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.

The vast majority of clinical small molecule multi-kinase inhibitors (mKI) report abject failures in targeting cancers with high stem cell contents like high-grade glioma and colorectal cancers. The FDA-approved mKIs to date ablate receptor tyrosine kinase signaling but do not target the paradoxical WNT signaling which is a key survival driver for the self-renewing cancer stem cells. The WNT pathway enhances cancer plasticity and triggers relapse of highly heterogenous tumours. Using de novo synthesis and structure-activity-relationship (SAR) studies with blood-brain-barrier (BBB) penetrant mKI scaffolds, we designed a highly potent and selective small molecule inhibitor of PI3Kα, PDGFR/KIT, and the WNT pathway denoted Dyr726. Dyr726 is superior to clinical mKIs and inhibits PI3K-AKT-mTOR and WNT-pathway signaling at multiple nodes thereby impeding proliferation, invasion, and tumour growth. Phospho-proteomic, structural, and target engagement analyses, combined with in vitro, in vivo efficacy, and pharmacokinetic studies reveal that Dyr726 is a brain-penetrant small molecule which effectively reduces tumour volume and extends survival of murine orthotopic models. Our current work establishes a first-in-class brain penetrant small molecule mKI which simultaneously antagonize the PI3K-AKT-mTOR and WNT pathways in preclinical cancer stem cell cultures, adult and pediatric primary organoids, and orthotopic murine models with positive efficacy in combination with clinical standard of care.

There are unmet clinical needs of bladder cancer associated with its late detection, limited treatment options, and high mortality rate. In response to this, we present the results of the in-vitro assessment of photodynamic polymers, prior to their integration into a wireless biomedical implant for in-situ photodynamic therapy (PDT). PDT selectively activates light-sensitive photosensitizers, to kill cancer cells without the adverse effects associated with systemic therapies, such as chemotherapy. PDT offers significant advantages in hypoxic tumors, including oxygen-independent cytotoxicity known to impede the curative efficiency of radiotherapy. Accordingly, we report on the design methodology and microfabrication process of an in-situ PDT wireless microsystem as a novel treatment modality. We present the preliminary results from three benzothiadiazole-based polymers that are evaluated as solid support photosensitizers for the inhibition of cancer growth in three cell lines. Cell lines represent ovarian, prostate and bladder cancer, the reduction in cell number was assessed with Sulforhodamine B assays. Following Polyamide, aminophenyl- (PA-ABT) treatment, cells were subjected to a range of 420 nm light exposures and fluorimetry was used to determine cell growth. PA-ABT was shown to cause growth inhibition only at higher concentrations in all tested cell lines. PA-ABT can be considered as a promising candidate for in-situ PDT treatment due to its inhibitory effects in cancer cell lines open new possibilities for mono or combination light-based therapy in cancer treatment for previously inaccessible cancers.

Background: Patients with glioblastoma who are elderly or have poor performance status (PS) experience particularly poor clinical outcomes. At the time of study initiation, these patients were treated with short-course radiotherapy (40 Gy in 15 fractions). Olaparib is an oral inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) that is well tolerated as a single agent but exacerbates acute radiation toxicity in extracranial sites. Preclinical data predicted that PARP inhibitors would enhance radiosensitivity in glioblastoma without exacerbating adverse effects on the normal brain.Methods: Phase I of the PARADIGM trial was a 3+3 dose escalation study testing olaparib in combination with radiotherapy (40 Gy 15 fractions) in patients with newly diagnosed glioblastoma who were unsuitable for radical treatment either because they were aged 70 or over (WHO PS 0-1) or aged 18-69 with PS 2. The primary outcome was the recommended phase 2 dose (RP2D) of olaparib. Secondary endpoints included safety and tolerability, overall survival (OS) and progression free survival (PFS). Effects on cognitive function were assessed by mini-mental state examination (MMSE).Results: Of 16 eligible patients (56.25% male, median age 71.5 [range 44-78 years], 75% PS 0-1), one dose-limiting toxicity was reported (grade 3 agitation). Maximum tolerated dose was not reached and the RP2D was determined as 200 mg twice daily. Median OS and PFS were 10.8 months (80% CI: 7.3-11.4) and 5.5 months (80% CI: 3.9-5.9) respectively. MMSE plots indicated that cognitive function was not adversely affected by the olaparib-radiotherapy combination.Conclusions: Olaparib can be safely combined with hypofractionated brain radiotherapy and is well tolerated in patients unsuitable for radical chemoradiation. These results enabled initiation of a randomised phase II study and support future trials of PARP inhibitors in combination with radiotherapy for patients with brain tumors.

PURPOSE:Patients with glioblastoma who are older or have poor performance status (PS) experience particularly poor clinical outcomes. At the time of study initiation, these patients were treated with short-course radiation therapy (40 Gy in 15 fractions). Olaparib is an oral inhibitor of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) that is well tolerated as a single agent but exacerbates acute radiation toxicity in extracranial sites. Preclinical data predicted that PARP inhibitors would enhance radiosensitivity in glioblastoma without exacerbating adverse effects on the normal brain. METHODS AND MATERIALS:Phase 1 of the PARADIGM trial was a 3+3 dose-escalation study testing olaparib in combination with radiation therapy (40 Gy 15 fractions) in patients with newly diagnosed glioblastoma who were unsuitable for radical treatment either because they were aged 70 years or older (World Health Organization PS 0-1) or aged 18 to 69 years with PS 2. The primary outcome was the recommended phase 2 dose of olaparib. Secondary endpoints included safety and tolerability, overall survival, and progression-free survival. Effects on cognitive function were assessed via the Mini Mental State Examination. RESULTS:Of 16 eligible patients (56.25% male; median age, 71.5 years [range, 44-78]; 75% PS 0-1), 1 dose-limiting toxicity was reported (grade 3 agitation). Maximum tolerated dose was not reached and the recommended phase 2 dose was determined as 200 mg twice daily. Median overall survival and progression-free survival were 10.8 months (80% CI, 7.3-11.4) and 5.5 months (80% CI, 3.9-5.9), respectively. Mini Mental State Examination plots indicated that cognitive function was not adversely affected by the olaparib-radiation therapy combination. CONCLUSIONS:Olaparib can be safely combined with hypofractionated brain radiation therapy and is well tolerated in patients unsuitable for radical chemoradiation. These results enabled initiation of a randomized phase 2 study and support future trials of PARP inhibitors in combination with radiation therapy for patients with brain tumors.

Abstract Glioblastoma (GBM) remains a formidable challenge in the realm of oncology, primarily due to the limitations of the standard of care therapies. Photodynamic therapy (PDT), which exploits the light-activated potential of 5-aminolevulinic acid (5-ALA), emerges as a promising avenue to combat GBM. In this study, primary patient-derived cell lines, Ox5 core, and G7, served as critical subjects, exposed to light excitation at wavelengths of 410 nm, 528 nm, and 810 nm, both with and without 5-ALA (protoporphyrin IX). The central objective was to rigorously assess the efficacy of PDT, either with or without 5-ALA, in reducing cell viability and curtailing clonogenic expansion. This evaluation was performed within the context of the existing standard of care for GBM, encompassing Temozolomide (TMZ) and Radiotherapy. Our results unveiled striking insights into the potential of PDT as an innovative therapeutic strategy. Notably, Ox5 cells exhibited a remarkable reduction in viability, exceeding 70%, following exposure to PDT with 410 nm light and 5-ALA at a concentration of 50μM. A similar, albeit slightly less profound, effect was observed in G7 cells, with a viability decrease of more than 53%. These outcomes, meticulously quantified through the MTT viability assay (n=4), were reinforced by statistical significance, as indicated by p-values falling below 0.05 and, lower 0.001. Furthermore, clonogenic assays employing G7 cells provided an additional layer of evidence, demonstrating a substantial inhibition of clonogenic potential following PDT treatment with 410 nm light and 5-ALA. Immunohistochemistry techniques, particularly focusing on the cleaved caspase-3 marker, in both Ox5 core and G7 cells, underscoring the mechanistic basis of the observed cell death. In stark contrast, the conventional standard of care, involving TMZ monotherapy, was tested, and the results across four biological replicates were less significant. The relative viability of Ox5 and G7 cells exhibited no statistical significance, with p-values exceeding 0.05, implying a limited impact of this regimen on cell viability. Moreover, clonogenic assays conducted under the standard of care protocol (TMZ+/-Radiotherapy) unveiled the persistence of colonies, even at higher radiation doses of 4 and 6 Gy.In summary, PDT, specifically when implemented with 410 nm light and 5-ALA, represents a breakthrough strategy that leads to substantial cell death in both Ox5 and G7 cells. This compelling alternative therapy shines a hopeful light on the horizon of GBM treatment, providing a potentially transformative approach that can augment the current standard of care. Citation Format: Nazar Vasyliv, Karin Williams. Photodynamic therapy improves therapeutic management of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7593.

