A deep convolutional neural network for segmentation of whole-slide pathology images identifies novel tumour cell-perivascular niche interactions that are associated with poor survival in glioblastoma.

BACKGROUND: Glioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. However, a spatial characterisation of gene signatures and the cell types expressing these in different tumour locations is still lacking. METHODS: We have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA). Correlation analysis between segmentation results from tumour images together with matched RNA expression data was performed to identify genetic signatures that are specific to different tumour regions. RESULTS: We found that spatially resolved gene signatures were strongly correlated with survival in patients with defined genetic mutations. Further in silico cell ontology analysis along with single-cell RNA sequencing data from resected glioblastoma tissue samples showed that these tumour regions had different gene signatures, whose expression was driven by different cell types in the regional tumour microenvironment. Our results further pointed to a key role for interactions between microglia/pericytes/monocytes and tumour cells that occur in the IT and CTmvp regions, which may contribute to poor patient survival. CONCLUSIONS: This work identified key histopathological features that correlate with patient survival and detected spatially associated genetic signatures that contribute to tumour-stroma interactions and which should be investigated as new targets in glioblastoma. The source codes and datasets used are available in GitHub: https://github.com/amin20/GBM_WSSM .

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA).

Epithelial-mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate 'indirect' miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon-Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-ß or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.

A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma.

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6-9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.

Bevacizumab-Induced Hypertension in Glioblastoma Patients and Its Potential as a Modulator of Treatment Response.

Glioblastoma invasion is the primary mechanism responsible for its dismal prognosis and is the direct result of interactions between glioblastoma cells and the tumor vasculature. The dysregulated microvasculature in glioblastoma tumors and vessels co-opted from surrounding brain tissue support rapid tumor growth and are utilized as pathways for invasive cancer cells. Attempts to target the glioblastoma vasculature with antiangiogenic agents (eg, bevacizumab) have nonetheless shown limited and inconsistent efficacy, and the underlying causes of such heterogeneous responses remain unknown. Several studies have identified that patients with glioblastoma who develop hypertension following treatment with bevacizumab show significant improvement in overall survival compared with normotensive nonresponders. Here we review these findings and discuss the potential of hypertension as a biomarker for glioblastoma treatment response in individual patients and the role of hypertension as a modulator of interactions between tumor cells and cells in the perivascular niche. We suggest that a better understanding of the actions of bevacizumab and hypertension at the cellular level will contribute to developing more effective personalized therapies that address glioblastoma tumor cell invasion.

3D-printed microplate inserts for long term high-resolution imaging of live brain organoids.

BACKGROUND: Organoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2 mm of size, which need to be constantly agitated to allow proper oxygenation. The culture conditions are, therefore, not suitable for whole-brain organoid live imaging, required to study developmental processes and disease progression within physiologically relevant time frames (i.e. days, weeks, months). RESULTS: Here we designed 3D-printed microplate inserts adaptable to standard 24 multi-well plates, which allow the growth of multiple organoids in pre-defined and fixed XYZ coordinates. This innovation facilitates high-resolution imaging of whole-cerebral organoids, allowing precise assessment of organoid growth and morphology, as well as cell tracking within the organoids, over long periods. We applied this technology to track neocortex development through neuronal progenitors in brain organoids, as well as the movement of patient-derived glioblastoma stem cells within healthy brain organoids. CONCLUSIONS: This new bioengineering platform constitutes a significant advance that permits long term detailed analysis of whole-brain organoids using multimodal inverted fluorescence microscopy.

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy.

OBJECTIVES: Targeted immunotherapies such as chimeric antigen receptor (CAR)-T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples. METHODS: Glioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets. Short-term cultures of glioma neural stem (GNS) cells were compared to cultures of healthy astrocytes and neurons using flow cytometry. Glioblastoma tissues were dissociated and analysed by high-parameter flow cytometry and single-cell transcriptomics (scRNAseq). RESULTS: Compared to normal brain, FAP was overexpressed at the gene and protein level in a large percentage of glioblastoma tissues, with highest levels of expression associated with poorer prognosis. FAP was also overexpressed in several paediatric brain cancers. FAP was commonly expressed by cultured GNS cells but absent from normal neurons and astrocytes. Within glioblastoma tissues, the strongest expression of FAP was around blood vessels. In fact, almost every tumor vessel was highlighted by FAP expression, whereas normal tissue vessels and cultured endothelial cells (ECs) lacked expression. Single-cell analyses of dissociated tumors facilitated a detailed characterisation of the main cellular components of the glioblastoma microenvironment and revealed that vessel-localised FAP is because of expression on both ECs and pericytes. CONCLUSION: Fibroblast activation protein is expressed by multiple cell types within glioblastoma, highlighting it as an ideal immunotherapy antigen to target destruction of both tumor cells and their supporting vascular network.

Combinatorial Targeting by MicroRNAs Co-ordinates Post-transcriptional Control of EMT.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, functioning in part by facilitating the degradation of target mRNAs. They have an established role in controlling epithelial-mesenchymal transition (EMT), a reversible phenotypic program underlying normal and pathological processes. Many studies demonstrate the role of individual miRNAs using overexpression at levels greatly exceeding physiological abundance. This can influence transcripts with relatively poor targeting and may in part explain why over 130 different miRNAs are directly implicated as EMT regulators. Analyzing a human mammary cell model of EMT we found evidence that a set of miRNAs, including the miR-200 and miR-182/183 family members, co-operate in post-transcriptional regulation, both reinforcing and buffering transcriptional output. Investigating this, we demonstrate that combinatorial treatment altered cellular phenotype with miRNA concentrations much closer to endogenous levels and with less off-target effects. This suggests that co-operative targeting by miRNAs is important for their physiological function and future work classifying miRNAs should consider such combinatorial effects.

A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.