We demonstrate multiple DC defects in patients with brain tumors. This includes a profound reduction in the frequency of multiple DC subsets, diminished activation marker expression, and reduced Flt3L levels in cancer patients with brain tumors compared to those without. We also demonstrate reduced intra-tumoral DCs in brain compared to lung tumors. This is the first time DC subsets have been fully characterized in a range of brain tumor patients. Importantly, corticosteroid usage was closely associated with DC defects, highlighting the adverse effects of a standard symptomatic treatment on these critical immune cells. However, tumors located within the brain also directly contribute to DC defects. Finally, we identified several mouse brain tumor models that replicate key observations in patients and may be used to further understand this endogenous DC deficiency and to develop approaches to restore DCs, ultimately leading to new combination immunotherapies for the treatment of brain cancers.

High-grade glioma (HGG) cells reactivate neurodevelopmental programs regulated by ion channels to drive tumor progression. The activity of voltage-gated sodium channels (VGSCs) is fundamental to development, a target of blood-brain barrier (BBB)-permeable FDA-approved drugs, and aids tumor advancement in several cancers. However, the contribution of VGSC activity to HGG pathology remains unknown. Using single-cell and spatial transcriptomics, proteomics, and immunohistochemistry, we profiled the expression landscape of the VGSC family in patient tumors from two HGGs: adult glioblastoma and pediatric diffuse midline glioma (DMG). We further validated VGSC expression and function in HGG patient-derived cell lines using RNA, protein, and electrophysiological analyses, and assessed the anticancer efficacy of VGSC-modulating drugs in vitro through cell viability and invasion assays. VGSCs α subunits targeted by different classes of VGSC-drugs are differentially expressed within DMG and glioblastoma. Overall, VGSCs that are sensitive to the neurotoxin, tetrodotoxin (TTX), and in normal physiology are expressed in the nervous system were upregulated by invasive HGG cells at the leading edge of DMG and glioblastoma tumors. Whereas the TTX-insensitive cardiac VGSC NaV1.5 was distinctly more abundant within the cellular tumor of the DMG microenvironment. VGSC-expressing HGG cells within both microenvironments receive oncogenic glutamatergic inputs from surrounding neurons. RNA, protein and electrophysiological analysis of patient-derived HGG cells supported our in vivo findings, where NaV1.5 plays a significant role in DMG cell lines, conducting TTX-insensitive transient and persistent sodium currents. Overall, VGSC-targeting drugs had limited anticancer efficacy; however, GS967 a persistent current blocker, significantly inhibited the invasiveness of a DMG cell line by 33

BACKGROUND:Ion channel activity underlying biological processes that drive high-grade gliomas (HGG) is largely unknown. We aimed to determine the networking of ion channel genes and validate their expression within HGG patient tumors, to identify ion channel-targeting drugs that would inhibit tumor-promoting processes. METHODS:We used weighted gene co-expression network analysis (WGCNA) of RNAseq data to identify ion channel gene hubs in diffuse midline glioma (DMG) and glioblastoma. Using scRNA-seq, spatial transcriptomics, and immunohistochemistry, we characterized the expression of identified hubs within patient tumors, validating their role by testing the efficacy of ion channel inhibitors alone or in combination with radiation and temozolomide on the growth and invasion of patient-derived glioblastoma explant organoids (GBOs). RESULTS:Network analysis revealed a preserved HGG "neuronal regulation" module, containing the greatest number of ion channels, with its corresponding genes concentrated at the tumor's leading edge. Hubs within this module included γ-Aminobutyric-acid type A receptor (GABAAR) genes GABRA1 (α1) and GABRG2 (γ2), which immunohistochemically colocalized with GABAergic synaptic markers at the leading edge. GBOs failed to retain this synaptic architecture but expressed a glioblastoma hub GABRA5 (α5), a component of extrasynaptic GABAARs. S44819, an α5-GABAAR antagonist strongly inhibited GBO invasion, with GABA(A)-compound 1b, a partial antagonist of GABAARs, robustly inhibiting GBO proliferation and invasion. Moreover, combined with standard-of-care (SOC) regimens, the anti-invasive properties of both compounds were enhanced in GBOs. CONCLUSIONS:Our co-expression network analysis identified key ion channels at the leading edge in HGGs, which can be targeted by GABAAR-acting drugs to disrupt tumor progression.