Supplementary Figure 1 from Tissue-Specific Consequences of Cyclin D1 Overexpression in Prostate Cancer Progression

The file contains 4 supplementary tables. Table S1 lists the primary antibodies used in the study. Table S2 provides the primers used to characterize AR-V expression. Table S3 provides the primers used to characterize epithelial and stem cell marker expression. Table S4 summarizes and compares the characteristics of CSC-like AR(-) HPET and HuSLCs with that of the standard AR(+) PCa cell line, LNCaP.

The file contains 6 supplementary figures. Figure S1 shows that AR protein expression is also induced following treatment with additional proteasomal inhibitors, MG115 and Epoxomicine. Figure S2 compares the levels of MDM2 expression, demonstrating that CSC-like HuSLC and HPET cells express higher levels of MDM2 as compared to standard PCa cell lines. Figure S3 demonstrates that HPET and HuSLCs only express AR-fl mRNA but not AR-V mRNA and this is confirmed in Figure S4 where HPET and HuSLCs only express AR-fl protein but not AR-V protein. Figure S5 provides information that induction of AR promotes expression of additional AR target genes, namely AR-regulated HES1 and HEY1 genes. Figure S6 compares p53 expression in CSC-like HPET and HuSLCs to that of LNCaP cells.

Abstract Transforming growth factor-β (TGF-β) is a pleiotropic growth factor with actions that are dependent on circumstances, including dose, target cell type, and context. TGF-β can elicit both growth-promoting and growth-suppressive activities. In normal tissues, TGF-β generally acts to restrict growth and maintain differentiation. However, during tumorigenesis, changes in TGF-β expression and cellular responses can promote tumorigenesis. The present study examines the effects of TGF-β on the nontumorigenic human prostatic epithelial cell line BPH1 and on three derivative tumorigenic sublines BPH1CAFTD1, BPH1CAFTD3, and BPH1CAFTD5. The data show that TGF-β has different effects on the nontumorigenic and tumorigenic cells. The nontumorigenic cells are growth inhibited by TGF-β. In contrast, the tumorigenic sublines are not growth inhibited but instead undergo an epithelial to mesenchymal transformation (EMT) in response to TGF-β. The tumorigenic lines show constitutively elevated levels of phosphorylated Akt, which modulates their response to TGF-β by blocking Smad3 and p21 nuclear translocation. On TGF-β stimulation of the tumorigenic sublines, the activated Akt allows the cell to escape cell cycle arrest. The phosphatidylinositol 3-kinase/Akt pathway is also involved in TGF-β-induced EMT, defined here by induction of vimentin expression and enhanced cellular motility. In vivo, tumorigenic cells with constitutively active TGF-β signaling show increased invasion with EMT, which express vimentin, located specifically at the invasive front of the tumor. These data indicate that following malignant transformation TGF-β can play a direct role in promoting prostatic cancer and further that these responses are context specific in vivo. (Cancer Res 2006; 66(16): 8007-16)

