Prostate cancer is a growing global health challenge. To identify new ways to improve patient care, researchers need a variety of preclinical models that faithfully recapitulate human tumours across the disease continuum, from initiation to metastasis. These complementary models include primary cultures of prostate epithelial cells (PrECs), co-cultures, patient-derived explants (PDEs), patient-derived organoids (PDOs) and patient-derived xenografts (PDXs). Collectively, these models enable researchers to study tumour biology and therapeutic responses in clinically relevant contexts. Yet, there is still a need to improve the fidelity of preclinical models to human tumours by integrating diverse cell types from the tumour microenvironment and mimicking biomechanical features. By improving culture methods with matrix components that resemble the tumour microenvironment and new formulations of media that imitate human plasma, in vitro models will more accurately reflect human physiology, nutrient availability, and metabolism. In time this may reduce the reliance on animal testing through organ-on-chip and related techniques. These more complex models are suited to more detailed experimental readouts, including single-cell and spatial analyses. Intravital imaging also enables dynamic visualisation of cell-cell interactions and treatment responses in vivo. Collectively, these approaches are facilitating a shift towards sophisticated models that capture patients' tumour heterogeneity, different cellular niches, and provide opportunities to carefully study tumorigenesis, metastasis, lineage plasticity, and therapy resistance. In this review, we discuss the current progress and future directions for patient-derived models of prostate cancer, highlighting how they can be generated, refined, characterised and shared to accelerate the worldwide effort in translational research.

Glycolysis, commonly used by malignant tumors for energy production, results in acidification of the tumor microenvironment (TME) through the secretion and accumulation of lactic acid. Acidosis is a potent inhibitor of immune cell function and may therefore affect T-cell infiltration and the efficacy of immunotherapy. This study aimed to characterize the metabolic tumor microenvironment and its association with lymphocyte distribution in patients with advanced melanoma treated with immune checkpoint blockade (ICB) therapies. Pre-treatment formalin-fixed, paraffin-embedded metastatic melanoma specimens from 45 patients treated with anti-PD-1 ± anti-CTLA-4 ICB were included in this study. Patients with progression-free survival (PFS) ≥ 6mo were categorized as responders (n = 23), while non-responders had a PFS < 6mo (n = 22). Two custom multiplex immunofluorescence panels were developed to evaluate the expression and distribution of markers of a hypoxic microenvironment (CA9 and HIF1α), glycolysis (GLUT1 and GLUT3) and vessels (CD31) in relation to melanocytes (SOX10) and T lymphocytes (CD3). GLUT1 + melanoma regions contained significantly lower proportions of CD3+ T-cells than GLUT1- regions (p < 0.0001). Responders displayed significantly higher proportions of intratumoral T-cells expressing GLUT1 (p = 0.049) and GLUT3 (p = 0.043) compared to non-responders. CD3+ T-cells co-expressing hypoxia-associated markers were present in higher proportions significantly closer to GLUT1+ melanoma cells in responders compared to non-responders (p < 0.05). Patients with higher proportions of CD3+ T-cells and CD3+CA9+ T-cells within the 20 µm distance to GLUT1+ melanoma cells had significantly longer progression-free survival (p = 0.0133 and p = 0.0378, respectively). Together, these findings support the hypothesis that the presence of glycolysis in melanoma (as inferred by increased GLUT expression) may affect the ability of T-cells to infiltrate tumors and function effectively. The results also suggest that the overall proportion and spatial distribution of GLUT+ T-cells, including those displaying evidence of adaptation to a hypoxic/acidic TME, may be relevant for responses to ICB therapy.

