Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations

Molecular and Genetic Determinants of Glioma Cell Invasion.

A diffusely invasive nature is a major obstacle in treating a malignant brain tumor, "diffuse glioma", which prevents neurooncologists from surgically removing the tumor cells even in combination with chemotherapy and radiation. Recently updated classification of diffuse gliomas based on distinct genetic and epigenetic features has culminated in a multilayered diagnostic approach to combine histologic phenotypes and molecular genotypes in an integrated diagnosis. However, it is still a work in progress to decipher how the genetic aberrations contribute to the aggressive nature of gliomas including their highly invasive capacity. Here we depict a set of recent discoveries involving molecular genetic determinants of the infiltrating nature of glioma cells, especially focusing on genetic mutations in receptor tyrosine kinase pathways and metabolic reprogramming downstream of common cancer mutations. The specific biology of glioma cell invasion provides an opportunity to explore the genotype-phenotype correlation in cancer and develop novel glioma-specific therapeutic strategies for this devastating disease

Hypoxic Cell Waves around Necrotic Cores in Glioblastoma: A Biomathematical Model and its Therapeutic Implications

Glioblastoma is a rapidly evolving high-grade astrocytoma that is distinguished pathologically from lower grade gliomas by the presence of necrosis and microvascular hiperplasia. Necrotic areas are typically surrounded by hypercellular regions known as "pseudopalisades" originated by local tumor vessel occlusions that induce collective cellular migration events. This leads to the formation of waves of tumor cells actively migrating away from central hypoxia. We present a mathematical model that incorporates the interplay among two tumor cell phenotypes, a necrotic core and the oxygen distribution. Our simulations reveal the formation of a traveling wave of tumor cells that reproduces the observed histologic patterns of pseudopalisades. Additional simulations of the model equations show that preventing the collapse of tumor microvessels leads to slower glioma invasion, a fact that might be exploited for therapeutic purposes.Comment: 29 pages, 9 figure

Reactive astrocytes potentiate tumor aggressiveness in a murine glioma resection and recurrence model

Surgical resection is a universal component of glioma therapy. Little is known about the postoperative microenvironment due to limited preclinical models. Thus, we sought to develop a glioma resection and recurrence model in syngeneic immune-competent mice to understand how surgical resection influences tumor biology and the local microenvironment

Rho-associated kinase signalling and the cancer microenvironment: novel biological implications and therapeutic opportunities

The Rho/ROCK pathway is involved in numerous pivotal cellular processes that have made it an area of intense study in cancer medicine, however, Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors are yet to make an appearance in the clinical cancer setting. Their performance as an anti-cancer therapy has been varied in pre-clinical studies, however, they have been shown to be effective vasodilators in the treatment of hypertension and post-ischaemic stroke vasospasm. This review addresses the various roles the Rho/ROCK pathway plays in angiogenesis, tumour vascular tone and reciprocal feedback from the tumour microenvironment and explores the potential utility of ROCK inhibitors as effective vascular normalising agents. ROCK inhibitors may potentially enhance the delivery and efficacy of chemotherapy agents and improve the effectiveness of radiotherapy. As such, repurposing of these agents as adjuncts to standard treatments may significantly improve outcomes for patients with cancer. A deeper understanding of the controlled and dynamic regulation of the key components of the Rho pathway may lead to effective use of the Rho/ROCK inhibitors in the clinical management of cancer

Surveilling the Distinctive Vascular and Metabolic Features of Tumor Progression and Response to Therapy

