Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate.

BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

Radiating the prostate bed in relapsed oligometastatic prostate cancer: How comprehensive should we be?

PURPOSE: A subset of patients with high-risk pathological features at radical prostatectomy recur with oligometastatic disease. The aim of this study is to investigate the rate of prostate bed recurrence, with or without history of prostate bed irradiation (PBRT), in oligometastatic prostate cancer (OMPC) patients after metastasis-directed therapy (MDT). METHODS: We performed a retrospective analysis of hormone-sensitive OMPC patients treated initially with curative-intent radical prostatectomy followed by disease recurrence and metastasis-directed stereotactic ablative radiotherapy (SABR) at our institution. Prostate bed recurrence rates were compared between patients who had PBRT at any point (i.e., before oligometastatic diagnosis or concurrently with MDT) versus those with no history of PBRT. RESULTS: Seventy-seven patients were included, and 68.8% had received PBRT. There were no significant differences in baseline characteristics between those who had received and had not received PBRT. There were five prostate bed recurrences following MDT, specifically with a 24-month cumulative incidence of 30.4% in patients who did not have PBRT and 2.4% in those who did (p = 0.03). Three of the five recurrences were isolated to the prostate bed at time of recurrence. CONCLUSIONS: Relapsed oligometastatic prostate cancer patients who have not received maximal local consolidative therapy to the prostate bed may have higher rates of local failure. Prospective studies are warranted investigating when prostate bed irradiation should be considered for patients after radical prostatectomy who ultimately have oligometastatic prostate cancer.

Longitudinal measurement of subcutaneous and intratibial human prostate cancer xenograft growth and response to ionizing radiation by plasma Alu and LINE-1 ctDNA: A comparison to standard methods.

BACKGROUND: Current preclinical models of metastatic prostate cancer (PCa) require sophisticated technologies and/or genetically engineered cells for the noninvasive monitoring of tumors in remote sites, such as bone. Recent developments in circulating tumor DNA (ctDNA) analysis provide an alternative method for noninvasive tumor monitoring at a low cost. Here, we sought to evaluate human Alu and LINE-1 ctDNA for the longitudinal measurement of subcutaneous and intratibial human PCa xenograft growth and response to ionizing radiation (IR) through comparison with standard slide caliper and bioluminescence measurements. MATERIAL AND METHODS: Intratibial and subcutaneous xenografts were established in male athymic nude mice using LNCaP cells that stably express firefly luciferase. A subset of tumors was treated with a single dose of IR (CT-guided focal IR, 6 Gy). Tumor measurements were simultaneously taken by slide caliper (subcutaneous only), in vivo bioluminescence imaging, and quantitative real-time PCR (qPCR) of human-specific Alu and LINE-1 ctDNA for several weeks. RESULTS: Levels of ctDNA and bioluminescence increased concordantly with subcutaneous and intratibial tumor growth. A statistically significant correlation (Spearman) was observed between ctDNA and subcutaneous tumor volume (LINE-1, r = .94 and Alu, r = .95, p < .0001), ctDNA and bioluminescence (LINE-1, r = .66 and Alu, r = .60, p < .002), and bioluminescence and tumor volume (r = .66, p = .0003). Bioluminescence and ctDNA were also significantly correlated in intratibial tumors (LINE-1, r = .82 and Alu, r = .81, p < .0001). Following external beam IR, the tumor responses varied briefly by method of measurement, but followed a similar trend. Statistically significant correlations were maintained between ctDNA and slide caliper measurement in irradiated subcutaneous tumors (LINE-1, r = .64 and Alu, r = .44, p < .02), and ctDNA and bioluminescence in intratibial tumors (LINE-1, r = .55, p = .018). CONCLUSIONS: Real-time qPCR of circulating human Alu and LINE-1 DNA provides an accurate measurement of subcutaneous and intratibial xenograft burden that is comparable with conventional bioluminescence imaging and slide caliper measurement. Transient differences in measurements were observed following tumor-targeted IR, but overall all measurements mirrored tumor growth and response.

A phase II randomized trial of RAdium-223 dichloride and SABR Versus SABR for oligomEtastatic prostate caNcerS (RAVENS).

