P02.10.A CHARACTERIZATION OF THE TUMOR MICROENVIRONMENT IN PERIPHERAL AREAS OF GLIOBLASTOMA USING MULTIPLEX SEQUENTIAL PROTOCOLS.

Abstract BACKGROUND Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults, with a high recurrence rate even after standard treatment, which includes surgery, radiotherapy and chemotherapy. This recurrence is associated with the tumor’s ability to infiltrate brain tissue beyond the margins visible on neuroimaging, making complete resection nearly impossible. In a prospective study, conducted in collaboration with Hospital Universitario 12 de Octubre, we analyzed a cohort of radiologically-guided biopsies, including regions of tumor infiltration, vasogenic edema and even apparently normal areas. Using DAB immunohistochemistry for MIB-1, we detected proliferative tumor cells not only in the tumor core but also in the non-infiltrated peripheral zone. This finding prompted us to further characterize the tumor microenvironment in these areas. MATERIAL AND METHODS We developed a multiplex sequential immunofluorescence (mIF) protocol based on the cyclic elimination of antibodies using 2-mercaptoethanol and SDS. Although pre-designed commercially panels exist for this type of approach, we designed custom panels focused on studying peripheral GBM regions, including specific markers for tumor, immune, glial and endothelial cells. This method enables detailed spatial characterization using confocal microscopy. In parallel, and with the aim of increasing accessibility of this technology, we developed a complementary protocol based on multiplex sequential immunohistochemistry with chromogens (mIC), compatible with conventional light microscopy. In this case, chromogen removal between rounds is performed by washing in absolute ethanol, and antibody stripping is achieved using citrate buffer pH 6. This process allows for the complete removal of previous markers and the application of new rounds of chromogenic staining on the same sample. RESULTS These two approaches complement each other depending on the analytical needs. While mIF enables higher multiplexing capacity and spatial resolution, its implementation requires specialized equipment. In contrast, mIC offers a simpler and more affordable solution, suitable for standard laboratory settings, though with reduced capacity for simultaneous labeling. CONCLUSION This combined approach allows for a more precise study of peripheral GBM areas, providing key insights into its biology and potential mechanisms of recurrence.