The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes ( Bmp4 , Crim1 , Degs1 , Gja1 , Itgav , Mfap3l , Pdpn , Ptgds , Rbp1 , Rnf13 , Rpe65 , Slc4a2 , Sulf1 and Ttr ) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (p adj ≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 ( Lcn2 ) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b . Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

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.

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.

PURPOSE. The orbit displays unique vulnerability to inflammatory conditions. The most prevalent of these conditions, thyroid eye disease (TED), occurs in up to 50% of patients with Graves' disease (GD). Whereas the pathology of both TED and GD is driven by autoantibodies, it is unclear why symptoms manifest specifically in the orbit. METHODS. We performed retinoic acid treatment on both normal and TED patient-derived orbital fibroblasts (OFs) followed by mRNA and protein isolation, quantitative realtime polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, RNA sequencing, and Western blot analyses. RESULTS. Both normal and TED patient-derived OFs display robust induction of monocyte chemoattractant protein 1 (MCP-1) upon retinoid treatment; TED OFs secrete significantly more MCP-1 than normal OFs. In addition, pretreatment of OFs with thiophenecarboxamide (TPCA-1) inhibits retinoid-induced MCP-1 induction, suggesting an NF-kappa B (nuclear factor kappa-light-chain-enhancer of activated B cells)-dependent mechanism. We also found that treatment with cholecalciferol (vitamin D3) mitigates MCP-1 induction, likely because of competition between retinoic acid receptors (RARs) and vitamin D receptors (VDR) for their common binding partner retinoid nuclear receptors (RXRs). CONCLUSIONS. Retinoids that naturally accumulate in orbital adipose tissue can act on orbital fibroblasts to induce the expression of inflammation-associated genes. These data suggest a potential role for retinoids in sensitizing the orbit to inflammation.

Severely damaged adult zebrafish extraocular muscles (EOMs) regenerate through dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. Members of the Twist family of basic helix-loop-helix transcription factors (TFs) are key regulators of the epithelial-mesenchymal transition (EMT) and are also involved in craniofacial development in humans and animal models. During zebrafish embryogenesis, twist family members (twist1a, twist1b, twist2, and twist3) function to regulate craniofacial skeletal development. Because of their roles as master regulators of stem cell biology, we hypothesized that twist TFs regulate adult EOM repair and regeneration. In this study, utilizing an adult zebrafish EOM regeneration model, we demonstrate that inhibiting twist3 function using translation-blocking morpholino oligonucleotides (MOs) impairs muscle regeneration by reducing myocyte dedifferentiation and proliferation in the regenerating muscle. This supports our hypothesis that twist TFs are involved in the early steps of dedifferentiation and highlights the importance of twist3 during EOM regeneration.

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.

Zebrafish extraocular muscles regenerate after severe injury. Injured myocytes dedifferentiate to a mesenchymal progenitor state and reenter the cell cycle to proliferate, migrate, and redifferentiate into functional muscles. A dedifferentiation process that begins with a multinucleated syncytial myofiber filled with sarcomeres and ends with proliferating mononucleated myoblasts must include significant remodeling of the protein machinery and organelle content of the cell. It turns out that autophagy plays a key role early in this process, to degrade the sarcomeres as well as the excess nuclei of the syncytial multinucleated myofibers. Because of the robustness of the zebrafish reprogramming process, and its relative synchrony, it can serve as a useful in vivo model for studying the biology of autophagy. In this chapter, we describe the surgical muscle injury model as well as the experimental protocols for assessing and manipulating autophagy activation.

Purpose: Genomic reprogramming and cellular dedifferentiation are critical to the success of de novo tissue regeneration in lower vertebrates such as zebrafish and axolotl. In tissue regeneration following injury or disease, differentiated cells must retain lineage while assuming a progenitor-like identity in order to repopulate the damaged tissue. Understanding the epigenetic regulation of programmed cellular dedifferentiation provides unique insights into the biology of stem cells and cancer and may lead to novel approaches for treating human degenerative conditions. Methods: Using a zebrafish in vivo model of adult muscle regeneration, we utilized chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to characterize early changes in epigenetic signals, focusing on three well-studied histone modifications—histone H3 trimethylated at lysine 4 (H3K4me3), and histone H3 trimethylated or acetylated at lysine 27 (H3K27me3 and H3K27Ac, respectively). Results: We discovered that zebrafish myocytes undergo a global, rapid, and transient program to drive genomic remodeling. The timing of these epigenetic changes suggests that genomic reprogramming itself represents a distinct sequence of events, with predetermined checkpoints, to generate cells capable of de novo regeneration. Importantly, we uncovered subsets of genes that maintain epigenetic marks paradoxical to changes in expression, underscoring the complexity of epigenetic reprogramming. Conclusions: Within our model, histone modifications previously associated with gene expression act for the most part as expected, with exceptions suggesting that zebrafish chromatin maintains an easily editable state with a number of genes paradoxically marked for transcriptional activity despite downregulation.

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.

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

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.