BACKGROUND: Metastasis directed therapy (MDT) for patients with oligometastatic disease is associated with improvements in progression free survival (PFS) and overall survival (OS) compared to systemic therapy alone. Additionally, within a prostate-cancer-specific cohort, MDT is able to forestall initiation of androgen deprivation therapy (ADT) in men with hormone-sensitive, oligometastatic prostate cancer (HSOPCa) compared to observation. While MDT appears to be safe and effective in HSOPCa, a large percentage of men will eventually have disease recurrence. Patterns of failure in HSOPCa demonstrate patients tend to have recurrence in the bone following MDT, raising the question of sub-clinically-apparent osseous disease. Radium-223 dichloride is a radiopharmaceutical with structural similarity to calcium, allowing it to be taken up by bone where it emits alpha particles, and therefore might have utility in the treatment of micrometastatic osseous disease. Therefore, the primary goal of the phase II RAVENS trial is to evaluate the efficacy of MDT + radium-223 dichloride in prolonging progression free survival in men with HSOPCa. METHODS: Patients with HSOPCa and 3 or less metastases with at least 1 bone metastasis will be randomized 1:1 to stereotactic ablative radiation (SABR, also known as stereotactic body radiation therapy (SBRT)) alone vs SABR + radium-223 dichloride with a minimization algorithm to balance assignment by institution, primary intervention, prior hormonal therapy, and PSA doubling time. SABR is delivered in one to five fractions and patients in the SABR + radium-223 dichloride arm will receive six infusions of radium-223 dichloride at four-week intervals. The primary end point is progression free survival. The secondary clinical endpoints include toxicity and quality of life assessments, local control at 12 months, locoregional progression, time to distant progression, time to new metastasis, and duration of response. DISCUSSION: The RAVENS trial will be the first described phase II, non-blinded, randomized study to compare SABR +/- radium-223 dichloride in patients with HSOPCa and 3 or less metastases with at least one bone metastasis. The primary hypothesis is that SABR + radium-223 dichloride will increase median progression-free survival from 10 months in the SABR arm to 20 months in the SABR + radium-223 dichloride arm. TRIAL REGISTRATIONS: Clinicaltrials.gov. Identifier: NCT04037358. Date of Registration: July 30, 2019. Date of First Participant Enrolled: August 9, 2019. Date of Last Approved Amendment: October 16, 2019. Protocol Version: Version 5.

Magnetic nanoparticle hyperthermia for treating locally advanced unresectable and borderline resectable pancreatic cancers: the role of tumor size and eddy-current heating.

PURPOSE: Tumor volume largely determines the success of local control of borderline resectable and locally advanced pancreatic cancer with current therapy. We hypothesized that a tumor-mass normalized dose of magnetic nanoparticle hyperthermia (MNPH) with alternating magnetic fields (AMFs) reduces the effect of tumor volume for treatment. METHODS: 18 female athymic nude mice bearing subcutaneous MiaPaCa02 human xenograft tumors were treated with MNPH following intratumor injections of 5.5 mg Fe/g tumor of an aqueous suspension of magnetic iron-oxide nanoparticles. Mice were randomly divided into control (n = 5) and treated groups having small (0.15 ± 0.03 cm3, n = 4) or large (0.30 ± 0.06 cm3, n = 5) tumors. We assessed the clinical feasibility of this approach and of pulsed AMF to minimize eddy current heating using a finite-element method to solve a bioheat equation for a human-scale multilayer model. RESULTS: Compared to the control group, both small and large MiaPaCa02 subcutaneous tumors showed statistically significant growth inhibition. Conversely, there was no significant difference in tumor growth between large and small tumors. Both computational and xenograft models demonstrated higher maximum tumor temperatures for large tumors compared to small tumors. Computational modeling demonstrates that pulsed AMF can minimize nonspecific eddy current heating. CONCLUSIONS: MNPH provides an advantage to treat large tumors because the MION dose can be adjusted to increase power. Pulsed AMF, with adjusted treatment time, can enhance MNPH in challenging cases such as low MION dose in the target tissue and/or large patients by minimizing nonspecific eddy current heating without sacrificing thermal dose to the target. Nanoparticle heterogeneity in tumors remains a challenge for continued research.

Design and construction of a Maxwell-type induction coil for magnetic nanoparticle hyperthermia.

