Abstract Spatial organization of normal tissues is key for their function, yet the role of distribution of distinct clones of cancer cells in tumor in not usually studied as a potential vulnerability in cancer. This is of particular interest in glioblastoma (GBM), most aggressive brain tumor, in which extensive intratumor heterogeneity hampers effectiveness of treatment. Multiregion sampling has been used in multiple studies to uncover evolutionary trajectories of the tumor. To better understand tumor evolution and the complexity of cancer ecosystem in GBM, we performed profiling of biopsies collected from MRI-defined regions of the tumors, spanning the surface, tumor core and deep tumor margins, which drive local recurrence after surgery. To test how spatially distinct biopsies differ in their cancer cell composition and how heterogeneous the tumor microenvironment is in these samples, we took advantage of 5-ALA metabolite fluorescence, which accumulates in GBM cells. 5-ALA administration prior to the surgery allows the surgeon to visualize cancer under blue light. We used it to separate cancer cells (predominantly 5-ALAhigh) from tumor microenvironment (5-ALAlow) and subjected the fractions to bulk and single cell transcriptomics, respectively. We found a gradient of cellular states associated with location in the tumor, with the deep margin samples having more 5-ALAlow cancer cells and generally less defined phenotype. We have also developed neurosphere cultures from our collection of spatially distant biopsies. Despite the selection pressure of the in vitro culture conditions, cells from different regions of the tumor adopted different morphologies, suggesting retention of some of their differential features. The differences between cultures were reflected in metabolism, proliferation, and differential sensitivity to a drug panel. Whole exome sequencing and transcriptomic analysis were also performed to identify the selection bottleneck of culture conditions, establish evolutionary trajectory of the distinct regions and neurosphere cultures, and test how strongly our cultures represent both the 5-ALAhigh and 5-ALAlow subpopulations of GBM cells. Our study identified properties associated with different spatial locations within GBM tumors, which will help uncover the cellular and microenvironmental dependencies that could lead to novel therapeutic targets for this highly heterogeneous tumor. Citation Format: Roberto Salatino, Ugoma Onubogu, Benjamin Oakes, Oszkar Szentirmai, Michalina Janiszewska. Multiregion biopsies and corresponding neurosphere cultures reveal spatial divergence in glioblastoma evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR014.