Radioresistance and Transcriptional Reprograming of Invasive Glioblastoma Cells

Zili Tang,Ivana Dokic,Maximilian Knoll,Federica Ciamarone,Christian Schwager,Carmen Klein,Gina Cebulla,Dirk C Hoffmann,Julian Schlegel,Philipp Seidel,Christiane Rutenberg,Stephan Brons,Christel Herold-Mende,Wolfgang Wick,Jürgen Debus,Dieter Lemke,Amir Abdollahi

doi:10.1016/j.ijrobp.2021.09.017

Abstract

Infiltrative growth pattern is a hallmark of glioblastoma (GBM). Radiation therapy aims to eradicate microscopic residual GBM cells after surgical removal of the visible tumor bulk. However, in-field recurrences remain the major pattern of therapy failure. We hypothesized that the radiosensitivity of peripheral invasive tumor cells (peri) may differ from the predominantly investigated tumor bulk. Invasive GBM populations were generated via debulking of the visible tumor core and serial orthotopic transplantation of peri cells, and sustained proinvasive phenotype of peri cells was confirmed in vitro by scratch assay and time lapse imaging. In parallel, invasive GBM cells were selected by transwell assay and from peri cells of patient-derived 3-dimensional spheroid cultures. Transcriptome analysis deciphered a GBM invasion-associated gene signature, and functional involvement of key pathways was validated by pharmacologic inhibition. Compared with the bulk cells, invasive GBM populations acquired a radioresistant phenotype characterized by increased cell survival, reduced cell apoptosis, and enhanced DNA double-strand break repair proficiency. Transcriptome analysis revealed a reprograming of invasive cells toward augmented activation of epidermal growth factor receptor- and nuclear factor-κB-related pathways, whereas metabolic processes were downregulated. An invasive GBM score derived from this transcriptional fingerprint correlated well with patient outcome. Inhibition of epidermal growth factor receptor and nuclear factor-κB signaling resensitized invasive cells to irradiation. Invasive cells were eradicated with similar efficacy by particle therapy with carbon ions. Our data indicate that invasive tumor cells constitute a phenotypically distinct and highly radioresistant GBM subpopulation with prognostic impact that may be vulnerable to targeted therapy and carbon ions.

Highlights

Radiation therapy is the mainstay for treatment of glioblastoma (GBM), the most common and malignant primary brain tumor in adults
In vivo tumors formed by orthotopic implementation of U87 cells were surgically debulked, dissociated, and ex vivo cultured under puromycin selection pressure to generate “bulk cells.”
Disseminated tumor cells infiltrating surrounding normal tissue in the tumor periphery (“peri cells,” see Fig. E1) were ex vivo enriched after tissue dissociation and puromycin selection

Summary

Introduction

Radiation therapy is the mainstay for treatment of glioblastoma (GBM), the most common and malignant primary brain tumor in adults. The sequence of therapy comprises surgical debulking followed by ionizing radiation (IR) and alkylating chemotherapy with temozolomide, with the intention to eradicate residual infiltrative tumor cells invading adjacent normal tissue. Despite this multimodal strategy, patients with newly diagnosed GBM have a median survival of approximately 14 months.[1,2] The predominant pattern of recurrence of GBM is within the infiltrative zone surrounding the resected tumor bulk, where single or groups of tumor cells reside even in pathologically complete resected tumors.[3,4] It is conceivable that recurrent tumors are formed from a rare population of residual infiltrative cells that we called recurrent tumor initiating cells (R-TIC). The clinically radioresistant phenotype of GBM conflicts with in vitro radiobiological studies demonstrating that the radiosensitivity of GBM cells is similar to that of clinically radiosensitive tumors such as Hodgkin lymphoma.[8]

Methods

Results

Conclusion