Development of spatial-integrative pre-clinical models is needed for glioblastoma, which are heterogenous tumors with poor prognosis. Here, we present an optimized protocol to generate three-dimensional ex vivo explant slice glioma model from orthotopic tumors, genetically engineered mouse models, and fresh patient-derived specimens. We describe a step-by-step workflow for tissue acquisition, dissection, and sectioning of 300-μm tumor slices maintaining cell viability. The explant slice model allows the integration of confocal time-lapse imaging with spatial analysis for studying migration, invasion, and tumor microenvironment, making it a valuable platform for testing effective treatment modalities. For complete details on the use and execution of this protocol, please refer to Comba et al. (2022).1.

Background High-grade gliomas are fatal with universally poor prognosis. Initiation of effective cancer immune responses requires functional immune cells, particularly afferent antigen-presenting cells, which are typically absent from the brain parenchyma. To overcome this limitation, two adenoviral vectors expressing HSV1-TK and Flt3L were combined to target human gliomas. This first-in-human trial assessed safety, cytotoxicity, and recruitment of immune cells to the brain, in support of a future phase 1b/2 clinical trial. Methods Treatment-naïve high-grade glioma adult patients received injections of adenoviral vectors expressing HSV1-TK and Flt3L into the tumor bed, following maximal safe resection, at six escalating doses ranging from a total of 1.1×10 10 to a maximum of 2×10 11 viral particles. This was followed by two 14-day courses of Valacyclovir and standard upfront chemoradiation. Key inclusion criteria were age between 18 to 75, KPS≥70, and treatment-naïve possible high-grade glioma amenable to gross total resection. Patients were consented pre-operatively, and definitive enrollment occurred intraoperatively upon pathology confirmation of malignant glioma. Findings The treatment was well-tolerated without dose-limiting toxicity in patients with high grade glioma (n=17) (including 3 of the Gliosarcoma variant), or Anaplastic Ependymoma (n=1). The maximal-tolerated dose was not reached. The median overall survival was 21.3 months (95%CI: 11.1, 26.1) compared to 14.6 months with standard-of-care, with seven patients surviving for >2 years, three patients surviving for >3 years, and one patient still alive 57 months after enrollment. Tissue from subsequent re-resections from eight subjects showed elevated markers for CD3 + , CD8 + T cells, and plasmacytoid dendritic cells (pDCs), suggesting the potential stimulation of anti-glioma immunity. Additionally, we detected biological activity from both viral vectors: (i) an increase in serum levels of Flt3L two weeks after vector administration, and (ii) expression of HSV1-TK in neurons, astrocytes, and SOX2+ cells in brain tumor samples up to 17 months post-vector injection into the brain. Interpretation Use of two adenoviral vectors expressing HSV1-TK and Flt3L appears to be both safe and feasible. Promising evidence from multiplex immunocytochemical analyses shows the presence of the expected immune infiltration, i.e., pDCs, along with persistent vector expression lasting up to 17 months post-injection. Moreover, the two-year survival rate of 38.8% compared to 19.6% with standard-of-care is promising, suggesting that this approach warrants further investigation in a phase 1b/2 clinical trial. Funding Funded in part by Phase One Foundation, Los Angeles, CA, The Board of Governors at Cedars-Sinai Medical Center, and The Rogel Cancer Center at The University of Michigan; clinicaltrials.gov: NCT01811992 .

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.

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.

