[This corrects the article on p. 1112 in vol. 17, PMID: 41970562.].

The ability to identify tumour tissue in a label-free, contactless, and real-time manner is much needed in tumour resection surgery. Current techniques cause interruptions to surgical flow and have high false positive rates, which can cause collateral damage to healthy brain tissue. We propose laser light scattering techniques, such as diffuse correlation spectroscopy and laser speckle contrast imaging, to image mechanical stiffness differences in the brain’s surface associated with tumour tissue. We validate the optimal processing technique quantitatively with a controlled experiment in which paraformaldehyde was used to induce a change in tissue stiffness in ex vivo mouse brains. We then demonstrate that the technique applies to tumour localisation using ex vivo mouse models with real tumours. Qualitative comparisons with magnetic resonance imaging indicate accurate tumour localisation using only surface stiffness changes to underlying tumours. We also demonstrate sub-millimetre precision when imaging brain slices.

Background:Glioblastomas (GBM) are highly invasive tumors with marginal regions comprising unresectable functional brain infiltrated by tumor cells. Effective drug delivery to these regions is crucial, but lack of understanding of the structural and functional characteristics of their vasculature is impeding drug development. We aimed to develop a bespoke analytical pipeline that could be used to characterize molecular and morphological features of the blood-brain barrier within marginal regions of GBM by analyzing image descriptors extracted from multiplex colorimetric imaging of human samples. Methods:Multiplex immunohistochemical consecutive staining of key vascular antigens was performed on human samples of GBM and adjacent brain to determine the morphology and composition of blood vessels. A neural network was utilized to segment the vessels, and multiple image descriptors extracted to characterize and classify them with a Linear Discriminant model. Results:Multiplexed immunohistochemistry was optimized for vessel related-antigens CD31, laminin, claudin-5, smooth muscle actin, platelet-derived growth factor beta, and glial fibrillary acidic protein. Multiple parameters analyzed from the segmented blood vessels were modeled into four distinct categories, linked to region-specific molecular and morphological factors. Margin regions exhibited the lowest vessel density and a heterogenous mix of vessels with some unique to the region and others similar to tumor core or normal brain vessels. Conclusions:We established a multiplex immunohistochemical staining protocol and pipeline to identify blood vessels and analyze their composition. The pipeline and the preliminary quantitative data it generated will facilitate more comprehensive characterization of margin-specific blood vessels, with implications for drug development.

BACKGROUND:Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking.METHODS:Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging.RESULTS:High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging.CONCLUSIONS:We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

Summary of the properties of BDP-9066

Supplementary figure S1 displays examples of flow cytometry gating for FACS sorted cell populations, abrogation of Chk1 phosphorylation by VE-821 in GSC and patterns of γ-H2AX staining produced by replication stress.

Supplemental Table 1 Summary table of in vivo growth characteristics of E2, G7, R10, R15, R24, R9 and S2 GSC primary GBM cultures after intracranial injection in CD1 nude mice. Supplemental Table 2 List of primary antibodies utilised Supplemental Table 3 List of secondary antibodies utilised

Confirmation of tumour volume (MRI), levels of circulating BDP-9066 and CT guided RT

Supplementary Figure S5 shows additional immunofluorescent staining images and quantification in human GBM samples

Supplementary Figure S3 shows additional immunofluorescent staining and conventional IHC in GBM intracranial xenografts.

Supplementary Figure S4 shows additional images of immunofluorescent staining in GBM intracranial xenografts.

Supplementary Figure S2 shows additional data obtained during aphidicolin studies detailed in Fig 1 and additional data detailing expression of genes associated with ongoing and stalled replication forks from figure 3.

Legends for supplementary figures and tables

AIMS Glioma stem cells (GSC) show high levels of DNA replication stress and exhibit abnormal S phase in comparison to differentiated, non GSC tumour cells. We investigated the DNA replication phenotypes of GSC and mechanism of GSC specific cytotoxicity following DNA replication stress response inhibition with combined ATR/PARP inhibition (CAiPi). METHOD Paired GSC enriched (‘GSC’) and GSC deplete differentiated (‘bulk’) populations were cultured from resected GBM specimens and maintained in neurobasal media with growth factors or serum containing media respectively. Cell viability, neurosphere and clonogenic assays were used to assess cytotoxicity whilst DNA replication was interrogated utilising immunofluorescent repair foci, flow cytometry and DNA fibre assay. CAiPi consisted of VE821 (ATRi) with Olaparib (PARPi). RESULTS GSC show reduced DNA replication rates and alterations in cell cycle profile and demonstrate extreme sensitivity to CAiPi. CAiPi resulted in significantly elevated levels of 53BP1 nuclear bodies in GSC indicating genomic under- replication, however did not preferentially reduce fork speed compared to non-GSC. Analysis of replication fork structures revealed an increase in new origin firing in GSC dependent upon PARP trapping. GSC cytotoxicity was reduced by roscovitine induced inhibition of origin firing, suggesting an important role of dysregulated origin firing in CAiPi response. CAiPi is also potently radiosensitising by clonogenic assay. CONCLUSION Taken together these data suggest that GSC are vulnerable to dysregulation of origin firing which results in under-replication of their genome and reliance upon G1 cell cycle resolution of DNA damage. We propose that CAiPi is an attractive therapeutic strategy for translation to the clinic.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.

