Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response

doi: 10.1073/pnas.2013644117

Figure Lengend Snippet: The effect of CXCL12-stimulated CXCR4 on chemokine receptor-mediated migration of human immune cells. ( Left ) The coexpression of CXCR4 with ( A ) CXCR1, ( B ) CXCR3, ( C ) CXCR5, ( D ) CXCR6, and ( E ) CCR2 on human immune cell lines was evaluated by flow cytometry after staining with antibodies specific for the relevant chemokine receptors. Gray peaks indicate isotype controls. ( Center ) The effect of CXCL12-stimulation of CXCR4 on the chemotactic responses of A CXCR1-coexpressing Jurkat T lymphoblastoid cells to CXCL8, ( B ) CXCR3-coexpressing HSB2DP T lymphoblastoid cells to CXCL10, ( C ) CXCR5-coexpressing Raji B lymphoblastoid cells to CXCL13, ( D ) CXCR6-coexpressing Jurkat T lymphoblastoid cells to CXCL16, and ( E ) CCR2-coexpressing Molm13 monocytoid cells to CCL2 was assessed by including CXCL12 in the upper chamber (blue) and the other chemokines in the lower chamber (red) in the Boyden two-chamber assay. ( Right ) The chemotaxis assays were performed with the five cell lines when the placement of the chemokines in the Boyden chambers was reversed. Bar diagrams display mean and SEM (n = 3-4). Statistical analysis by Student’s t test: *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The concentration of each chemokine used is as following: CXCL8 (R&D Systems, 208-IL), 20 ng/mL; CXCL10 (R&D Systems, 266-IP), 1,000 ng/mL; CXCL13 (R&D Systems, 801-CX), 1,000 ng/mL; CXCL16 (R&D Systems, 976-CX), 50 ng/mL; CCL2 (R&D Systems, 279-MC), 200 ng/mL

Techniques: Migration, Flow Cytometry, Staining, Boyden Chamber Assay, Chemotaxis Assay

Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: Restricted normal T cell expression of CXCR5 and upregulation of CXCL13 in non-small cell lung cancer (NSCLC) (A) The expression of CXCL13 in patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) using the online tool of GEPIA. (B) CXCL13 protein expressions in NSCLC tissues were confirmed by immunohistochemistry on two tissue microarray slides (NSC157 and LC20813b). The intensity of immunostaining was graded as follows: −, negative; +, weak; ++, moderate; or +++, strong. (C) Expression of CXCL13 by immunohistochemistry. The subpanels show negative expression of CXCL13 (−), weak (+), moderate (++), and strong (+++) expressions of CXCL13 in tumor tissues ( ×400). (D) ELISA quantification of the level of CXCL13 protein in plasma samples (healthy donors n = 34, NSCLC patient donors n = 95). Single dot represents individual plasma sample. Error bars represent mean ± SD. ∗∗∗p < 0.001. (E) FACS analysis of the expression of different chemokine receptors from resting and activated T cells. Single dot represents individual sample. Error bars represent mean ± SD for each T cell population (n = 12).

Article Snippet: The CXCL13 level in A549 and A549-CXCL13 cells was quantified by a CXCL13 ELISA kit (cat. no. EA800062; OriGene).

Techniques: Expressing, Immunohistochemistry, Microarray, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: Evaluation of the antitumor efficacy and chemotaxis migration of EGFR-CXCR5-CAR-T cells in vitro (A) Analysis of the cytotoxicity of EGFR-CXCR5-CAR-T cells against PC9, A549, and K562 cells. Tumor cell killing was measured via an IncuCyte assay over 48 h. SYTOX Green and CellTrace Far Red double-positive tumor cells (yellow) were calculated. Error bars represent mean ± SD for each time point. (B) Real-time cell killing image. Target cells were red, and CAR-T cells were green. (C) Cytokine production by CAR-T cells co-cultured with PC9 tumor cells. CAR-T cells were co-cultured 10:1 with tumor cells in 96-well plates for 20 h. Levels of IFN-γ and IL-2 in supernatant were determined by ELISA. Error bars represent mean ± SD for each group. (D) Chemotaxis migration of CAR-Ts toward various concentrations of recombinant human CXCL13 at different time courses of 4 h, 8 h, and 16 h. Error bars represent mean ± SD for each group (n = 3). ∗p < 0.05 derived via unpaired t test. (E) CAR-T cell proliferation assay with indicated CAR-T cells cocultured with various concentrations of recombinant human CXCL13.