Tumor mesenchymal transformation (MT) is a hallmark of high-grade gliomas. The mesenchymal state is associated with specific changes related to cell adhesion, migration, and the extracellular matrix. Collagen 1a1 (COL1A1) is a main component of the extracellular matrix in gliomas, and its expression correlates inversely with patient survival. However, the cellular and molecular mechanisms of the tumor-associated COL1A1 matrix in gliomas remains elusive. Our study integrates histopathological features, spatially resolved transcriptomics, cellular dynamics and microenvironment alterations associated with MT in high-grade gliomas. Using deep learning analysis of mouse and human glioma histological samples we identified that the density of areas of MT, named oncostreams, correlates with tumor aggressiveness. Spatial transcriptomics analysis, using laser capture microdissection, identified a signature enriched in extracellular matrix related proteins, in which COL1A1 appeared as a key determinant of mesenchymal organization. Correspondingly, human and mouse high-grade gliomas showed prominent alignment of collagen fibers along these mesenchymal fascicles and higher COL1A1 expression compared to low-grade gliomas. Moreover, RNA fluorescent multiplex assays identified at single cell level that different cells within glioma tumors contribute to COL1A1 expression, including neoplastic cells and perivascular non-neoplastic cells such as ACTA2+, CYR61+ and FAP+. Inhibition of COL1A1 using genetically engineered mouse models decreased areas of mesenchymal transformation and increased survival. COL1A1 downregulation impaired tumor cell proliferation and remodeled the tumor microenvironment by reducing CD68+ macrophages/microglia cells, CD31+ endothelial cells, ACTA2+, CYR61+ and FAP+ perivascular cells, and increased GFAP+ astrocytes infiltration withing the tumor mass. Further studies, using ex-vivo glioma explants demonstrated that CO1A1 downregulation decreased collective invasion of the normal brain, supporting its importance in tumor progression. We propose that COL1A1 expression is a valuable marker for diagnosis, and COL1A1 depletion within glioma tumors is a promising direct or complementary therapeutic approach to reprogram mesenchymal transformation, and halt tumor growth.

Background High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn's role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion. Methods We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8-/-, CD4-/-). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME. Results We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8-/- and CD4-/- immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME. Conclusions Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.

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.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Tumor heterogeneity is a hallmark of cancer and a determinant of malignant behavior. How tumor heterogeneity arises is thus of fundamental importance. Gliomas display oncostreams, self-organizing multicellular fascicles of elongated, aligned, collectively motile glioma cells, that establish dynamic heterogeneity throughout gliomas. Gliomas exhibit two collective motion patterns: streams, displaying bidirectional collective motion, and flocks, displaying unidirectional collective motion. Oncostreams function as highways to facilitate the intratumoral spread of tumoral and non-tumoral cells. Detailed quantitative and deep learning analysis of rodent and human gliomas uncovered that the density of oncostreams correlates positively with glioma aggressiveness. Our study establishes the self-organizing dynamic nature of gliomas, and its role in setting up dynamic tumor heterogeneity and consequently tumor malignant behavior.

Abstract 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.

Abstract High grade gliomas (HGG) such as glioblastoma lack effective treatment with poor prognosis of median overall survival (OS) around 14-16 months with standard of care. Initiation of effective immune response against cancer requires functional dendritic cells, which are absent from the central nervous system, resulting in lack of anti-HGG immune responses. An effective anti-glioma immune response can be achieved by combining glioma cytotoxicity with HSV1-TK and valacyclovir, and recruitment of dendritic cells to the brain with Flt3L. This dual approach makes endogenous tumor antigens available to infiltrating dendritic cells in its microenvironment by causing: (i) dendritic cells' infiltration of gliomas, (ii) CD8+, CD4+ T cell immune cytotoxicity and memory, and (iii) the systemic immune system to recognize tumor neoantigens. We report the first in human phase I dose escalation trial of adenoviral vectors expressing HSV1-TK and Flt3L (NCT01811992). Injection of dose escalated HSV1-TK and Flt3L adenovectors (range 1x10^9 vp - 1x10^11 vp) to the tumor bed post-resection of newly diagnosed HGG was followed by two cycles of 14-day course of valacyclovir starting 1-3 days and 10-12 weeks post-op combined with standard of care upfront radiation, concurrent and adjuvant temozolomide. Key inclusion criteria were ages 18-75, KPS ≥70, and suspected newly diagnosed HGG amenable to gross total resection. Enrollment and vector injection occurred after frozen pathology confirmed HGG. Out of 18 patients, six are still alive. The primary endpoint of maximal tolerated dose was not reached and the experimental treatment was well tolerated without dose limiting toxicity. The secondary endpoint of OS is promising with median of 21.9 months (range 5.4-52.7). Five out of six patients (83%) who had re-resection at the time of suspected radiographic progression had treatment effect rather than true progression, and increase in markers for dendritic cells, CD4+ T cells, and macrophages were noted, indicating successful immunity recruitment consistent with pre-clinical findings. Updated survival data, as well as comparison to matched controls, and detailed toxicities will be presented at the time of the meeting. In conclusion, the use of dual adenoviral vectors expressing Flt3L and HSV1-TK is safe and well tolerated in newly diagnosed HGG patients. Our results indicate promising preliminary survival outcome and histological evidence of immune infiltration. Future studies to assess treatment efficacy is warranted. Citation Format: Pedro Lowenstein, Daniel Orringer, Yoshie Umemura, Oren Sagher, Jason Heth, Shawn Hervey-Jumper, Aaron Mammoser, Denise Leung, Ted Lawrence, Mishell Kim, Daniel Wahl, Paul McKeever, Sandra Camelo-Piragua, Andrew Lieberman, Sriram Venneti, Kait Verbal, Karen Sagher, Patrick Dunn, Daniel Zamler, Andrea Comba, David Altshuler, Lili Zhao, Karin Muraszko, Larry Junck, Maria G. Castro. First in human phase I trial of adenoviral vectors expressing Flt3L and HSV1-TK to treat newly diagnosed high-grade glioma by reprogramming the brain immune system [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 CT105.

