Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response

doi: 10.1136/jitc-2022-004712

Figure Lengend Snippet: Targeting CD47 decreases melanoma tumor burden in vivo. (A) Schematic of the in vivo syngeneic mouse melanoma treatment regimen. C57Bl/6 mice were injected subcutaneously with B16 melanoma cells into the hind flank. Once tumors reached 100 mm 3 , intraperitoneal injections of 10 uM CD47 morpholino (CD47(-)) or saline began once a week for 3 weeks. (B) Tumor volume was determined throughout the study (LW /2). (C) Mice were euthanized at the end of the study (day 21) or when tumor volume reached 1500 mm 3 where tumor weight was determined (*p < 0.05, n=6–8/group). (D) Representative PAI imaging for tumor sO along with Power Doppler in 3D-Mode to determine tumor vascularity. (E) Tumor sO was quantified with the Vevo 2100 LAZR software tools while (F) tumor vascularity was quantified with Vevo CQ software of WT and CD47 targeted tumors (*p<0.05, n=4–5/group). (G) Representative immunohistochemistry images of tumor sections for (H) carbonic anhydrase, (I) intratumoral cytotoxic (yellow cells due to colocalization of CD3+ (red, APC) and CD8+ (green, FITC) T cells and (J) granzyme B (*p<0.05, n=4–5/group). Images were obtained at 20x magnification with the Olympus BX43 microscope and quantified using the PerkinElmer Mantra and inform analysis. (K) Gene expression of perforin-1 was determined through RT-qPCR of tumors (*p<0.05, n=4–5/group). PAI, photoacoustic imaging; WT, wild type.

Article Snippet: Subcutaneous injections of 5×10 5 mouse B16 or YUMM1.7 melanoma cells occurred into the outer hind limb of 6–8 weeks old C57Bl/6 mice (Jackson Laboratory).

Techniques: In Vivo, Injection, Saline, Imaging, Software, Immunohistochemistry, Microscopy, Gene Expression, Quantitative RT-PCR

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response

doi: 10.1136/jitc-2022-004712

Figure Lengend Snippet: CD47 expression regulates cytotoxic T cell activation, proliferation and antitumor function. (A, B) Pmel-1 CD8+ T cells were stained with CFSE stain to determine if CD47 expression and TSP1 exposure impacted their activation and proliferation through flow cytometry. The BD LSRFortessa X-20 Analyzer was used to acquire data, while FCS Express was used for analysis (*p<0.05, n=3). To determine if CD47 expression and TSP1 exposure impact CD8+ T cell antitumor function, effector (E) Pmel-1 CD8+ T cells were cocultured with target (T) B16 melanoma cells after stimulation with IL-2 and gp100 peptide. Target cell viability was dynamically monitored by surface impedance and presented as (C, D) normalized cell index and (E, F) percent of cytolysis at 18 hours after adding effector Pmel-1 CD8+ T cells (*p<0.05, n=3). CFSE, carboxyfluorescein succinimidyl ester; TSP1, thrombospondin-1.

Article Snippet: Subcutaneous injections of 5×10 5 mouse B16 or YUMM1.7 melanoma cells occurred into the outer hind limb of 6–8 weeks old C57Bl/6 mice (Jackson Laboratory).

Techniques: Expressing, Activation Assay, Staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response

doi: 10.1136/jitc-2022-004712

Figure Lengend Snippet: Targeting CD47 in combination with anti-PD-1 therapy decreases melanoma tumor burden in vivo. (A) Schematic of the melanoma patient cohort therapy regimen. (B, C) scRNA-seq was performed through a 10x genomics system on patient PBMC pre-anti-PD-1 and post-anti-PD-1 therapy (*p<0.05, n=16 (4/group)). (D) Circulating TSP1 was determined through ELISA of human patient plasma pre and post-anti-PD-1 therapy (*p<0.05, n=11–16/group). (E) Patient PBMC stained with antibodies for human CD3, CD8, and CD47 to determine CD47 expression on T cells (F) pre-anti-PD-1 and (G) post-anti-PD-1 therapy through flow cytometry. Data were acquired using BD LSRFortessa X-20 Analyzer and analyzed using FCS Express (*p<0.05, n=24). (H) Schematic of in vivo syngeneic mouse melanoma combination treatment regimen. C57Bl/6 mice were injected subcutaneously with B16 melanoma cells into the outer hind limb. Mice received alternating day intraperitoneal treatments of 10 µM CD47(-) and/or 200 µg anti-PD-1 or their controls over 6 days. (I, J) Tumors were measured three times a week to determine tumor volume (LW 2 /2). (*p<0.05, n=5). PBMC, peripheral blood mononuclear cells.

Article Snippet: Subcutaneous injections of 5×10 5 mouse B16 or YUMM1.7 melanoma cells occurred into the outer hind limb of 6–8 weeks old C57Bl/6 mice (Jackson Laboratory).

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