Introduction and Objectives Pulmonary fibroblasts respond to environmental signals triggered by injury or infections, shaping subsequent responses in the lung. Fibroblasts may contribute to enhanced immune protection, or chronic pathogenic inflammation and fibrosis. We hypothesise that molecules upregulated by lung fibroblasts early following influenza A virus (IAV) infection and bleomycin-induced injury persist, in order to generate/maintain immune memory, via altered stromal-immune cell communication. Methods To address this, we performed RNA-seq on FACS sorted lung fibroblasts from naïve animals and at early (day 10) and late time points (day 40) following intranasal IAV infection. Transcriptional changes were compared with the bleomycin model of early lung injury (publicly available RNA-seq). The functional profile and location of injury altered lung stromal and immune cells was determined using flow cytometry and immunohistochemistry. Results Analysis of differentially expressed genes demonstrated an enrichment in cell cycle and extracellular matrix genes at day 10 post IAV infection (FDR < 0.05), consistent with fibroblast activation profiles in the bleomycin-injured lung. Three distinct lung fibroblast populations were identified using flow cytometry: damage-responsive (DRF), interferon-responsive (IRF), and antigen-presenting fibroblasts (APF). DRF were significantly elevated in both models at day 10 post injury, while IRF were only detectable in the IAV lung. Interestingly, APF were reduced in the bleomycin lung compared to naïve controls. Furthermore, immunohistochemistry demonstrated that expression of the immunomodulatory molecule, podoplanin, was found in close proximity to immune cell infiltrates in the lung in both models. Conclusions These data have important implications for understanding the altered communications between immune and stromal cells during and following subsequent lung infections and injury/fibrotic responses.

Abstract AIMS Gliomas have high levels of DNA replication stress and clinical trials of inhibitors of the key replication stress response protein ATR (ataxia telangiectasia and rad 3 related protein) as radiosensitisers are planned. We aimed to investigate the effect of ATR inhibition on the ability of glioma cells to infiltrate and invade the brain. METHOD Live cell imaging in a panel of primary glioma cultures following siRNA or pharmacological inhibition of ATR. Invasion following treatment of murine orthotopic gliomas was determined by immunohistochemistry. Intravital imaging of GFP expressing murine orthotopic xenografts via an intracranial window model of glioma was undertaken. RESULTS Invading margins of human glioma samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated reduced cell velocity following ATR inhibition (Berzosertib/BAY1895344) or siRNA. Cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures which engulf high molecular weight dextran, compatible with blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed sequestration of integrins within macropinosomes and reduced integrin cycling. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further in vivo study demonstrated a reduction in invading tumour cells. CONCLUSION We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin recycling in GBM cells which results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.

Abstract BACKGROUND Evidence suggests treatment resistant glioma stem cells (GSCs) drive glioblastoma (GBM) recurrence. Current treatments fail to eradicate GSC and novel GSC targeting therapies are a priority. GSC exhibit elevated DNA replication stress (RS) versus non GSC tumour cells driving constitutive DNA damage response (DDR) activation and efficient DNA repair. We previously demonstrated that targeting RS response with combined ATR and PARP inhibition (CAiPi) (VE821 and Olaparib) provides potent GSC specific cytotoxicity. In this study we investigated the underlying DDR phenotype which determines this vulnerability. RESULTS Paired GSC enriched (‘GSC’) and GSC depleted, differentiated (‘bulk’) populations were cultured from GBM specimens in neurobasal media with growth factors or serum containing media respectively. GSC exhibited reduced survival following exposure to CAiPi versus bulk. CAiPi significantly increased 53BP1 G1 phase nuclear bodies (53BP1NBs) in GSC, which are known to shield under-replicated DNA in actively transcribed genes. Mapping the genomic distribution of endogenously occurring replication dependent DNA double strand breaks via Breaks Ligation In Situ Sequencing (BLISS), revealed reduced intragenic DSB in long actively transcribed genes in GSC versus bulk at baseline, suggesting a reliance upon transcription coupled DSB repair in GSC. RNA seq demonstrated CAiPi-induced transcriptomic alterations in GSC including replication regulation and initiation. DNA fibre assay showed that CAiPi increased GSC new origin firing which correlated with PARP trapping. GSCs were rescued from CAiPi by roscovitine induced inhibition of excess origin firing. CAiPi is potently radiosensitizing by clonogenic assay and we demonstrated murine blood brain barrier penetration of CAiPi utilising VE822 and pamiparib in vivo. CONCLUSION Dysregulation of origin firing by CAiPi exposes a GSC specific vulnerability which results in DNA under-replication and abrogation of proficient DNA repair seen at long actively transcribed genes and has potential to be clinically translated as a GSC specific cytotoxic therapy.

Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour and remains incurable despite decades of research. GBM are characterised by highly infiltrative growth patterns that contribute to the profound cognitive and neurological symptoms experienced by patients, and to inevitable recurrence following treatment. Novel treatments that reduce infiltration of the healthy brain have potential to ameliorate clinical symptoms and improve survival. Here, we report a novel role of the Ataxia telangiectasia and Rad 3 related kinase (ATR) in supporting the invasive properties of GBM cells through the regulation of macropinocytosis-driven internalisation of integrin adhesion receptors. We demonstrate that inhibition of ATR opposes GBM migration in vitro, and correspondingly reduces infiltrative behaviour in orthotopic mouse models. These results indicate that ATR inhibition, in addition to its use as a radiosensitiser, may be effective in reducing GBM infiltration and its associated symptoms.