Abstract 1273: Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes
Authors: Nassour-Caswell Lauren C. , Eustace Nicholas J. , Stackhouse Christian T. , Alrefai Hasan , Hicks Patricia H. , Schanel Taylor L. , Anderson Joshua C. , Beierle Andee M. , Willey Christopher D. 
Published: April 2023

Abstract
Background: Glioblastoma (GBM) has a median survival of &lt;2 years and generally recurs within 6 months of treatment due to the development of chemo- and radiotherapy (RT) resistance. Tunneling nanotubes (TNTs) serve as intercellular conduits for establishing robust, tumor-promoting networks within the hypoxic tumor microenvironment. TNTs are provoked by hypoxia and can passage organelles like mitochondria, i.e., between astrocytes and stem-like brain tumor-initiating cells (BTICs). This is theorized to expand resistance properties to other cell types, as well as facilitate metabolic rescue in damaged cancer cells. We investigated the mitochondrial uptake (MU) abilities of RT-sensitive or RT-resistant BTICs from normal human astrocytes (NHAs) under hypoxic conditions.
Methods: We used a Cytation5 Cell Imager to quantify MU by BTICs from NHAs in direct contact under normal (20%) or hypoxic oxygen tensions (5%). We obtained RT-sensitive (JX14P) patient-derived xenograft BTICs and generated a paired acquired-resistant (JX14P-RT) line. This was achieved by implanting primary tumors into flanks of athymic nude mice and serially treating with 6 fractions of 2Gy over 14 days for multiple passages until the median doubling time was halved. Cells were plated at a 1:1 ratio on Geltrex for 18h and exposed to 5% or 20% oxygen in serum-free media. NHAs were pre-labeled with a GFP-mitochondria tracker and BTICs were infected with a mCherry lentivirus. BTIC-MU was determined by quantifying double-positive cells in whole-well images. Viability was determined using CellTiterGlo, n = 4.
Results: Time-lapse imaging revealed GFP-mitochondria transfer from NHAs to BTIC cells via TNTs stimulated by hypoxic conditions. We measured overall MU and cell viability in both BTIC lines. JX14P co-cultured with NHAs trended toward an increased MU (cell fraction) in hypoxia (Hyp) compared to Normoxia (Norm) (Norm = 32.61 ± 13, Hyp = 44.83 ± 5, P&gt;0.167). JX14P exhibit higher cell viability (RLU) in hypoxia when mono- or co-cultured with NHAs (Mono: Norm = 10275 ± 901, Hyp = 12599 ± 579, P&lt;0.0039, Co: Norm = 5415 ± 664, Hyp = 8341 ± 700, P&lt;0.0001). JX14P-RT co-cultured with NHAs show a trend for more MU in hypoxia compared to Normoxia (Norm = 25.90 ± 12, Hyp = 38.12 ± 12, P&gt;0.167). Compared to monoculture, JX14PRT exhibits higher cell viability in co-culture with NHAs under hypoxia (Mono: Norm = 9721 ± 255, Hyp = 10011 ± 1462, P&gt;0.998, Co: Norm = 4928 ± 664, Hyp = 7805 ± 944, P&lt;0.005).
Conclusions: RT-sensitive or -resistant BTICs cocultured with NHAs exhibit increased cell viability under acute hypoxia compared to Normoxia with a trend toward increased MU. Results indicate a potential protective effect following direct interaction with NHAs under hypoxia. We are further exploring metabolic changes in each cell type following mitochondrial exchange.
Citation Format: Lauren C. Nassour-Caswell, Nicholas J. Eustace, Christian T. Stackhouse, Hasan Alrefai, Patricia H. Hicks, Taylor L. Schanel, Joshua C. Anderson, Andee M. Beierle, Christopher D. Willey. Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1273.