OBJECTIVES/SPECIFIC AIMS: Oncostreams represent a novel growth pattern of GBM. In this study we uncovered the cellular and molecular mechanism that regulates the oncostreams function in GBM growth and invasion. METHODS/STUDY POPULATION: We studied oncostreams organization and function using genetically engineered mouse gliomas models (GEMM), mouse primary patient derived GBM model and human glioma biopsies. We evaluated the molecular landscape of oncostreams by laser capture microdissection (LCM) followed by RNA-Sequencing and bioinformatics analysis. RESULTS/ANTICIPATED RESULTS: Oncostreams are multicellular structures of 10-20 cells wide and 2-400 μm long. They are distributed throughout the tumors in mouse and human GBM. Oncostreams are heterogeneous structures positive for GFAP, Nestin, Olig2 and Iba1 cells and negative for Neurofilament. Using GEMM we found a negative correlation between oncostream density and animal survival. Moreover, examination of patient’s glioma biopsies evidenced that oncostreams are present in high grade but no in low grade gliomas. This suggests that oncostreams may play a role in tumor malignancy. Our data also indicated that oncostreams aid local invasion of normal brain. Transcriptome analysis of oncostreams revealed 43 differentially expressed (DE) genes. Functional enrichment analysis of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented GO biological process. Network analysis indicated that Col1a1, ACTA2, MMP9 and MMP10 are primary target genes. These genes were also overexpressed in more malignant tumors (WT-IDH) compared to the less malignant (IDH1- R132H) tumors. Confocal time lapse imagining of 3D tumor slices demonstrated that oncostreams display a collective motion pattern within gliomas that has not been seen before. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, oncostreams are anatomically and molecularly distinctive, regulate glioma growth and invasion, display collective motion and are regulated by the extracellular matrix. We propose oncostreams as novel pathological markers valuable for diagnosis, prognosis and designing therapeutics for GBM patients.

Abstract Mesenchymal gliomas are the most aggressive tumors that carry the worst prognosis. The origins of mesenchymal cells within brain tumors, remains poorly understood. They could originate either from invading mesenchymal cells, from perivascular smooth muscle actin+ cells, or from a mesenchymal transformation of tumor cells. Identifying the origin and function of mesenchymal cells within gliomas is essential as these cells contribute to increased glioma aggressiveness and tumor progression. In this study we used human biopsies and implantable and genetically engineered mouse models (GEMM) of GBM to study tumor mesenchymal transformation. GBM implantable models were used to analyze the molecular landscape by laser microdissection followed by RNA-Seq and bioinformatics analysis. Time lapse confocal imagining was implemented to analyze GBM cells dynamics. Our results indicate the existence of a complex intratumoral and peritumoral dynamic organization of glioma cells (i.e., Oncostreams). Multicellular structures of elongated cells compatible with mesenchymal differentiation. These structures play important roles in intratumoral movements, peritumoral invasion of normal brain, and overall glioma progression. We also show that oncostreams are molecularly distinct and display increased expression of mesenchymal genes such as Col1a1. Knocking down of Col1a1 in a GEMM of aggressive gliomas reduced tumor progression and significantly increased animal survival. Histological examination confirmed absence of Col1a1, and absence of morphologically identifiable oncostreams. Our results show that tumor cells, especially within oncostreams, display a fibroblastic-like morphology and express proteins typical of mesenchymal cells. The knockout of Col1a1 from tumoral cells eliminated oncostreams from tumors and delayed tumor progression. These data suggest that tumor cells expressing mesenchymal genes regulate the organization of mesenchymal multicellular structures, and determine glioma progression. We propose that inhibiting mesenchymal transformation of glioma cells will assist in the treatment of glioblastoma.

Abstract There is currently much excitement for the use of immunotherapies in cancer. In spite of positive results using checkpoint inhibitors in melanoma and CAR T cells in leukemias, these strategies have not yet achieved robust clinical responses in human gliomas. A powerful inhibitory microenvironment is thought to be the culprit. Mechanisms that determine the inhibitory microenvironment remain poorly understood. Herein we demonstrate that FYN, a downstream target of receptor tyrosine kinases signaling, inhibits the anti-glioma immune response. We utilized genetically engineered mouse models (GEMM) of glioma based on the Sleeping Beauty Transposon method. We examined the activities of FYN in NP (N-ras + shp53), NPA (NP + shATRX), and NPD (NP + PDGF overexpression) tumors. We also generated FYN knockdown glioma stem cells in vitro to induce gliomas in immune-competent and immune-deficient mice of varying genetic backgrounds (NSG, CD8-/-). Flow cytometry was used to characterize immune cells within the glioma microenvironment. Our results show that FYN knockdown in NP, NPA, or NPD GEMM of glioma reduced tumor progression and increased survival by 25–77%. GSEA analysis of differential expressed genes of WT vs. FYN knockdown gliomas revealed enrichments of gene ontologies related to immune functions. In NSG and CD8-/- immune-deficient mice, FYN knockdown failed to inhibits tumor growth and increase animal survival. These results suggest that FYN enhances tumor progression through changes in anti-glioma immune activation. Examination of tumor immune infiltrates by flow cytometry indicate a 50–70% reduction in powerful immune inhibitory myeloid derived cells (MDSCs) with no changes in the total level of CD8+ and CD4+ cells. Our results show for the first time that FYN reduces anti-glioma immune responses, likely through a reduction in inhibitory MDSCs. The specific inhibition of FYN exclusively within glioma cells, could improve the efficacy of anti-glioma immunotherapies.

An inadequate immune response is increasingly recognized as a central element in the pathogenesis of high-grade glioma. Based on our prior work, we hypothesized that dendritic cell dysfunction plays a key role in poor anti-brain tumor immunity. To stimulate a robust immune response against high-grade glioma, we developed a strategy to recruit dendritic cells to the brain and induce tumor cytotoxicity. We chose Flt3L to attract dendritic cells to the brain and HSV1-TK (plus valacyclovir) to kill tumor cells and make antigens available to dendritic cells. Studies in animal models of glioma reveal that Flt3L and HSV1-TK combination therapy results in infiltration of gliomas by dendritic cells, generation of immune cytotoxicity and memory, and the recognition of tumor neoantigens by immune cells. Based on compelling preclinical data, we executed a first-in-human phase I dose escalation trial of HSV1-TK or Flt3L dual adenoviral therapy for the treatment of newly diagnosed malignant gliomas. Vectors were injected into the tumor cavity following resection. The trial consisted of a dose escalation of both vectors, starting at 1 × 10∧9 iu and increasing to 1 × 10∧11 iu through a total of 6 combinations administered to 6 cohorts of 3 patients each. Two cycles of 14 d each of valacyclovir were administered to activate HSV1-TK cytotoxicity on postoperative days 1 and 56. All patients received Stupp protocol chemoradiation. The experimental treatment was well tolerated, and at this time the MTD has not been reached. Additionally, preliminary analysis suggests that the dual-vector therapy provides a potent survival advantage when evaluated against contemporary and historical controls. Updated progression free survival and overall survival, AEs, and SAEs will be communicated at the time of presentation. Flt3L/HSV1-TK dual vector immunotherapy is well tolerated and may prolong survival in high-grade glioma patients. Further evaluation in a larger-scale, multi-center trial is justified.

