Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an actionable target to disrupt tumor progression

Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. Col1a1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of Col1a1 eliminates oncostreams, reprograms the malignant histopathological phenotype, reduces expression of the mesenchymal associated genes, induces changes in the tumor microenvironment and prolongs animal survival. Oncostreams represent a pathological marker of potential value for diagnosis, prognosis, and treatment.

Code Availability: The analysis of oncostreams in mouse and human glioma tissue was performed using U-Net architecture to provide semantic segmentation of specimens using deep learning. Public GitHub repository for the project code can be found at https://github.com/MLNeurosurg/DeepStreams. Analysis of glioma cells dynamics was performed using the Julia Programing Language. Link for this project Script and their dependencies can be found at public GitHub repository https://github.com/smotsch/analysis_glioma. Following standards of the field, including power analysis, sample sizes were estimated which were capable of yielding statistically significant differences. For in vivo studies, at least n"3 mice were utilized. To verify the data obtained, and estimate the median survival at least n"3 biological replicates were chosen on the basis of previously published studies (PMID: 26936505, PMID: 30760578, and PMID: 34586841). The number of replicates used are mentioned in the corresponding figure legends. For sleeping beauty survival experiments, at least n"9 were used, to detect a hazard ratio of 0.714 using log-rank test corresponding to at least 40% increase in median survival/improvement. In cases where sample size was less than three due to experimental limitations, statistical differences were not presented.
No data were excluded from the analyses.
Replicates were used in all experiments as noted in the manuscript and figure legends. All experiments were repeated at least three times with reproducible results.
All samples such as cells or animals were randomly allocated into experimental groups.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. We were not blinded to group allocations during data collection and analysis of ex vivo and in vivo experiments involving various experimental groups. In order to perform the imaging and dynamic analysis ex-vivo and in-vivo, it was imperative to know the assigned experimental group. In case of image analysis, experimental images were blinded during selection of the imaging. Validation by immunohistochemistry-immunofluorescence staining of human neural stem cell transplanted into rat brain and SH-SY5Y cells. Citation from manufacturer is listed at: https://www.emdmillipore.com/US/en/product/Anti-Nuclei-Antibody-clone-235-1,MM_NF-MAB1281 Validation by immunohistochemistry-immunofluorescence staining of human ovarian cancer tissue, human melanoma xenograft mouse model, and human embryonic stem cells differentiated into mesoderm. Citation from manufacturer is listed at: https://www.abcam.com/n-cadherin-antibody-intercellular-junction-marker-ab18203.html • Anti-Nestin [Novus, Cat# NB100-1604, Host# Chicken, Dilution# 1:800]: Validation by immunohistochemistry-immunofluorescence staining of 3T3 cells, adult dentate gyrus of the hippocampal formation, and A7R5 neuroblastoma cells. Citation from manufacturer is listed at: https://www.novusbio.com/PDFs/NB100-1604.pdf • Anti Sox2 [Invitrogen,Host# Mouse,Dilution# 1:200]: Validation by immunohistochemistry-immunofluorescence staining of H9 embryonic stem cells grown for a few days on Matrigelcoated chamber slides, H9 embryonic stem cells grown for a few days on Matrigel-coated chamber slides, and human neural stem cells derived from PD-3 iPSCs. Citation from manufacturer is listed at: https://www.thermofisher.com/order/genome-database/dataSheetPdf? producttype=antibody&productsubtype=antibody_primary&productId=MA1-014&version=223 • Anti-Iba1 Antibody [EPR16588] [Abcam, Cat# ab178846, Host# Rabbit, Dilution# 1:500]: Validation by immunohistochemistry-immunofluorescence staining of rat normal brain, human normal hippocampus, and mouse microglia cells. Citation from manufacturer is listed at: https://www.abcam.com/iba1-antibody-epr16588-ab178846.html