Abstract PARP inhibitors (PARPi) enhance radiation sensitivity in multiple cancer models, both in vitro and in vivo. Our observation that the radiosensitizing properties of PARPi are most pronounced in rapidly proliferating cells is reflected in early phase clinical trial data showing exacerbation of acute radiation toxicity in rapidly proliferating tissues such as oropharyngeal and esophageal mucosa. Lack of radiosensitization in late responding, slowly proliferating normal tissues indicates that PARPi may be more effectively combined with radiation therapy (RT) in patients with brain tumors. We are therefore evaluating the oral PARPi olaparib in combination with RT and/or temozolomide (TMZ) in the treatment of glioblastoma (GBM), the most prevalent and most aggressive primary brain tumor. Patients with GBM experience very poor outcomes in terms of median survival (c.1 year) and neurocognitive decline caused primarily by RT. Olaparib was initially evaluated in combination with daily low-dose TMZ in patients with recurrent GBM in the OPARATIC trial. Pharmacokinetic studies revealed that olaparib penetrates both core and margin regions of GBM, indicating that the BBB is significantly disrupted throughout these tumors. Olaparib could be safely combined with daily TMZ (75 mg/m2), but intermittent olaparib dosing (150 mg three days per week) was required to avoid dose-limiting hematological toxicity. Early phase testing of the olaparib-radiotherapy combination is now underway in three populations of patients with newly diagnosed GBM. Patients aged >65 with MGMT unmethylated GBM are being recruited to a randomized, placebo-controlled phase II study (PARADIGM) after a phase I dose escalation study showed that olaparib (200 mg twice daily) was extremely well tolerated when combined with brain irradiation (40 Gray in 15#). Good performance status patients aged <70 are being recruited to two parallel phase I dose escalation studies: patients with MGMT unmethylated tumors are receiving daily olaparib with RT (60 Gy in 30#) without TMZ, while patients with MGMT methylated tumors are receiving intermittent olaparib with standard chemoradiation (60 Gy). The impact of PARPi on RT induced neurotoxicity is being investigated in preclinical studies. In vitro data show that PARPi reduce proliferation of neural stem cells and protect them against RT induced apoptosis, while in vivo studies support the emerging concept that RT induced neuroinflammation is important in the pathogenesis of neurotoxicity. Importantly, preliminary PET and immunohistochemical studies have shown robust anti-neuroinflammatory effects of PARPi in this context. Ongoing experiments are defining the roles of microglia, astrocytes and neurogenesis in this phenomenon. These diverse data sets provide support for our hypothesis that combining PARPi with RT has potential to improve outcomes for GBM patients by enhancing tumor control while simultaneously suppressing neuroinflammation and alleviating RT related neurocognitive decline. Citation Format: Anthony J. Chalmers, Rodrigo Gutierrez-Quintana, David J. Walker, Karin Williams, Duncan Forster, Mark R. Jackson, Sarah Derby, Jon Stobo, Lorna Sweeting, Caroline Kelly, Stephen Durant, Kaye J. Williams. Enhancing the therapeutic ratio for glioblastoma by combining radiation therapy with PARP inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-006.

PURPOSE:Low-dose whole lung radiation therapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection, and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling, and mechanisms of action. METHODS AND MATERIALS:Female C57BL/6 mice were treated with intranasal bleomycin sulfate (7.5 or 11.25 units/kg, day 0) and then exposed to whole lung radiation therapy (0.5, 1.0, or 1.5 Gy, or sham; day 3). Bodyweight was measured daily, and lung tissue was harvested for histology and flow cytometry on day 10. Computed tomography lung imaging was performed before radiation (day 3) and pre-endpoint (day 10). RESULTS:Bleomycin caused pneumonitis of variable severity, which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight, and a proportion of these mice exhibited less severe histopathologic lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. In addition, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells (DCs), and neutrophil-DC hybrids. Overall, bleomycin-treated mice exhibited significantly higher percentages of nonaerated lung in left than right lungs, and LDLR (1.0 Gy) limited further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not improve bodyweight, flow cytometric, or radiologic readouts of bleomycin-induced pneumonitis. CONCLUSIONS:Our data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose, and provide evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ DCs, and neutrophil-DC hybrids.

Background The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide (TMZ) chemotherapy in preclinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side effects of TMZ. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib and assess the safety and tolerability of its combination with TMZ. Methods Preclinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose TMZ in a 3 + 3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumor core and tumor margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results Olaparib was a substrate for multidrug resistance protein 1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients; median, 496 nM) and 21/21 tumor margin specimens (9 patients; median, 512.3 nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150 mg (3 days/week) with TMZ 75 mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression free at 6 months. Olaparib radiosensitized 6 glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusion Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better preclinical models.

Abstract Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR−) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR− CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance. Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.

This chapter focuses on the role of estrogens and their roles in cancer with an emphasis on prostate biology. It is now apparent that estrogens, both those synthesized by the body and those from our environment, target estrogen-responsive tissues at all stages of development and maturation. Little is known about the mechanisms involved in estrogen stimulation of carcinogenesis and less is known about how to prevent or treat cancer through estrogenic pathways. To better understand how estrogens mediate their carcinogenic effects, the respective roles of estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β) must be elucidated in the epithelial and stromal cells that constitute each tissue. Lastly, the significance of estrogen receptor (ER) signaling during various ontogenic periods must be determined. Answers to these questions will further our understanding of the mechanisms of estrogen/ER …