All tissues _differentially_expressed_genes

BACKGROUND:Diffuse intrinsic pontine gliomas (DIPGs) and other H3K27M-mutated diffuse midline gliomas (DMGs) are brain tumors that primarily affect children. Radiotherapy is the standard of care but only provides only temporary symptomatic relief due to radioresistance. Although hypoxia is a major driver of radioresistance in other tumors, there is no definitive evidence that DIPGs are hypoxic. Diffuse intrinsic pontine gliomas often contain histone mutations, which alter tumor metabolism and are also associated with radioresistance. Our objective was to identify the metabolic profiles of DIPG cells, detect hypoxia signatures, and uncover metabolism-linked mechanisms of radioresistance to improve tumor radiosensitivity. METHODS:Using DIPG models combined with clinical datasets, we examined mitochondrial metabolism and signatures of hypoxia. We explored DIPG reliance on mitochondrial metabolism using extracellular flux assays and targeted metabolomics. In vitro and in vivo models were used to explore the mechanisms of targeting mitochondrial bioenergetics and hypoxia for radiosensitization. Treatment-induced transcriptomics and metabolomics were also investigated. RESULTS:Comprehensive analyses of DIPG cells show signatures of enhanced oxidative phosphorylation (OXPHOS). We also identified increased expression of specific OXPHOS-related genes and signatures of hypoxia gene expression in datasets obtained from DIPG patients. We found the presence of hypoxia in orthotopic mouse models bearing DIPG tumors. These findings enabled us to develop a proof-of-concept treatment strategy to enhance radiosensitivity of DIPGs in vitro and in animal models. CONCLUSIONS:Diffuse intrinsic pontine glioma cells rely on mitochondrial metabolism for growth, and targeting mitochondria disrupts bioenergetics, alleviates hypoxia, and enhances radiosensitivity. These findings warrant further exploration of OXPHOS inhibition as a radiosensitizing strategy for DIPG treatment.

Although c-Jun N-terminal Kinase (JNK) represents an attractive anti-cancer target, its pleiotropic functionality limits the use of direct JNK inhibitors. Here, we identify a distinct subcellular pattern of JNK activity as a therapeutic vulnerability in breast cancer, where cytoplasmic JNK activity predicts poor survival outcomes, is elevated in triple-negative breast cancers (TNBC) and is essential for metastatic outgrowth. Mechanistic analyses reveal cytoplasmic JNK acts through multiple mechanisms, with downstream targets involved in cellular metabolism and cytoskeletal regulation. On this basis, we leveraged actin-based phenotypic drug-screening and identified K12, an indirect but selective inhibitor of cytoplasmic JNK that blocks TNBC metastatic outgrowth in vivo . We reveal that K12 inhibits glutaminase-1 and the pyruvate dehydrogenase complex, and that this poly-pharmacology overcomes pyruvate anaplerosis, a known resistance mechanism of existing glutaminase inhibitors. These findings demonstrate the potential of selectively targeting the oncogenic function of JNK, offering new treatment options for early-stage metastatic TNBC. ### Competing Interest Statement The authors have declared no competing interest.

Upfront standard-of-care treatment for glioblastoma (GBM) has not evolved for two decades; prognosis remains poor, particularly for patients with MGMT-unmethylated GBM who generally derive limited benefit from temozolomide (TMZ). Immunotherapies, including vaccine approaches, shows great promise in a variety of cancers, with efficacy observed even in the setting of sizeable cerebral metastases (e.g. melanoma). The discovery of CMV antigen expression in GBM, but not in the normal brain, presents a unique opportunity to harness these immunogenic viral proteins as tumour-specific targets. In an 11 GBM patients’ trial, targeting CMV antigens using at least 3 patient-derived dendritic cell vaccines resulted in a median overall survival of 41.1 months warranting further evaluation. An alternative approach using an “off-the-shelf” CMV-specific peptide vaccine (PEP-CMV vaccine) has also been tested in adult and paediatric clinical trials (PERFORMANCE and PRIME, respectively). This is a Phase-I trial of 26 patients with newly diagnosed, MGMT-unmethylated, CMV-seropositive GBM in Australia (INTERROGATE-GBM; NCT06132438) across two treatment sites. At 4 weeks (+/-2 weeks) following standard concurrent radiotherapy and TMZ, participants receive tetanus-diphtheria booster/pre-conditioning and one cycle of adjuvant TMZ for lymphodepletion followed by PEP-CMV vaccine given fortnightly for 3 vaccines then monthly from vaccine 4 onwards, up to 12 vaccines. MRI performed every 8 weeks. Primary objectives are to evaluate feasibility and safety. Secondary objective is to quantitate specific immune responses to PEP-CMV vaccine by ELISpot. Exploratory objectives include progression-free and overall survival compared to historical controls (VERTU and PERFORMANCE), radiographic response (RANO 2.0) in patients with residual disease, and profiling of systemic and tumour-associated immune responses at progression/recurrence. Recruitment commenced June 2024. Up to 6 June 2025, 28 patients have been pre-screened and 12 enrolled. Among those enrolled, 5 patients have progressive disease; no patients have withdrawn from the study due to safety concerns.

EGFR is overexpressed in several cancers and hence EGFR-targeted theranostics is a promising approach to manage cancers, with widespread applicability. When nanoceria, which possesses intrinsic anticancer properties, is conjugated with EGFR-targeted fluorophore-tagged ligands, this nanoformulation can both image tumors and kill them through ROS-mediated cell destruction. Further, targeting enhances the cellular uptake of nanoparticles through EGFR-mediated endocytosis. The present work evaluates the in vitro theranostic performance of FITC-tagged EGF-functionalized nanoceria on EGFR-positive cancers. Three EGFR-positive cell lines were used for the study: MDA-MB-231, PANC-1 and HeLa. The EGFR-binding specificity of the EGF-functionalized nanoparticles was confirmed using western blot analysis. The therapeutic and diagnostic activities of the theranostic nanoparticles were confirmed, the former by cell viability assays and ROS measurements and the latter by confocal imaging. The results demonstrate significant ROS elevation levels for the treated cells and hence the suitability of the particles for therapeutic applications. The nanoparticles also are capable of detection using fluorescence imaging following 5 minutes of treatment, thus confirming the applicability for imaging. Hemolysis assay studies revealed excellent hemocompatibility of the nanoparticles, confirming their suitability for in vivo applications.

This files contains all supplementary data supporting the development and validation of AAnet

Abstract Diffuse midline glioma (DMG) is a lethal pediatric and adolescent high-grade glioma. DMG patients harbor a cold tumor immune microenvironment (TIME), similar to adult glioblastoma patients, who also suffer from systemic lymphopenia. In glioblastoma, lymphopenia is linked to T-cell sequestration in the bone marrow, caused by T-cell internalization of S1PR1, limiting trafficking. The DRD2 antagonist/ClpP agonist ONC201 is currently in clinical trials for DMG and has reported immunomodulatory effects in other cancers, thus we aimed to investigate ONC201’s immunomodulatory effects in DMG. Flow cytometry of peripheral blood from DMG patients at diagnosis showed low levels of lymphocytes (CD4+, CD8+, and NK cells) compared to the reference range (n = 9). This was also seen in immunocompetent C57BL/6 DMG-engrafted mice (known as ‘PPK’ harboring PdgfraD842V, DNp53, and H3f3aK27M mutations) compared to tumor-naïve, sham-engrafted mice (n = 3). Vehicle-treated PPK-engrafted mice showed increased numbers of T cells in the bone marrow compared to sham-engrafted mice (log2FC:-1.28, p-value:0.0367), suggesting T-cell sequestration. ONC201 treatment (125 mg/kg B.I.W.) increased S1PR1 surface expression on T cells (log2FC:0.73, p-value=0.0005), reducing sequestration in the bone marrow. Immunohistochemistry identified increased levels of tumor-infiltrating lymphocytes (TILs) within the TIME of ONC201-treated mice, including CD45+ and CD3+ T cells (log2FC:2.38 and 1.94, p-value:0.0275 and 0.0103). ONC201 also promoted increased expression of the MHC I subunit B2M (log2FC:1.67, p-value:0.0281), suggesting enhanced antigen presentation. Despite warming the TIME, no survival benefit was seen using immunocompetent mice (n = 10). scRNAseq of PPK tumors +/-ONC201 identified increased infiltration of immunosuppressive myeloid-derived macrophages (MDMs). These MDMs showed upregulation of CD74, known to be expressed on tumor-associated macrophages favoring a tumor-permissive TIME, revealing a possible therapeutic vulnerability. In conclusion, DMG tumors contribute to systemic lymphopenia, partially reversed by ONC201. While ONC201 promotes antigen presentation and TIL recruitment, killing efficacy may be limited by tumor-promoting CD74+ MDMs, highlighting future combination strategies.

Diffuse midline glioma (DMG) is a universally fatal high-grade glioma. Its immunologically cold tumor immune microenvironment (TIME) presents a major barrier to durable anti-tumor responses. Dordaviprone (ONC201) is a brain-penetrant DRD2 antagonist and ClpP agonist that disrupts mitochondrial complex II (CII) activity; currently under clinical evaluation for DMG. In melanoma, CII inhibition has been shown to enhance MHC presentation and T cell–mediated killing. Here, we investigated the immunomodulatory effects of dordaviprone in DMG mouse models, patient tumor tissues, and blood samples. Immunocompetent DMG-PPK mice (PdgfraD842V, Trp53DN, H3f3aK27M mutations) were treated with dordaviprone (125 mg/kg, twice weekly) for 1 week (acute) or 3 weeks (chronic) and compared to naïve, sham (orthotopic surgery without tumor engraftment), and vehicle-treated controls. Murine tumors were analyzed by single-cell multiome (scRNA-seq and scATAC-seq) and spatial transcriptomics. Murine blood and bone marrow (BM) and human blood samples were profiled by scRNA-seq and flow cytometry. In models, dordaviprone promoted microglial activation and macrophage infiltration within the TIME. Spatial transcriptomics identified increased expression of the MHC-I subunit B2m in tumor cells and tumor-infiltrating Cd8+ T cells following dordaviprone treatment. Flow cytometry of murine and human peripheral blood confirmed CD4+ and CD8+ lymphopenia at baseline. Dordaviprone increased circulating lymphocytes in models (log2 fold change=1.03; patient data pending), reversing DMG-induced CD3+ T cell sequestration in the BM. Immunohistochemistry confirmed elevated levels of B2M (p=0.0281) and tumor-infiltrating lymphocytes (TILs), including CD45+ (p=0.0275) and CD3+ T cells (p=0.0103) in dordaviprone-treated mice. Despite enhanced TIL recruitment, dordaviprone did not improve survival (n=10), likely due to persistent immunosuppressive myeloid activity, i.e. Havcr2 (TIM-3), Cd74, Cd274 (PD-L1). This study demonstrates that DMG tumors contribute to systemic lymphopenia, partially reversed by dordaviprone. While supporting its immunomodulatory effects, including enhanced antigen presentation and TIL recruitment, our findings highlight future combination strategies to leverage dordaviprone-induced immunoreactive TIME.

Mitochondrial oxidative phosphorylation (OXPHOS) is a therapeutic vulnerability in glycolysis-deficient cancers. Here we show that inhibiting OXPHOS similarly suppresses the proliferation and tumorigenicity of glycolytically competent colorectal cancer (CRC) cells in vitro and in patient-derived CRC xenografts. While the increased glycolytic activity rapidly replenished the ATP pool, it did not restore the reduced production of aspartate upon OXPHOS inhibition. This shortage in aspartate, in turn, caused nucleotide deficiencies, leading to S phase cell cycle arrest, replication fork stalling, and enrichment of the p53 pathway, manifestations of replication stress. The addition of purine nucleobases adenine and guanine along with the pyrimidine nucleoside uridine restored replication fork progression and cell proliferation, whereas the supplementation of exogenous aspartate recovered the nucleotide pool, demonstrating a causal role of the aspartate shortage in OXPHOS inhibition-induced nucleotide deficiencies and consequently replication stress and reductions in proliferation. Moreover, we demonstrate that glutamic-oxaloacetic transaminase 1 (GOT1) is critical for maintaining the minimum aspartate pool when OXPHOS is inhibited, as knockdown of GOT1 further reduced aspartate levels and rendered CRC cells more sensitive to OXPHOS inhibition both in vitro and in vivo. These results propose GOT1 targeting as a potential avenue to sensitize cancer cells to OXPHOS inhibitors, thus lowering the necessary doses to efficiently inhibit cancer growth while alleviating their adverse effects.

Identifying functionally important cell states and structure within heterogeneous tumors remains a significant biological and computational challenge. Current clustering- or trajectory-based models are ill-equipped to address the notion that cancer cells reside along a phenotypic continuum. We present Archetypal Analysis network (AAnet), a neural network that learns archetypal states within a phenotypic continuum in single-cell data. Unlike traditional archetypal analysis, AAnet learns archetypes (AT) in a simplex-shaped neural network latent space. Using preclinical and clinical models of breast cancer, AAnet resolves distinct cell states and processes, including cell proliferation, hypoxia, metabolism, and immune interactions. Primary tumor ATs are recapitulated in matched liver, lung, and lymph node metastases. Spatial transcriptomics reveals archetypal organization within the tumor and intra-archetypal mirroring between cancer and adjacent stromal cells. AAnet identifies GLUT3 within the hypoxic AT that proves critical for tumor growth and metastasis. AAnet is a powerful tool, capturing complex, functional cell states from multimodal data. SIGNIFICANCE:Defining critical cell states among cells that reside along a phenotypic continuum is a current biological and computational challenge. In this study, we present AAnet, a neural network that learns archetypal cell states of cancer cells. AAnet defines discrete spatially localized ATs that resolve intratumoral heterogeneity.