INTRODUCTION:Pituitary neuroendocrine tumours (PitNETs) are common accounting for 10 to 25 % of all intracranial tumours. This project describes the feasibility of developing a novel membrane-based biomarker that could be used for fluorescent guided surgery. The aim was to catalogue the differential expression of membrane proteins between non-functional PitNETs and pituitary glands. METHODOLOGY:Ten pituitary gland tissue specimens were obtained from the National Institute of Health (NIH) NeuroBio-Bank and twenty non-functional PitNETs were obtained from the Northwestern University Nervous System Tumour Bank. Mass spectrometry analysis using an Orbitrap Fusion Lumos Tribrid Mass Spectrometer linked to a Dionex Ultimate 3000 UPLC system was undertaken. Data Dependent Acquisition Mass Spectrometry and Data Independent Acquisition Mass Spectrometry was then completed. Pathway enrichment analysis was performed using clusterProfiler v4.6.0. Functional enrichment analysis was conducted using Gene Ontology terms and Reactome pathways. RESULTS:Differential expression analysis between the two groups revealed a total of 2110 significant differently expressed proteins (DEPs), with 1387 of these also having a Log2 fold change either greater than 1, or less than -1. Of the 2110 DEPs, 925 were upregulated in tumours compared to control, while 1185 were downregulated. CONCLUSION:We have demonstrated a proteomic comparison between non-functional PitNETs and normal pituitary glands. These results demonstrate differences consistent with contemporary literature but shows that NOTCH3 and PTPRJ are up-regulated in non-functional PitNETs compared to pituitary glands.

Abstract Improved preclinical tools are urgently needed to translate new brain cancer treatments. Patient-derived brain tumor explant organoids (GBOs) offer promise for studying tumor cells in a relevant human tumor microenvironment and predicting patient responses to therapy. However, generating GBOs is time-consuming, costly, and technically challenging, hindering the creation of comprehensive biobanks covering the spectrum of brain tumor heterogeneity. This work presents a semi-automated method for producing GBOs, involving tumor tissue processing, size selection, and same-day cryopreservation, reducing generation time to less than 1 hour. We established a biobank of 24 GBOs from 33 samples (11 males and 13 females) with primary (n=13, GBO Yield (GY)>90%) and recurrent (n=5, GY>90%) glioblastomas, high-grade gliomas (HGG, n=2, GY≤50%), and low-grade gliomas (LGG, n=2 with GY>90%, n=2 with GY≤50%). Using GBO size and propidium iodide as readouts, we used this biobank to compare anticancer activities of novel idronoxil-conjugated benzopyran compounds (NX786, NX904/904E1) against Bortezomib (100% cell death reference). NX786 and NX904/904E1 reduced GBO growth (≥50%) in all primary glioblastoma and one (of two) recurrent glioblastoma GBOs with milder effects in LGG GBOs. Two (of four) primary glioblastoma, one (of two) recurrent glioblastoma, and one LGG GBOs exhibited significant cell death (>60%) in response to NX904/904E1. In contrast, NX786 induced cell death in one (of three) primary glioblastoma (34% cell death) and one (of two) recurrent glioblastoma (71% cell death) GBOs but not in LGG GBOs, together showcasing varied responses across different brain tumors. Highly-passaged GBOs lacking the non-malignant tumor microenvironment were more susceptible to these treatments, underlining the tumor microenvironment’s critical role in responses to anticancer agents. We provide a new method for efficient processing and cryopreserving GBOs, enabling the establishment of large biobanks for patient-specific preclinical drug testing across brain tumor subgroups in a clinically relevant human 3D model.

S1: Comparison of the Incucyte FLR and Operetta High Content Imaging System for detection of apoptosis in PC-3 cells induced by Paclitaxel. S2: Nuclear fragmentation index of PC-3 cells in the presence and absence of ectopically expressed p53 in response to MET and VPA. S3: p53 knock-down in LNCaP cells using siRNA. S4: Efficiency of GFP-p53 and GFP-only plasmid transfection in PC-3 cells. S5: Apoptosis in PC-3 cells in response to MET and VPA in the presence and absence of Enzalutamide.

