Metabolism-based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential

Abstract

Background Glioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporo-spatially, a contributor to therapy resistance. Fluorescence guided neurosurgical resection utilizes administration of 5-aminolevulinic acid (5ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5ALA based method. Methods We have isolated the critical invasive GBM cell population by developing 5ALA-based metabolic fluorescence activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background “normal brain” signal to confound data. The population was studied using RNAseq, rtPCR and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors. Results RNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5ALA positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient derived cells but not the 5ALA negative cells. Conclusions Identification of unique molecular features in the invasive GBM population offer hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor sub-populations based upon intrinsic metabolic properties.

Spatial-transcriptomics reveals unique defining molecular features of fluorescence-sorted 5-aminolevulinic acid+ infiltrative tumor cells associated with glioblastoma recurrence and poor survival

“…Apart from temporal plasticity, GBM exhibits spatial heterogeneity and a growing body of evidence suggests that a collection of tissue from a single site fails to capture the spatial heterogeneity and transcriptional dynamics of GBM cells 14 . Tissues isolated from the resected tumor margin where GBM penetrates into the normal brain parenchyma, harbors distinct genomic and transcriptomic profiles in contrast to tissue removed from tumor core and enhancing rim regions 14 . Recurrence of GBM is initiated at the invasive margin within 2cm of the resected region after surgery 15 .…”

“…A viable solution emerged from the use of 5-aminolevulinic acid (5ALA) during GBM neurosurgery, reported to increase the percentages of complete resection from 36% to 65% 16 . 5ALA -a porphyrin -is metabolized to a fluorescent metabolite protoporphyrin IX (PpIX) by cells in which the heme biosynthetic pathway is activated, such as GBM cells but not non-neoplastic cells 14 . GBM cells exhibit a marked accumulation of PpIX due to their inability to incorporate iron into the protoporphyrin core because of the absence of ferrochelatase enzyme activity.…”

“…GBM cells exhibit a marked accumulation of PpIX due to their inability to incorporate iron into the protoporphyrin core because of the absence of ferrochelatase enzyme activity. The accumulated PpIX in GBM cells show a maximum excitement by blue light at 400-410 nm with an emission light peak at 635-704 nm exhibiting a pink fluorescence corresponding to regions of viable tumor cells 14 . The necrotic tumor core does not emit fluorescence owing to their deficiency of active heme metabolism; in contrast to the invasive tumor margin which fluoresces brightly and eventually fades with the decreasing number of tumor cells in the periphery 17 .…”

“…The necrotic tumor core does not emit fluorescence owing to their deficiency of active heme metabolism; in contrast to the invasive tumor margin which fluoresces brightly and eventually fades with the decreasing number of tumor cells in the periphery 17 . We have shown that invasive margins from primary tissue harboring the infiltrative 5ALA+ tumor cells can be purified from background non-neoplastic cells through fluorescence-activated cell sorting (FACS) 14 .…”

“…Intra-tumor surgical sampling, aided by FACS-isolation of 5ALA+ and 5ALAsubpopulations from invasive margin tissue, can serve as an ideal resource to untangle the intricacy of cellular and transcriptional heterogeneity within GBM. We have demonstrated that analysis of spatial transcriptomics within this sampling framework can identify non-canonical molecular factors associated with GBM infiltration, such as SERPINE1 14 . However, deep profiling of transcriptomic landscapes of spatially distinct tumor core, rim, invasive margin, and 5ALA sorted cells, is required to elucidate the spatial transcriptional heterogeneity and unique molecular signatures of 5ALA+ cells.…”