Microscopy and omics are complementary approaches to probe cellular molecular states in health and disease, combining granularity with scalability. However, integrating both imaging- and sequencing-based assays on the same cell has proven challenging. This study demonstrates a new approach called SpectralSeq that combines hyperspectral autofluorescence imaging with transcriptomics on the same cell. SpectralSeq is applied to Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and identifies a subpopulation of cells exhibiting bright autofluorescence rings at the plasma membrane in optical channel 13 (λex = 431 nm, λem = 594 nm). Correlating the presence of a ring with the gene expression in the same cell indicates that ringed cells show higher expression of apoptosis-related genes and lower expression of ATP production genes. Furthermore, correlation of cell morphology with gene expression reveals downregulation of multiple spliceosome members in larger MCF-7 cells. Multiple genes exhibit consistent expression across cell sizes but varied exon usage. Finally, correlation between gene expression and fluorescence within the spectral range of nicotinamide adenine dinucleotide hydrogen (NADH) provides insights into the metabolic states of MCF-7 cells. Overall, SpectralSeq links optical spectrum with internal molecular states, offering a single streamlined workflow for single-cell resolution studies integrating spectral, morphological, and transcriptomic analyses.

Identifying functionally important cell states and structure within a heterogeneous tumor remains a significant biological and computational challenge. Moreover, current clustering or trajectory-based computational models are ill-equipped to address the notion that cancer cells reside along a phenotypic continuum. To address this, we present Archetypal Analysis network (AAnet), a neural network that learns key archetypal cell states within a phenotypic continuum of cell states in single-cell data. Applied to single-cell RNA sequencing data from pre-clinical models and a cohort of 34 clinical breast cancers, AAnet identifies archetypes that resolve distinct biological cell states and processes, including cell proliferation, hypoxia, metabolism and immune interactions. Notably, archetypes identified in primary tumors are recapitulated in matched liver, lung and lymph node metastases, demonstrating that a significant component of intratumoral heterogeneity is driven by cell intrinsic properties. Using spatial transcriptomics as orthogonal validation, AAnet-derived archetypes show discrete spatial organization within tumors, supporting their distinct archetypal biology. We further reveal that ligand:receptor cross-talk between cancer and adjacent stromal cells contributes to intra-archetypal biological mimicry. Finally, we use AAnet archetype identifiers to validate GLUT3 as a critical mediator of a hypoxic cell archetype harboring a cancer stem cell population, which we validate in human triple-negative breast cancer specimens. AAnet is a powerful tool to reveal functional cell states within complex samples from multimodal single-cell data. ### Competing Interest Statement The authors have declared no competing interest.

AbstractTriple-negative breast cancer (TNBC) metabolism and cell growth uniquely rely on glutamine uptake by the transporter ASCT2. Despite previous data reporting cell growth inhibition after ASCT2 knockdown, we here show that ASCT2 CRISPR knockout is tolerated by TNBC cell lines. Despite the loss of a glutamine transporter and low rate of glutamine uptake, intracellular glutamine steady-state levels were increased in ASCT2 knockout compared to control cells. Proteomics analysis revealed upregulation of macropinocytosis, reduction in glutamine efflux and increased glutamine synthesis in ASCT2 knockout cells. Deletion of ASCT2 in the TNBC cell line HCC1806 induced a strong increase in macropinocytosis across five ASCT2 knockout clones, compared to a modest increase in ASCT2 knockdown. In contrast, ASCT2 knockout impaired cell proliferation in the non-macropinocytic HCC1569 breast cancer cells. These data identify macropinocytosis as a critical secondary glutamine acquisition pathway in TNBC and a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine metabolism for their growth, providing a rationale for targeting of more downstream glutamine metabolism components.

