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Glioblastomas multiforme (GBMs) are the most lethal tumors of the brain. Tumoral mesenchymal transformation is a hallmark of GBMs associated with alterations in cellular morphology and dynamic organization. However, little is known about the mechanisms that control this pathological process. Here, we report a comprehensive spatiotemporal study integrating novel intra-tumoral histopathological structures, ‘oncostreams’, with tumor dynamic properties, microenvironment assets and spatial molecular features. Cellular analyses of genetic engineered mouse models of glioma identified that oncostreams are heterogenous structures formed by elongated and aligned neoplastic cells enriched in non-neoplastic cells such as ACTA2+ mesenchymal like cells and CD68+ tumor associated microglia/macrophages (TAM). Deep learning analysis of H&E glioma histological samples from mouse and human gliomas identified that oncostream density correlates with tumor aggressiveness. To determine whether oncostreams fascicles are characterized by a specific gene expression profile, we performed transcriptomic analysis using laser capture microdissection coupled to RNA-sequencing. We found that oncostreams are defined by a transcriptomic signature enriched in mesenchymal genes. Network analyses identified that COL1A1 is a critical gene that regulates oncostream organization and function. Correspondingly, human and mouse high-grade gliomas with high oncostream densities showed prominent alignment of collagen fibers along these fascicles and higher COL1A1 expression compared to low-grade gliomas. To evaluate the functional role of COL1A1 in oncostream formation we generated a COL1A1-deficient GEMM of glioma. We observed that COL1A1 inhibition decreased oncostream formation, impaired tumor cell proliferation and remodeled the tumor microenvironment by diminishing CD68+ TAM cells, CD31+ endothelial vascular proliferation and ACTA2+ perivascular mesenchymal cells, thus increasing animal survival. Further studies, using time lapse confocal imaging in ex vivo glioma explants, and intravital imaging in vivo demonstrated that oncostreams are organized collective dynamic structures present at the tumor core and the invasive tumor border. Oncostreams dynamics increased the intra-tumoral spread of cells within the tumor and foster glioma aggressiveness through collective invasion of the normal brain parenchyma. The analysis of glioma invasion in COL1A1 knockdown tumors exhibited a reduction in collective migration patterns, strongly supporting its importance in tumor progression. We propose that oncostreams represent a novel pathological marker of potential value for diagnosis and COL1A1 depletion within oncostreams is a promising approach and reprogram mesenchymal transformation to reduce the tumor malignancy. Citation Format: Andrea Comba, Syed Faisal, Patrick J. Dunn, Anna E. Argento, Todd C. Hollon, Wajd N. Al-Holou, Maria L. Varela, Daniel B. Zamler, Gunnar L. Quass, Pierre F. Apostolides, Christine E. Brown, Phillip E. E. Kish, Alon Kahana, Celina G. Kleer, Sebastien Motsch, Maria G. Castro, Pedro R. Lowenstein. Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2476.

Intratumoral heterogeneity is a hallmark of high grade gliomas. However, whether heterogeneity is static or dynamic remains unknown. Here we demonstrate that gliomas’ core and border regions display areas of self-organized collective motion, which we have termed oncostreams. Histologically oncostreams appear as multicellular fascicles of elongated and aligned glioma cells with mesenchymal-like morphology, and their density correlates positively with tumor malignant behavior. Using time lapse confocal imaging of organotypic brain slices of experimental glioma we discovered oncostream dynamics, namely, as two self-organizing patterns of collective motion: streams (bidirectional motion) and flocks (unidirectional motion). Oncostreams were also present in human glioblastoma multiforme, and could also be detected using objective artificial indigent approaches. They were present in 47% of TCGA-GBM grade IV tumor tissues, in 8.6 % of TCGA-LGG grade III, and were absent in TCGA-LGG grade II. Oncostreams are heterogeneous, contain GFAP+ cells, Iba1+ microglia/macrophages cells, and ACTA2+ mesenchymal cells aligned along Sox2+ tumor cells. We propose that oncostreams function as highways possibly stimulating the spread of slower-moving glioma cells and/or non-tumor cells throughout the tumor mass, reinforcing a potential role of oncostreams in determining spatial heterogeneity and remodeling of the tumor microenvironment. To analyze the molecular landscape of oncostreams we used laser capture microdissection coupled to RNA-Seq and bioinformatics analysis: we detected the existence of genetic networks specific to oncostreams. Oncostreams were aligned along highly expressed extracellular matrix proteins, among them Col1a1 was the most differentially expressed. Genetic inhibition of Col1a1 dissembled oncostreams, decreased glioma heterogeneity and improved animal survival. We propose that oncostreams dynamics will be a promising avenue to understand glioma behavior, and treat these malignant tumors. Citation Format: Andrea Comba, Sebastien Motsch, Patrick Dunn, Todd Hollon, Daniel Zamler, Anna Argento, Alon Kahana, Phillip Kish, Maria Castro, Pedro Lowenstein. The dynamic tumor microenvironment: Oncostreams are self-organizing structures that modulate glioma progression and treatment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR005.