GBM remains the deadliest primary malignant brain tumor. Given the importance of invading cells, less attention has been paid to the tumor mass, even if such a mass eventually kills the patient. We previously demonstrated that human and mouse transplantable or GEMM gliomas display regular anatomical multicellular structures containing elongated cells which we named ‘oncostreams’. Oncostreams are 10–20 cells wide, 2–400 um long, and are distributed throughout the tumors. Furthermore, we uncovered a negative correlation between oncostream density and animal survival suggesting that oncostreams play a role in tumor malignancy. Further data indicate that oncostreams aid local invasion of normal brain. Co-implantation experiments demonstrated that oncostreams facilitate the intratumoral spread of slow migrating cells. To determine a possible molecular distinctiveness of oncostreams we used laser capture microdissection followed by RNA-Seq, bioinformatics and network analysis. Evaluation of the transcriptome demonstrated differential expression (DE) of genes between oncostreams and adjacent tumor. Functional enrichment of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented gene ontologies. Network analysis indicated that Col1a1, ACTA2, MMP9, MMP10 and ADAMTS2, genes important for cell migration and ECM interactions, are part of these networks. IHC and PCR were used to validate RNA-Seq expression changes. To understand the cellular dynamics in our system we used time lapse imagining and evaluated the results using high level statistical analyses. The following tests, i.e., velocity distribution, pair-wise correlation of local position, velocity correlation, etc. were used to characterize the dynamics of cellular intratumoral motility. These data demonstrate the existence of collective motion within glioma tumors. Taken together, our results show that oncostreams, move by collective motion, are anatomically and molecularly distinct, reveal the existence of glioma self-organization, and regulate glioma growth and invasion. Targeting oncostreams is a candidate therapeutic strategy.

Background:Glioblastoma (GBM) is an aggressive and highly vascular tumor with median survival below 2 years. Despite advances in surgery, radiotherapy, and chemotherapy, survival has improved modestly. To combat glioma vascular proliferation, anti-angiogenic agents targeting vascular endothelial growth factor (VEGF) were introduced. Preclinically these agents were effective, yet they did not improve overall survival in phase III trials. We tested the hypothesis that ganciclovir (GCV)-mediated killing of proliferating endothelial cells expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) would have direct antitumor effects, and whether vessel ablation would affect the antitumor activity of anti-VEGF antibodies and radiotherapy.Methods:Proliferating endothelial cells were eliminated using GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK (in Tie2-TK-IRES-GFP mice). Syngeneic NRAS/p53 (NP) gliomas were implanted into the brains of Tie2-TK-IRES-GFP mice. Endothelial proliferation activates the Tie2 promoter and HSV1-TK expression. Administration of GCV kills proliferating tumor endothelial cells and slows tumor growth. The effects of endothelial cell ablation on anti-angiogenic therapy were examined using anti-VEGF antibodies or irradiation.Results:GCV administration reduced tumor growth and vascular density, increased tumor apoptosis, and prolonged survival. Anti-VEGF antibodies or irradiation also prolonged survival. Surprisingly, combining GCV with irradiation, or with anti-VEGF antibodies, reduced their individual therapeutic effects.Conclusion:GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK, anti-VEGF antibodies, or irradiation all reduced growth of a murine glioma. However, elimination of microvascular proliferation decreased the efficacy of anti-VEGF or irradiation therapy. We conclude that, in our model, the integrity of proliferating vessels is necessary for the antiglioma effects of anti-VEGF and radiation therapy.