Abstract OBJECTIVE Gliomas, particularly glioblastoma which is the most common and deadly type, frequently express androgen receptors (AR). We systematically characterized AR expression in low- and high-grade gliomas to investigate whether disrupting AR signaling with readily available blood-brain-barrier penetrant drugs could improve treatment. METHODS Low- and high-grade glioma samples were stained for AR by immunohistochemistry, with AR-expression correlated to grade and sex. Localization of AR and the influence of dihydrotestosterone (DHT) and anti-androgens in glioblastoma cell line (U251) were determined by immunofluorescence and immunoblot. Building on previous in vitro data, we used an orthotopic AR-positive patient-derived model (RN1) to test the efficacy of seviteronel, a blood-brain-barrier penetrant anti-androgen. RESULTS Two thirds of biopsy samples were positive for AR expression by IHC (n=25/39). AR expression correlated with increasing tumor grade (p<0.05) but was similar between sexes (p=0.94). Anti-androgens enzalutamide and seviteronel prevented DHT-induced nuclear translocation of AR; however, seviteronel did so to a lesser extent, as measured by immunofluorescence and immunoblot. In the mouse model, seviteronel significantly improved survival compared to vehicle (n=12, 36.0 vs 27.5 days, p=0.03), and demonstrated reduced Ki67 expression in tumors at the endpoint (68.9% vs 85.0%, p<0.001). Combining seviteronel with temozolomide limited tumor growth as measured by bioluminescence imaging, but did not significantly extend survival compared to temozolomide alone (n=16, 63.5 vs 56.5 days, p=0.37). Adding seviteronel to radiotherapy did not extend survival compared to radiotherapy alone (n=16, 33.0 vs 39.0 days, p=0.19). CONCLUSIONS Targeting AR signaling with blood-brain-barrier penetrant anti-androgens may represent a promising biomarker-directed therapeutic strategy. The mechanisms underlying the effects of seviteronel are being further investigated through affinity-purification mass spectrometry analysis and CUT&RUN sequencing of untreated and treated glioma cells. Additional animal experiments are underway to examine the role of biological sex and the glioma immune microenvironment on the efficacy of anti-androgens in vivo.

e14016 Background: Glioblastoma is a lethal brain cancer. Clinical patterns of disease suggest that androgens influence glioblastoma risk and prognosis, and androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. Thus, potent brain-penetrant anti-androgen therapies may be an effective treatment strategy in glioblastoma. Methods: Clinical samples and patient-derived glioma xenografts (PDXs) were assessed for AR expression by immunohistochemistry. Glioblastoma cell lines and patient-derived models maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Change in plasticity marker ZEB1 was measured using immunofluorescence. Stem cell function was assessed using a tumoursphere assay. In an intracranial AR positive glioblastoma PDX model (RN1) in an immunocompromised mouse, the most effective drug in vitro, seviteronel, was tested alone and in combination with temozolomide or radiation. Tumor growth was monitored with weekly bioluminescent imaging, with survival as the primary endpoint. Results: Cytoplasmic AR staining was present in ~55% of glioblastoma samples using immunohistochemistry; this biomarker has shown to be predictive in triple-negative breast cancer. AR positive model RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC 50 ) after 96 h (enzalutamide 52 µM, abiraterone 12 µM, seviteronel 7 µM). AR negative model WK1 was inhibited though with higher IC 50 values after 96 h (enzalutamide 63 µM, seviteronel 21 µM). Cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgens (normalized mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). Tumoursphere assays demonstrated that anti-androgens inhibit the tumor-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. In mice intracranially implanted with RN1 cells, seviteronel improved overall survival compared to vehicle alone (36 vs 27.5 days, p=0.03). There was no observed benefit when seviteronel was added to radiotherapy (33 vs 39 days, p=0.19). Adding seviteronel to temozolomide limited tumor growth by IVIS but did not significantly improve overall survival compared to temozolomide alone (63.5 vs 56.5 days, p=0.37). Conclusions: Targeting AR with brain penetrant anti-androgen drugs may be a promising biomarker-directed therapeutic strategy for glioblastoma. To address the limitations of our study, further experiments to optimize translational treatment protocols, address the role of biological sex and examine the role of the immune microenvironment are underway.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin β1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.