Article Snippet: The CXCL13 level in A549 and A549-CXCL13 cells was quantified by a CXCL13 ELISA kit (cat. no. EA800062; OriGene).

Techniques: Chemotaxis Assay, Migration, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Derivative Assay, Proliferation Assay

Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: In vivo tracking of the migration of 89 Zr-oxine-labeled CAR-T to A549 and A549-CXCL13 tumors using micro-PET/CT scan (A) EGFR expression in the A549 cell line stably expressing the CXCL13 gene (A549-CXCL13) after lentiviral transduction and selection. (B) Increased secretion of CXCL13 generated by A549-CXCL13 cells. ∗∗∗p < 0.001. (C) The effects of 89 Zr-oxine labeling on T cell proliferation. (D) Whole-body PET imaging, quantitative PET analysis, and biodistribution of 89 Zr-labeled T cells in tumor-bearing mice. 89 Zr-labeled mock T cells, 89 Zr-EGFR-CAR-T cells, or 89 Zr-EGFR-CXCR5-CAR-T cells were tail-vein injected into NSG mice inoculated with A549 tumor cells at the left and A549-CXCL13 tumor cells at the right side. Isotopic distribution of 89 Zr was quantified and plotted in a coronal horizon map at different time points of 2, 24, 72, and 168 h post-injection. The red and green circles represent the A549 tumor region and A549-CXCL13 tumor region, respectively. (E) Accumulated isotope signaling in A549 tumor region (green circle) and A549-CXCL13 tumor region (red circle). The percentage injection dose rate ([%ID]/g value) was calculated. Error bars represent mean ± SD for each group (n = 3).

Article Snippet: The CXCL13 level in A549 and A549-CXCL13 cells was quantified by a CXCL13 ELISA kit (cat. no. EA800062; OriGene).

Techniques: In Vivo, Migration, Labeling, Micro-PET, Computed Tomography, Expressing, Stable Transfection, Transduction, Selection, Generated, Imaging, Injection

Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: Antitumor efficacy of CAR-T cells in vivo (A) Serial bioluminescence imaging of NSG mice injected subcutaneously with A549 luc cells on the left flank and A549 luc -CXCL13 cells on the right flank. 10 days after tumor engraftment, the mice were injected with 5 × 10 5 CAR + T cells as indicated. n = 5 mice per group. Error bars represent mean ± SD for each time point (n = 5). (B) The tumor volume of the left tumors (A549 luc ) and right tumors (A549 luc -CXCL13) over 28 days was measured. Error bars represent mean ± SD for each time point (n = 5). (C) The copy number of CAR gene in the left and right tumor tissues was analyzed. ∗∗p < 0.01.

Article Snippet: The CXCL13 level in A549 and A549-CXCL13 cells was quantified by a CXCL13 ELISA kit (cat. no. EA800062; OriGene).

Techniques: In Vivo, Imaging, Injection

Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: Addition of CXCR5 facilitates T cell migration The chemokine CXCL13 is highly expressed in various tumors including lung carcinoma, whereas the classical CAR-T does not effectively infiltrate into tumor regions due to the absence of CXCR5 receptor expression. Chemotactic movement is a taxis in response to a chemical concentration gradient. When CAR-T cells are modified with the CXCR5 receptor, the motorized CAR-T cells could infiltrate into the tumor site along the gradient of CXCL13 to further clear the tumor cells.

Article Snippet: The CXCL13 level in A549 and A549-CXCL13 cells was quantified by a CXCL13 ELISA kit (cat. no. EA800062; OriGene).

Techniques: Migration, Expressing, Concentration Assay, Modification