E). Addition of increasing doses (10 to 800 ng/ml) of exogenous CXCL9 to young (day 7) hiPSC-ECs induces down-regulation of SIRT3 mRNA expression (F). Expression of the CXCL9 receptor, CXCR3, was measured in young cardiomyocytes derived from hiPSCs (hiPSC-CM) as well as in hiPSC-ECs, HUVEC cells, freshly isolated fibroblasts and hiPSCs. Elevated expression is observed in hiPSC-ECs, HUVEC cells but not in other cell types ( F ) suggesting that the endothelium but no other cell subsets is target of CXCL9 and potentially other CXCR3 ligands as well.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: Biomarker Discovery, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: To validate the association between CXCL9 expression and SIRT3 in hiPSC-ECs, CXCL9 was knockdown (KD) in hiPSCs using shRNA. Quantitative PCR data show significant knockdown of CXCL9 in hiPSCs following transfection with CXCL9-shRNA compared to scramble controls ( A ). hiPSC-ECs failed to show an increase in CXCL9 expression in CXCL9-KD cells as they were passaged when compared to scramble controls ( B ). On the contrary, CXCL9-KD hiPSC-ECs show reversal of their SIRT3 levels in serially passaged hiPSC-ECs when compared to scramble controls ( C ). Quantification of the number of capillary-like networks formed by scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubular networks even at later passages when compared to scramble controls that showed impaired tube formation at later passages of hiPSC-ECs ( D ). Line graph of percent relaxation of mouse thoracic aortic sections to Acetylcholine show impaired vascular reactivity to increasing concentrations of CXCL9, suggesting CXCL9 impairs vascular function ( E ). Significance of impaired vascular reactivity was determined by 2-way ANOVA, followed by a Bonferroni post-test, N=3, n=3-4, *p<0.05.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: Expressing, Knockdown, shRNA, Real-time Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: Representative images of capillary-like networks from scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubes even at later passages when compared to scramble that showed impaired tube formation towards later passages of hiPSC ECs.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: