Supplementary Material for: IMMUNE SCENARIO IDENTIFICATION COMBINING MULTIPLEXED, QUANTITATIVE, AND ADVANCED IMAGING ANALYSIS COULD BE RELEVANT IN IMMUNOTHERAPY AGAINST GLIOBLASTOMA AND GRADE 4 ASTROCYTOMA.

Introduction
In our research on understanding glioblastoma’s resistance mechanisms to immunotherapy, we extensively investigated through an innovative technology that enables the simultaneous assessment of multiple biomarkers. With this approach, we aim to gain deeper insights into the interplay between immunosuppressive cells (IC) and effector cells (EC).

Methods
It were studied 126 cases of glioblastoma employing tissue microarrays stained with a panel of immune infiltrate validated via multiplex immunofluorescence and quantified by advanced image analysis. All cases were categorized according to an EC/IC ratio and their respective medians. Statistical correlations between cell populations and with survival were calculated.

Results
M2 macrophages were the most abundant IC, followed by a variable number of EC and pro-tumoral activated microglia, and a scant quantity of FoxP3 cells. EC showed a statistically significant direct positive correlation with IC. The patients with tumors exhibiting an EC/IC ratio ≤0.063 displayed a significantly poorer outcome. Furthermore, in the context of incomplete surgical resection, significant differences were evident considering immune scenarios.

Conclusions
By integrating multiplex technology with advanced imaging analysis, we successfully identified 4 distinct immune scenarios in glioblastoma. We observed a favorable immune scenario characterized by a relatively high EC/IC ratio, which is especially evident in the clinical setting of incomplete tumor resection. This promising immune scenario holds significant potential for selecting suitable candidates for immunotherapy in glioblastoma.