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Despite maximal treatment with surgical resection, radiotherapy and temozolomide chemotherapy, prognosis is dismal with median survival around 15 months. GBMs are highly infiltrative tumors that invade into surrounding brain tissue, which makes defining the extent of tumor spread difficult and recurrence common. Radiological identification of GBMs with magnetic resonance imaging (MRI), using transvers (T2) or longitudinal (T1) relaxation contrasts, is a mainstay in the initial diagnosis as well as tracking therapeutic response in GBM. However, there is extreme variability in the structural appearance, size, metabolism, and genetic landscape of GBMs, making imaging characteristics highly heterogeneous and hard to define with tumor progression. Although T2-weighted and contrast-enhanced T1-weighted MRI provides anatomical details of the tumor architecture, these methods can be confounded by pseudoprogression and pseudoresponse in the context of therapy.The GBM microenvironment is characterized by immature vasculature and extracellular acidification due to a metabolic shift towards aerobic glycolysis (Warburg effect). The reduced extracellular pH (pHe) has been associated with promoting angiogenesis and invasion as well as creating an immunosuppressive environment. Given the important contribution of vascular changes and extracellular acidosis to shaping the tumor microenvironment, advanced MRI techniques are needed to better characterize the tumor microenvironment to provide more specific readouts of tumor progression and therapeutic response. Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) is a magnetic resonance spectroscopic imaging (MRSI) technique that utilizes the temperature and pH-dependent hyperfine shifts of paramagnetic agents (e.g., TmDOTP5-) for high resolution, three-dimensional, quantitative temperature and pHe mapping. BIRDS has been used to demonstrate the acidic pH in preclinical models of GBM, where the intratumoral space is highly acidified (pH\u3c6.8) in comparison to healthy brain tissue (pH~7.2) and acidic pH spread beyond the anatomically defined tumor core relates to the invasiveness of the tumors. However, a limitation of the BIRDS technique is the necessity of detectable (\u3e1 mM) levels of contrast agent, which are cleared rapidly by the kidney. To obviate need for surgical intervention (e.g., renal ligation) to stop rapid agent clearance, here we demonstrate that pharmacological inhibition of renal clearance of these agents using probenecid to allow for longitudinal imaging of pHe throughout tumor progression and show that acidosis develops early in tumor progression in human-derived GBM tumors (U87 and U251). Since other tomographic pHe mapping methods are non-quantitative and directly altering pHe in a specific tissue is difficult to implement, we looked to assess the BIRDS-based temperature measurements for verification of the quantitative BIRDS readout. A localized cooling system was used to induce hypothermia in sheep brain to levels suggested to be neuroprotective in hypoxic states. Quantitative temperature mapping using BIRDS showed significantly decreased cerebral temperatures with cooling over all defined brain regions and was in agreement with thermocouple measurements. While pHe is a useful metric, tumor vascularity also shapes tumor metabolism and the microenvironment. BIRDS can be combined with other imaging modalities such as dynamic contrast enhanced (DCE) MRI, which allows quantification of vascular parameters (e.g., permeability) through modeling the dynamic uptake of Gd3+-based contrast agents. Multiparametric characterization of the spatiotemporal changes in cellularity, vascularity and acidosis of U87 and U251 tumors throughout progression showed unique patterns that could be used to identify tumor features and differentiate between tumor types. Finally, pHe readouts have potential as a biomarker of therapeutic response. After finding an increase in pHe after treatment with temozolomide in U251 tumors, we used BIRDS longitudinally to demonstrate normalization of pHe in U87 tumors treated with sorafenib, a nonselective tyrosine kinase inhibitor. Both treatments slowed tumor progression and led to increases of pHe which establishes a role for pHe imaging as an early and sensitive marker of evaluating therapeutic response prior to observable changes in the tumor appearance on standard MRI. The potential of BIRDS is vast and not limited to GBM, or cancer in general. Additional work has demonstrated that an acidic pHe is not limited to preclinical tumor models, but is also found in patient-derived xenograft (PDX) models of metastatic melanoma in the brain. BIRDS can also be utilized in evaluating tumors in any organ, as BIRDS has also shown acidic pHe in models of liver cancer. In summary, this work further expands BIRDS into a broadly applicable longitudinal platform for characterization of the tumor microenvironment and may aid in evaluation of many targeted therapeutic strategies

Metabolism within the tumor microenvironment and its implication on cancer progression: an ongoing therapeutic target

Since reprogramming energy metabolism is considered a new hallmark of cancer, tumor metabolism is again in the spotlight of cancer research. Many studies have been carried out and many possible therapies have been developed in the last years. However, tumor cells are not alone. A series of extracellular components and stromal cells, such as endothelial cells, cancer-associated fibroblasts, tumor-associated macrophages and tumor-infiltrating T cells, surround tumor cells in the so-called tumor microenvironment. Metabolic features of these cells are being studied in deep in order to find relationships between metabolism within the tumor microenvironment and tumor progression. Moreover, it cannot be forgotten that tumor growth is able to modulate host metabolism and homeostasis, so that tumor microenvironment is not the whole story. Importantly, the metabolic switch in cancer is just a consequence of the flexibility and adaptability of metabolism and should not be surprising. Treatments of cancer patients with combined therapies including anti-tumor agents with those targeting stromal cell metabolism, anti-angiogenic drugs and/or immunotherapy are being developed as promising therapeutics.Mª Carmen Ocaña is recipient of a predoctoral FPU grant from the Spanish Ministry of Education, Culture and Sport. Supported by grants BIO2014-56092-R (MINECO and FEDER), P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

Molecular mechanisms of therapy resistance and recurrence in glioblastoma multiforme

