Spatial immunosampling of MRI-defined glioblastoma regions reveals immunologic fingerprint of non-contrast enhancing, infiltrative tumor margins

Glioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hi Tregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence. ### Competing Interest Statement NICO Corporation provided the NICO Myriad and Tissue Preservation System devices at no cost. MSA declares the following: Research funding from Seagen; Consultation for Bayer, Novocure, Kiyatec, Insightec, GSK, Xoft, Nuvation, Cellularity, SDP Oncology, Apollomics, Prelude, Janssen, Tocagen, Voyager Therapeutics, Viewray, Caris Lifesciences, Pyramid Biosciences, Anheart Therapeutics, Varian Medical Systems, Theraguix; Scientific Advisory Board for Cairn Therapeutics, Pyramid Biosciences, Modifi Biosciences; Stock shareholder of Mimivax, Cytodyn, MedInnovate Advisors LLC. The authors have declared that no other potential conflict of interest exists. ### Clinical Trial NCT04545177 ### Funding Statement This study was funded by: Case Comprehensive Cancer Center (JDL), Cleveland Clinic Lerner Research Institute (JDL), 5T32AI007024-40 (DCW), R35 NS127083 (JDL), K99 CA248611 (DB), Reza Khatib, MD Endowed Chair in Neurosurgery (AMM), F30 CA250254 (AL), Fernandez Family Foundation Endowed Chair in Cancer Research (MSA). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Cleveland Clinic gave ethical approval for this work. Case Comprehensive Cancer Center Protocol Review and Monitoring Committee gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors