ID #766 Combination of Radiation with anti-CD47 in Diffuse Midline Glioma
Nicole Tan, Yanbo Yang, Ehsan Tajik-Mansoury, Ashley Tetens, Chanel Hutchison, Claire Vanpouille-Box, Michael Koldobskiy, Sahaja Acharya, Mohammad Rezaee, Robyn GartrellAbstract
Background
Diffuse midline glioma (DMG) is a fatal tumor with H3K27M mutation primarily treated with radiation (RT). RT stimulates type I interferon (IFN1) and prior work showed IFN1 signalling in myeloid cells in DMG after RT, that was correlated to higher CD47, a “don’t-eat-me signal”. This study aims to evaluate potential for combination of CD47 with RT in DMG.
Methods
DMG cell line (KAPP) used to generate KAPP CD47 knockout (KAPPCD47KO) via CRISPR-Cas9. Expression of IFN1 (STING) and CD47 evaluated via western blot (WB). KAPP wild-type (KAPPWT) and KAPPCD47KO cells received RT at 10Gy in 1, 2 or 3 daily fractions using Small Animal Radiation Research Platform (SARRP) and compared to unirradiated cells (SHAM). Apoptosis evaluated with live-cell imaging of cleaved caspase 3 (cc3). RNA-sequencing (RNA-seq) was performed 24h after RT. Cells were co-cultured with bone-marrow derived macrophages (BMDM) and phagocytosis evaluated using flow cytometry. KAPPWT and KAPPCD47KO were injected into brainstem of C57BL/6 mice, MRI evaluated to confirm tumor presence prior to conformal RT at 3Gy x13 daily fractions (39Gy) comparing to SHAM. Another cohort of KAPPWT mice were treated with RT combined with anti-CD47 (aCD47) and followed for survival.
Results
At baseline, KAPPWT shows high expression of STING and no CD47 in KAPPCD47KO confirmed via WB. Both cell lines have more cc3 after RT compared to SHAM with more tumor growth in KAPPCD47KO compared to KAPPWT. RNA-seq shows increased expression of other “don’t-eat-me” signals in KAPPCD47KO cells with or without RT. KAPPCD47KO exhibited less phagocytosis when co-cultured with BMDM. RT improved survival in KAPP mice with KAPPCD47KO not improved over KAPPWT. Combination RT with aCD47 did not improve survival in mice compared to RT alone.
Conclusions
Combination of CD47 with RT does not improve tumor growth or survival in DMG. Further work will evaluate the effect of CD47 on the immune microenvironment in DMG after RT.