Purpose: We describe a modified Helmholtz induction coil, or Maxwell coil, that generates alternating magnetic fields (AMF) having field uniformity (≤10%) within a = 3000 cm3 volume of interest for magnetic hyperthermia research.Materials and methods: Two-dimensional finite element analysis (2D-FEA) was used for electromagnetic design of the induction coil set and to develop specifications for the required matching network. The matching network and induction coil set were fabricated using best available practices and connected to a 120 kW industrial induction heating power supply. System performance was evaluated by magnetic field mapping with a magnetic field probe, and tests were performed using gel phantoms.Results: Tests verified that the system generated a target peak AMF amplitude along the coil axis of ∼35 kA/m (peak) at a frequency of 150 ± 10 kHz while maintaining field uniformity to >90% of peak for a volume of ∼3000 cm3.Conclusions: The induction coil apparatus comprising three independent loops, i.e., Maxwell-type improves upon the performance of simple solenoid and Helmholtz coils by providing homogeneous flux density fields within a large volume while minimizing demands on power and stray fields. Experiments with gel phantoms and analytical calculations show that future translational research efforts should be devoted to developing strategies to reduce the impact of nonspecific tissue heating from eddy currents; and, that an inductor producing a homogeneous field has significant clinical potential for deep-tissue magnetic fluid hyperthermia.

A phase II randomized placebo-controlled double-blind study of salvage radiation therapy plus placebo versus SRT plus enzalutamide with high-risk PSA-recurrent prostate cancer after radical prostatectomy (SALV-ENZA).

BACKGROUND: In men with a rising PSA following radical prostatectomy, salvage radiation therapy (SRT) offers a second chance for cure. Hormonal therapy can be combined with SRT in order to increase prostate tumor control, albeit with associated higher rates of treatment side effects. This trial studies the effectiveness of SRT combined with hormonal therapy using a more potent anti-androgen with a favorable side effect profile. Enzalutamide, a next generation selective androgen receptor antagonist, is approved by the Food and Drug Administration for the treatment of metastatic castrate-resistant prostate cancer (CRPC) where it has been shown to improve overall survival in combination with androgen deprivation therapy. The primary objective of this study is to evaluate the efficacy of combination SRT and enzalutamide for freedom-from-PSA-progression. Secondary objectives include time to local recurrence within the radiation field, metastasis-free survival and safety as determined by frequency and severity of adverse events. METHODS/DESIGN: This is a randomized, double-blind, phase II, prospective, multicenter study in adult males with biochemically recurrent prostate cancer following radical prostatectomy. Following registration, enzalutamide 160 mg or placebo by mouth (PO) once daily will be administered for 6 months. Following two months of study drug, external beam radiotherapy to 66.6-70.2 Gray (Gy) will be administered to the prostate bed over 7-8 weeks while continuing daily placebo/enzalutamide. This is followed by two additional months of placebo/enzalutamide. DISCUSSION: The SALV-ENZA trial is the first phase II placebo-controlled double-blinded randomized study to test SRT in combination with a next generation androgen receptor antagonist in men with high-risk recurrent prostate cancer after radical prostatectomy. The primary hypothesis of this study is that clinical outcomes will be improved by the addition of enzalutamide compared to standard-of-care SRT alone and pave the path for phase III evaluation of this combination. TRIAL REGISTRATIONS: ClinicaltTrials.gov Identifier: NCT02203695 Date of Registration: 06/16/2014. Date of First Participant Enrollment: 04/16/2015.

Stereotactic ablative radiation therapy for oligometastatic prostate cancer delays time-to-next systemic treatment.

PURPOSE: Local ablative treatment to oligometastatic patients can result in long-term disease-free survival in some cancer patients. The importance of this treatment paradigm in prostate cancer is a rapidly evolving field. Herein, we report on the safety and preliminary clinical outcomes of a modern cohort of oligometastatic prostate cancer (OPC) patients treated with consolidative stereotactic ablative radiation (SABR). METHODS: Records of men with OPC who underwent consolidative SABR at our institution were reviewed. SABR was delivered in 1-5 fractions of 5-18 Gray. Kaplan-Meier estimates of local progression-free survival (LPFS), biochemical progression-free survival (bPFS; PSA nadir + 2), distant progression-free survival (DPFS), and time-to-next intervention (TTNI) were calculated. RESULTS: In total, 66 OPC patients were identified with consolidative SABR delivered to 134 metastases: 89 bone, 40 nodal, and 5 viscera. The majority of men (49/66) had hormone-sensitive prostate cancer (HSPC). Crude grade 1 and 2 acute toxicities were 36% and 11%, respectively, with no ≥ grade 3 toxicity. At 1 year, LPFS was 92% and bPFS and DPFS were 69%. Of the 18 men with HSPC who had deferred hormone therapy , 11 (56%) remain disease free following SABR (1-year ADT-FS was 78%). In 17 castration-resistant men, 11 had > 50% prostate-specific antigen (PSA) declines with 1-year TTNI of 30%. CONCLUSIONS: Consolidative SABR in OPC is feasible and well tolerated. The heterogeneity and small size of our series limit extrapolation of clinically meaningful outcomes following consolidative SABR in OPC, but our preliminary data suggest that this approach warrants continued prospective study.