Glioblastoma multiforme (GBM) is the most frequent and lethal tumor of the central nervous system. GBM are characterized by diffuse invasion and cellular heterogeneity which challenges treatment efficacy. Tumors with mesenchymal properties display the most aggressive phenotype. However, the biological function and molecular mechanisms underlying GBM mesenchymal transformation remain unknown. Analysis of mouse and human malignant gliomas revealed the presence of organized multicellular structures formed by elongated and aligned cells. These structures resemble areas of GBM mesenchymal transformation that we named Oncostreams. We determined the molecular signature underlying oncostreams function by performing laser capture microdissection followed by transcriptomic analysis. We found that oncostreams overexpressed Col1a1, ACTA2, MMP9, MMP10 and ADAMTS2 genes, all of them associated with regulation of extracellular matrix organization, collagen catabolic process and cellular migration pathways. Functional network analysis indicated that Col1a1 was a primary regulator gene. To analyze whether these structures display migratory properties we used time lapse imagining in a 3D organotypic glioma model. Morphological and statistical analysis revealed that oncostreams displayed a collective motion pattern, organized as streams or flocks. This dynamic patterns participate in local invasion and function as tumoral highways to facilitate the spread of several cells as intra-. Further analysis showed that oncostreams presence correlates with increased collagen expression and decreased animal survival. Patient's glioma biopsies also evidenced that these areas of Col1a1 overexpression were present in high grade but no in low-grade gliomas. We corroborated by immune-hystochemistry that Col1a1 were overexpressed and co-localized within GFP positive tumoral cells. Further, to analyze the origin and role of Col1a1 in glioma malignancy we generated genetically engineered mouse glioma models (GEMM) with Col1a1 downregulation. Interestingly, Col1a1 was retained only surrounding the blood vessel but was completed absent within the tumor parenchyma. We demonstrated that Col1a1 downregulation ablates oncostreams structures, reversed the malignant phenotype resembling low grade glioma histopathology and prolonged animal survival. This study reveals that oncostreams are organized dynamic structures that regulate glioma growth and invasion. They are areas of mesenchymal transformation defined by a molecular signature associated to extracellular matrix proteins expressed as Col1a1. Oncostreams with high expression of Col1a1 within glioma cells represent a novel potential targets for future translational development. Disruption of oncostreams will provide a new avenue to treat GBM. Citation Format: Andrea Comba, Patrick Dunn, Anna E. Argento, Padma Kadiyala, Sebastien Motsch, Daniel Zamler, Alon Kahana, Phillips E. Kish, Maria G. Castro, Pedro R. Lowenstein. Spatiotemporal analysis of gliomas: Dynamics of mesenchymal multicellular structures as novel target for tumor treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3950.

Cancer stem cells (CSCs) are those cells within a malignant tumor that can self-renew and initiate new tumors, with the ability to produce all the cell lineages that comprise the tumor. Recent studies have revealed that cancer treatments that target the tumor without addressing CSCs might reduce the tumor load but be unable to achieve a cure. Generation and maintenance of CSCs through genetic and epigenetic reprogramming is essential for tumor maintenance and resistance to therapy. Therefore, developing therapies targeted at CSCs is critical to achieving cures for cancer1. Yet the biology of CSCs, and particularly the process of cellular reprogramming, is still not well understood, in part because of the many mutations carried by these cells, only a few of which are deterministic. In order to develop a better understanding of the mechanistic underpinnings of adult cell dedifferentiation, we developed a zebrafish model that utilizes adult myocyte reprogramming in the context of muscle regeneration. Our model utilizes the lateral rectus muscle of the eye, which can recover from >50% myectomy by reprogramming the residual (i.e. “post-mitotic”) myocytes to reenter the cell cycle, remodel the extracellular matrix, proliferate, migrate and then redifferentiate into myocytes that reform a functional muscle (as determined by an optokinetic response). The proliferative burst of reprogrammed myocytes is particularly robust, with >70% of cells undergoing mitosis during the regeneration process. Such a robust in vivo model of cell dedifferentiation represents a unique opportunity to study the cellular, genetic and epigenetic processes involved. We find that the dedifferentiation process begins with activation of epigenetic regulators to remodel chromatin, followed by activation of autophagy to remodel the cytoplasm, followed by cell cycle reentry. Epigenetic changes include changes in DNA and histone methylation, along with activation of genes encoding transcription factors that are frequently found to be involved with oncogenesis, such as myc, jun, fos, and twist. A transcriptome analysis using deep sequencing reveals significant similarities between the genetic signatures of dedifferentiated zebrafish myocytes and human cancer cells. This analysis also identifies long non-coding RNAs that are induced early during the dedifferentiation process, and work is ongoing to annotate these lncRNAs with reference to human cancer. In summary, an in vivo zebrafish non-cancer model of cancer can provide unique mechanistic insights into the biology of CSCs. In addition, as an aquatic species this model is particularly well suited for drug screening, providing an innovative new approach to developing drugs that target reprogramming/dedifferentiating cells. Citation Format: Ke’ale Louie, Alfonso Saera-Vila, Phillip E. Kish, Alon Kahana. Zebrafish adult cell dedifferentiation as a noncancer model of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2541.