Glioblastoma multiforme (GBM) WHO grade 4 is the most malignant and frequent primary brain tumor with a five-year overall survival rate of 9.8%. Virtually every time, the standard treatment with surgery and radio-/chemotherapy applying temozolomide (TMZ) fails, and the tumor progresses, develops resistance mechanisms, and recurs. Thus, it is crucial to further expand the research field for a proper understanding of those mechanisms enabling deeper classifications, the assembly of combinatorial biomarkers, and the development of new therapeutic approaches to overcome therapy resistances. The first publication thematizes TMZ-resistance mechanisms based on the tumor’s changed metabolism and pH-value regulation. Carbonic anhydrase 2 (CA2) was previously identified as highly upregulated in TMZ-resistant glioblastoma stem-like cells (GSCs) as well as recurrent GBM tissue samples. Here, the metalloenzyme was functionally analyzed. Increased oxygen consumption and extracellular acidification rates were detected in CA2-overexpressing GBM cell lines induced via CRISPR/Cas9. This observation underlines the known function of the proton exchanger to enhance aerobic glycolysis (Warburg effect) and proton efflux, thereby leading to extracellular acidification and a pro-invasive and pro-inflammatory microenvironment in cancer. The pan-CA inhibitor acetazolamide (ACZ) repressed the oxygen consumption and extracellular acidification rates more efficiently than the more selective CA2 inhibitor brinzolamide (BRZ) in CA2-overexpressing GBM cells, indicating the crucial role of additional CA isozymes mediating tumor pH regulation and contributing to the Warburg effect. On the contrary, BRZ reduced infiltrative rates and augmented TMZ-induced autophagy causing cell death more efficiently than ACZ in CA2-overexpressing cells, CA2-highly expressing GSCs and TMZ-resistant GSCs. Those data provide evidence for a BRZ-induced pH shift to a physiological level, optimizing the conditions for an efficient TMZ treatment. In the second publication, signaling pathways contributing to tumor progression and resistance were examined for the influence of the disintegrin and metalloproteinase 8 (ADAM8). Next to its ability to cleave extracellular matrix proteins thereby shaping the tumor microenvironment, ADAM8 is characterized as a multi-domain enzyme and can bind to integrins thereby modulating various signaling pathways. Here, the mechanism of ADAM8 regulating miRNA expression profiles through intra- and extracellular signaling was investigated. Several dysregulated miRNAs were identified, comparing GBM cells with endogenous ADAM8 expression or a CRISPR/Cas9-induced ADAM8 knockout. In GBM, miR-181a-5p is described as a tumor-suppressor. Here, this miRNA was identified being upregulated in ADAM8 knockout cells. Mechanistically, ADAM8 was found to mediate miR-181a-5p repression via STAT3 and MAPK signaling. MiR-181a-5p mimic transfection suppressed GBM cell proliferation partially through the post-transcriptional inhibition of MEK1/2, ERK1/2, and CREB-1, indirectly targeting MMP9. Breaking down the ADAM8-mediated signaling involving miR-181a-5p, an ADAM8/STAT3/miR-181a-5p/osteopontin and ADAM8/ERK1/2/CREB-1/miR-181a-5p axis with a negative feedback loop targeting MMP9 was examined. Upon loss of ADAM8 expression, enriched miR-181a-5p levels were detected in extracellular vesicles. Thus, the ADAM8-mediated repression of miR-181a-5p could prevent surrounding cells from MMP9 repression, stimulating invasion and proliferation. Clinically, miR-181a-5p was detected in serum-EVs and downregulated in GBM tissue samples compared to ADAM8, which correlated with MMP9 mRNA expression. The versatile influences of ADAM8 on progression- and resistance-driving signaling pathways, also via miRNA regulation, justifies further experimental approaches targeting ADAM8 in GBM. Lastly, the miRNA expression patterns of three patient-derived GSCs compared to their differentiated astrocytic states were investigated. Thirty-one significantly changed miRNAs were identified, including ten highly dysregulated miRNAs involved in GBM progression and stem cell fate. Among them, miR-425-5p was highly expressed in GSCs, and miR-425-5p mimic transfection reduced the PTEN and GFAP protein expression in patient-derived GBM cell lines, suggesting to induce the GSC phenotype partially. A miRNA-target relationship KEGG enrichment analysis revealed a section of the dynamic miRNA/signaling network deciding about stem cell fate. Thus, we further classified the cellular subtype GSCs revealing potential targets to stimulate GSC differentiation, which could modulate the sensitivity towards GBM treatment strategies