rat igg 2a isotype control 2a3  (Bio X Cell)

Journal: Journal for Immunotherapy of Cancer

Article Title: Label-free metabolic imaging for sensitive and robust monitoring of anti-CD47 immunotherapy response in triple-negative breast cancer

doi: 10.1136/jitc-2022-005199

Figure Lengend Snippet: Imaging of the mechanisms of the CD47-SIRPα pathway in vivo unravels antibody-dependent cellular phagocytosis (ADCP). (A) Schematic diagram of intravital imaging procedure for imaging the mechanisms of the CD47-SIRPα pathway in vivo using triple-negative breast cancer models. (B) Representative images for illustration of the CD47-SIRPα pathway. After injection of anti-CD47-AF488 and F4/80-PE, anti-CD47-AF488 mAb (green) rapidly targeted NAD(P)H hi cancer cells (blue) indicated by yellow arrows. Engulfment of NAD(P)H hi and CD47-AF488+ cancer cells by F4/80-PE+ macrophages (red) was well observed (white arrows) presenting ADCP events. (C) Quantitative co-localization analysis using Pearson’s coefficient and Manders’ coefficients further elaborated imaging observation. (D, E) ADCP events occurred as rapid but short wave between F4/80-PE-labeled macrophages (red) and CD47-AF488 mAb-targeted cells (green). CD47 inhibitor-treated cancer cells were first overlapped (yellow arrows) with F4/80+ macrophages and then soon engulfed (white arrows) by the macrophages extensively. (F–J) CD47-AF488 mAb-targeted cells (green) depict metabolic phenotype of strong NAD(P)H expression (blue), (I) whereas no meaningful co-localization with FAD hi cells (yellow) (J). Analyses with Peason’s coefficient and Manders’ coefficients corroborated imaging results (F–H). (K) Real-time visualization of phagocytosis events (white arrows) with treatment of CD47 blockade was also achieved using NAD(P)H imaging. Scales, 20 µm. AF, Alexa Flour; CD47, cluster of differentiation 47; FAD, flavin adenine dinucleotide; FAD hi , cells high in FAD intensity; mAb, monoclonal antibody; NAD(P)H, reduced nicotinamide adenine dinucleotide (phosphate) hydrogen; NAD(P)H hi , cells high in NAD(P)H intensity; PE, phycoerythrin; SIRPα, signal regulatory protein α.

Article Snippet: Then, systemic intravenous (IV) injections of anti-mouse CD47 monoclonal antibodies (anti-CD47 mAbs, clone MIAP301, Rat IgG2a, κ, BioXcell, USA) and rat IgG 2a mAbs isotype control (clone 2A3, Rat IgG2a, κ, BioXcell, USA) were performed with 10 mg/kg daily for 5 days.

Techniques: Imaging, In Vivo, Injection, Labeling, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Label-free metabolic imaging for sensitive and robust monitoring of anti-CD47 immunotherapy response in triple-negative breast cancer

doi: 10.1136/jitc-2022-005199

Figure Lengend Snippet: Sensitive monitoring of anti-CD47 therapy response by dynamic metabolic imaging of FAD. (A, B) Label-free metabolic intravital imaging (LMII) of FAD demonstrates similar sensitive early response with NAD(P)H imaging after anti-CD47 mAb (MIAP301) treatment of a triple-negative breast cancer model (10 mg/kg intravenous injection daily for 5 days). (C) Quantification of average intensity shows statistically significant difference in FAD signals before and after MIAP301 treatment (n=16 images, p<0.0001). (D) Optical redox ratio was increased with tumor growth and decreased with anti-CD47 mAb treatment. (E) Kinetics of single-cell metabolism with FAD signal. Each colored small square represents the visible pixel intensity of FAD in each cancer cell. The 10,000-pixel intensities were randomly measured from the cells in different mice every day for 16 days (n=6 mice). Metabolic signal of FAD has been increased with tumor growth from day 1 to day 9 and dramatically decreased with start of anti-CD47 mAb treatment from day 10. (F, G) Conventional whole-body fluorescent glucose imaging with 2-DG 750 verified cancer treatment with significant decrease of glucose uptake after anti-CD47 mAb treatment (n=5 mice). LMII shows superior sensitivity to detect immunotherapy response (p<0.0001 in C) over fluorescent glucose imaging (p=0.035 in G). (H) Visual inspection of treatment effect by color anatomy images. (I) The signal from FAD+ cells was significantly decreased after MIAP301 treatment when compared with isotype control as determined by flow cytometry. 2-DG, 2-DeoxyGlucosone; CD47, cluster of differentiation 47; FAD, flavin adenine dinucleotide; mAb, monoclonal antibody; NAD(P)H, reduced nicotinamide adenine dinucleotide (phosphate) hydrogen.

Article Snippet: Then, systemic intravenous (IV) injections of anti-mouse CD47 monoclonal antibodies (anti-CD47 mAbs, clone MIAP301, Rat IgG2a, κ, BioXcell, USA) and rat IgG 2a mAbs isotype control (clone 2A3, Rat IgG2a, κ, BioXcell, USA) were performed with 10 mg/kg daily for 5 days.

Techniques: Imaging, Injection, Control, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Label-free metabolic imaging for sensitive and robust monitoring of anti-CD47 immunotherapy response in triple-negative breast cancer

doi: 10.1136/jitc-2022-005199

Figure Lengend Snippet: Sensitive monitoring of anti-CD47 therapy response by dynamic metabolic imaging of NAD(P)H. (A, B) Label-free metabolic intravital imaging (LMII) of NAD(P)H demonstrates sensitive early response at single-cell level after anti-CD47 mAb (MIAP301) treatment of a triple-negative breast cancer model (10 mg/kg intravenous injection daily for 5 days). (C) Quantification of average intensity shows statistically significant difference in NAD(P)H signals before and after MIAP301 treatment (n=16 images, p<0.0001). (D) Kinetics of single-cell metabolism with NAD(P)H signal. Each colored small square represents the visible pixel intensity of NAD(P)H in each cancer cell. The 10,000-pixel intensities were randomly measured from the cells in different mice every day for 16 days (n=6 mice). Metabolic signal of NAD(P)H has been increased with tumor growth from day 1 to day 9 and dramatically decreased with start of anti-CD47 mAb treatment from day 10. (E, F) Conventional whole-body bioluminescence imaging of 4T1_PB3R-RFP-luc tumor mice verified cancer treatment with increased signal before anti-CD47 therapy and decreased signal after the therapy compared with isotype control treatment (n=6 mice). LMII shows superior sensitivity to detect immunotherapy response (p<0.0001 in C) over bioluminescence imaging (p=0.0122 in F). (G) Flow cytometric analysis shows decrease of NAD(P)H and RFP signals with CD47 inhibitor treatment over isotype control treatment. (H) The signal from NAD(P)H+RFP+ cells was significantly decreased after MIAP301 treatment when compared with isotype control as determined by flow cytometry (n=11 mice, p=0.0043). CD47, cluster of differentiation 47; mAb, monoclonal antibody; NAD(P)H, reduced nicotinamide adenine dinucleotide (phosphate) hydrogen; RFP, red fluorescent protein.

Article Snippet: Then, systemic intravenous (IV) injections of anti-mouse CD47 monoclonal antibodies (anti-CD47 mAbs, clone MIAP301, Rat IgG2a, κ, BioXcell, USA) and rat IgG 2a mAbs isotype control (clone 2A3, Rat IgG2a, κ, BioXcell, USA) were performed with 10 mg/kg daily for 5 days.

Techniques: Imaging, Injection, Control, Flow Cytometry