924 Novel immune checkpoint interactions in Medulloblastoma and the TME

• Published in: Journal for immunotherapy of cancer 2023-11, Vol.11 (Suppl 1), p.A1027-A1027
• Main Authors: Martin, Allison M, Moziak, Kirsten, Munoz Perez, Natalia A, Galbo, Phillip M, Picarda, Elodie, Bell, Robert, Eberhart, Charles G, Zheng, Deyou, Zang, Xingxing
• Format: Article
• Language: English
• Subjects: Brain cancer; Flow cytometry; Immunotherapy; Regular and Young Investigator Award Abstracts; Tumors
• ISSN: 2051-1426
• DOI: 10.1136/jitc-2023-SITC2023.0924

BackgroundMedulloblastoma, the most common embryonal brain tumor of childhood, has a cold tumor immune microenvironment, harboring low lymphocytic infiltration and a paucity of tumoral PD-L1 expression.1 We and others have shown that this tumor is highly infiltrated with IBA-1 positive tumor associated microglia/macrophages that more frequently express PD-L1 than tumor cells. Despite this, agents disrupting the PD-1 pathway have been ineffective in treating patients with this tumor, suggesting that medulloblastoma may utilize alternate pathways to maintain a suppressive microenvironment.MethodsNanoString GeoMx digital spatial profiling was used to isolate IBA-1 expressing cells infiltrating 12 medulloblastoma tumor samples from a tumor issue microarray that were previously profiled for molecular subgroup. IBA-1+ cells versus other were evaluated for expression of 77 immunomodulatory proteins. mCB DNp53 MYC murine medulloblastoma cell line, which mimics recurrent/refractory disease, was implanted orthotopically into BL6/J mice and evaluated for immune cell infiltration by multicolor flow cytometry and immunohistochemistry.2 Lenticrisprv2 containing gRNA targeting B7-H3 was used to knock out B7-H3 in vitro. These cells were subsequently evaluated using InCucyte spheroid assays, flow cytometry, western blot and bulk RNA sequencing analyses.ResultsProteomic profiling of IBA-1+ fractions was significantly different than that of other cells, primarily consisting of synaptophysin positive tumor cells. In line with other studies, we find the immune checkpoint molecule B7-H3 to be enriched in the non-IBA-1+ fraction. IBA-1+ cells infiltrating SHH versus non-WNT/non-SHH molecular subgroups cluster separately, with differential protein expression between subgroups, suggesting influence of the molecular drivers on the immunobiology of these tumors. In addition to PD-L1, we find increased expression of CTLA-4, TIM-3, and VISTA within the IBA-1+ component. Similar to human tumors, our syngeneic murine medulloblastoma cell line, mCB DNp53 MYC has a paucity of infiltrating lymphocytes and increased infiltration of microglia/macrophages. Both lymphoid and myeloid cells express VISTA in the microenvironment of medulloblastoma tumor-bearing mice. This model also demonstrates tumoral expression of B7-H3 in vitro and in vivo. To further investigate the influence of B7-H3 on both medulloblastoma and its microenvironment, we used CRISP/Cas9 to remove B7-H3 in this model. VSIG3,