Cancers in the central nervous system resist therapies effective in other cancers, possibly due to the unique biochemistry of the human brain microenvironment composed of cerebrospinal fluid (CSF). However, the impact of CSF on cancer cells and therapeutic efficacy is unknown. Here, we examined the effect of human CSF on glioblastoma (GBM) tumors from 25 patients. We found that CSF induces tumor cell plasticity and resistance to standard GBM treatments (temozolomide and irradiation). We identified nuclear protein 1 (NUPR1), a transcription factor hampering ferroptosis, as a mediator of therapeutic resistance in CSF. NUPR1 inhibition with a repurposed antipsychotic, trifluoperazine, enhanced the killing of GBM cells resistant to chemoradiation in CSF. The same chemo-effective doses of trifluoperazine were safe for human neurons and astrocytes derived from pluripotent stem cells. These findings reveal that chemoradiation efficacy decreases in human CSF and suggest that combining trifluoperazine with standard care may improve the survival of patients with GBM.

Abstract A significant challenge in neuro-oncology is identifying the metabolic fuel sources that underpin glioblastoma malignancy. Metabolic reprogramming is a hallmark of tumorigenesis, providing malignant cells with an adaptation advantage to survive and grow under environmental stresses, including anti-cancer therapies. Incorporating single-cell RNA sequencing data from 56 glioblastoma patient biopsies, we have identified distinct subpopulations of malignant cells which diverge in their metabolic activities. These metabolic subpopulations are conserved across patient datasets but vary in abundance from patient to patient, contributing to glioblastoma heterogeneity. We are also employing spatial transcriptomics to build a map of the metabolic tumour landscape. This analysis showed that these malignant sub-populations occupy distinct spatial regions in the tumour landscape. Environmental influences like distance to blood vessels, which impact oxygen and nutrient supply, can vary significantly between regions. This suggests that as malignant cells colonise different regions of the tumour, they modify their metabolism as an adaption response. Importantly these niches vary in their compositions of native brain and immune cells, contributing to the diverse ecosystems inhabited by these malignant subpopulations. The next steps are to explore the specific metabolite dependencies of these tumour cells by mass spectrometry imaging and identify metabolite fluxes within the microenvironment that we can target therapeutically.

Legends of supplementary Figure S1, S2, S3, S4, and S5.

Trinucleotide repeat disorders comprise ~20 severe, inherited, human neuromuscular and neurodegenerative disorders, which result from an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington's disease (HD), results from expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Since non-coding RNAs have been implicated in the initiation and progression of many diseases, herein we focused on a circular RNA (circRNA) molecule arising from non-canonical splicing (backsplicing) of HTT pre-mRNA. The most abundant circRNA from HTT, circHTT(2-6), was found to be more highly expressed in the frontal cortex of HD patients, compared with healthy controls, and positively correlated with CAG repeat tract length. Furthermore, the mouse orthologue (mmu_circHTT(2-6)) was found to be enriched within the brain and specifically the striatum, a region enriched for medium spiny neurons that are preferentially lost in HD. Transgenic overexpression of circHTT(2-6) in two human cell lines-SH-SY5Y and HEK293-reduced cell proliferation and nuclear size without affecting cell cycle progression or cellular size, or altering the CAG repeat region length within HTT. CircHTT(2-6) overexpression did not alter total HTT protein levels, but reduced its nuclear localisation. As these phenotypic and genotypic changes resemble those observed in HD patients, our results suggest that circHTT(2-6) may play a functional role in the pathophysiology of this disease.

Advances in cellular reprogramming have radically increased the use of patient-derived cells for neurological research in vitro. However, adherence of human neurons on tissue cultureware is unreliable over the extended periods required for electrophysiological maturation. Adherence issues are particularly prominent for transferable glass coverslips, hindering imaging and electrophysiological assays. Here, we assessed thin-film plasma polymer treatments, polymeric factors, and extracellular matrix coatings for extending the adherence of human neuronal cultures on glass. We find that positive-charged, amine-based plasma polymers improve the adherence of a range of human brain cells. Diaminopropane (DAP) treatment with laminin-based coating optimally supports long-term maturation of fundamental ion channel properties and synaptic activity of human neurons. As proof of concept, we demonstrated that DAP-treated glass is ideal for live imaging, patch-clamping, and optogenetics. A DAP-treated glass surface reduces the technical variability of human neuronal models and enhances electrophysiological maturation, allowing more reliable discoveries of treatments for neurological and psychiatric disorders.

Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cell-based immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients. However, a major challenge for T-cell-based immunotherapy of glioblastoma and other solid cancers is T-cell infiltration into tumours. This process is mediated by chemokine-chemokine receptor and integrin-adhesion molecule interactions, yet the specific nature of the molecules that may facilitate T-cell homing into glioblastoma are unknown. Here, we have characterised chemokine receptor and integrin expression profiles of endogenous glioblastoma-infiltrating T cells, and the chemokine expression profile of glioblastoma-associated cells, by single-cell RNA-sequencing. Subsequently, chemokine receptors and integrins were validated at the protein level to reveal enrichment of receptors CCR2, CCR5, CXCR3, CXCR4, CXCR6, CD49a, and CD49d in glioblastoma-infiltrating T-cell populations relative to T cells in matched patient peripheral blood. Complementary chemokine ligand expression was then validated in glioblastoma biopsies and glioblastoma-derived primary cell cultures. Together, enriched expression of homing receptor-ligand pairs identified in this study implicate a potential role in mediating T-cell infiltration into glioblastoma. Importantly, our data characterising the migratory receptors on endogenous tumour-infiltrating T cells could be exploited to enhance the tumour-homing properties of future T-cell immunotherapies for glioblastoma.

Background Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited.Methods We profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control.Results Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors.Conclusions Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

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.

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 .

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.

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)—which occur in 30% of functional mRNA transcripts—have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs. In addition, when employing a bespoke bioinformatics pipeline for identifying RNA chimeras, called Hyb, we could also identify over 2000 ME-circRNAs which contain novel MEs at their backsplice junctions, that are uncalled by either CIRCexplorer2 or CIRI2. Analysis of circRNA-seq datasets from gliomas of varying clinical grades compared with matched control tissue has shown circRNAs have potential as prognostic markers for stratifying tumor from healthy tissue. Furthermore, the abundance of microexon-containing circRNAs (ME-circRNAs) between tumor and normal tissues is correlated with the expression of a splicing associated factor, Serine/arginine repetitive matrix 4 (SRRM4). Overexpressing SRRM4, known for regulating ME inclusion in mRNAs critical for neural differentiation, in human HEK293 cells resulted in the biogenesis of over 2000 novel ME-circRNAs, including ME-circEIF4G3, and changes in the abundance of many canonical circRNAs, including circSETDB2 and circLRBA. This shows SRRM4, in which its expression is correlated with poor prognosis in gliomas, acts as a bona fide circRNA biogenesis factor. Given the known roles of MEs and circRNAs in oncogenesis, the identification of these previously unrecognized ME-circRNAs further increases the complexity and functional purview of this non-coding RNA family.

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.

While radiotherapy is widely used in cancer treatment, the benefits can be limited by radiation-induced damage to neighboring healthy tissues. We previously demonstrated in mice that the anti-inflammatory compound dimethylaminoparthenolide (DMAPT) selectively induces radiosensitivity in prostate tumor tissue from transgenic adenocarcinoma of mouse prostate (TRAMP) mice, while simultaneously protecting healthy tissues from 6 Gy whole-body radiation-induced apoptosis. Here, we examined the radioprotective effect of DMAPT on fibrosis in normal tissues after a partial-body fractionated radiation protocol that more closely mimics the image-guided fractionated radiotherapy protocols used clinically. Male C57BL/6J mice, 16 weeks old, received 20 Gy fractionated doses of X rays (2 Gy daily fractions, five days/week for two weeks) or sham irradiation to the lower abdomen, with or without a prior 20 mGy dose to mimic an image dose. In addition, mice received thrice weekly DMAPT (100 mg/kg by oral gavage) or vehicle control from 15 weeks of age until time of analysis at 6 weeks postirradiation. In the absence of exposure to radiation, there were no significant differences observed in the tissues of DMAPT and vehicle-treated mice (P > 0.05). DMAPT treatment significantly reduced radiation-induced testis weight loss by 60.9% (P < 0.0001), protected against a decrease in the seminiferous tubule diameter by 42.1% (P < 0.0001) and largely preserved testis morphology. Inclusion of the image dose had no significant effect on testis mass, seminiferous tubule diameter or testis morphology. DMAPT reduced radiation-induced fibrosis in the corpus cavernous region of the penis (98.1% reduction, P = 0.009) and in the muscle layer around the bladder (80.1% reduction, P = 0.0001). There was also a trend towards reduced collagen infiltration into the submucosal and muscle layers in the rectum. These results suggest that DMAPT could be useful in providing protection from the radiation-induced side effects of impotence and infertility, urinary incontinence and fecal urgency resulting from prostate cancer radiotherapy. DMAPT is a very well-tolerated drug and can conveniently be delivered orally without strict time windows relative to radiation exposure. Protection of normal tissues by DMAPT could potentially be useful in radiotherapy of other cancer types as well.