Metastasis-Directed Therapy in Prostate Cancer. Why, When, and How?

Metastatic prostate cancer remains a life-limiting disease; while we have seen significant advances in systemic approaches which form the backbone of management, no curative paradigm yet exists. Metastasis-directed therapy (MDT) with stereotactic ablative radiotherapy (SABR) has emerged as a promising complementary technique for the management of low-volume metastatic prostate cancer. Herein we will review the rationale, potential benefits, and practical considerations associated with this approach.

Detectable end of radiation prostate specific antigen assists in identifying men with unfavorable intermediate-risk prostate cancer at high risk of distant recurrence and cancer-specific mortality.

BACKGROUND: Undetectable End of Radiation PSA (EOR-PSA) has been shown to predict improved survival in prostate cancer (PCa). While validating the unfavorable intermediate-risk (UIR) and favorable intermediate-risk (FIR) stratifications among Johns Hopkins PCa patients treated with radiotherapy, we examined whether EOR-PSA could further risk stratify UIR men for survival. METHODS: A total of 302 IR patients were identified in the Johns Hopkins PCa database (178 UIR, 124 FIR). Kaplan-Meier curves and multivariable analysis was performed via Cox regression for biochemical recurrence free survival (bRFS), distant metastasis free survival (DMFS), and overall survival (OS), while a competing risks model was used for PCa specific survival (PCSS). Among the 235 patients with known EOR-PSA values, we then stratified by EOR-PSA and performed the aforementioned analysis. RESULTS: The median follow-up time was 11.5 years (138 months). UIR was predictive of worse DMFS and PCSS (P = 0.008 and P = 0.023) on multivariable analysis (MVA). Increased radiation dose was significant for improved DMFS (P = 0.016) on MVA. EOR-PSA was excluded from the models because it did not trend towards significance as a continuous or binary variable due to interaction with UIR, and we were unable to converge a multivariable model with a variable to control for this interaction. However, when stratifying by detectable versus undetectable EOR-PSA, UIR had worse DMFS and PCSS among detectable EOR-PSA patients, but not undetectable patients. UIR was significant on MVA among detectable EOR-PSA patients for DMFS (P = 0.021) and PCSS (P = 0.033), while RT dose also predicted PCSS (P = 0.013). CONCLUSIONS: EOR-PSA can assist in predicting DMFS and PCSS among UIR patients, suggesting a clinically meaningful time point for considering intensification of treatment in clinical trials of intermediate-risk men.

Radical Prostatectomy, External Beam Radiotherapy, or External Beam Radiotherapy With Brachytherapy Boost and Disease Progression and Mortality in Patients With Gleason Score 9-10 Prostate Cancer.

Importance: The optimal treatment for Gleason score 9-10 prostate cancer is unknown. Objective: To compare clinical outcomes of patients with Gleason score 9-10 prostate cancer after definitive treatment. Design, Setting, and Participants: Retrospective cohort study in 12 tertiary centers (11 in the United States, 1 in Norway), with 1809 patients treated between 2000 and 2013. Exposures: Radical prostatectomy (RP), external beam radiotherapy (EBRT) with androgen deprivation therapy, or EBRT plus brachytherapy boost (EBRT+BT) with androgen deprivation therapy. Main Outcomes and Measures: The primary outcome was prostate cancer-specific mortality; distant metastasis-free survival and overall survival were secondary outcomes. Results: Of 1809 men, 639 underwent RP, 734 EBRT, and 436 EBRT+BT. Median ages were 61, 67.7, and 67.5 years; median follow-up was 4.2, 5.1, and 6.3 years, respectively. By 10 years, 91 RP, 186 EBRT, and 90 EBRT+BT patients had died. Adjusted 5-year prostate cancer-specific mortality rates were RP, 12% (95% CI, 8%-17%); EBRT, 13% (95% CI, 8%-19%); and EBRT+BT, 3% (95% CI, 1%-5%). EBRT+BT was associated with significantly lower prostate cancer-specific mortality than either RP or EBRT (cause-specific HRs of 0.38 [95% CI, 0.21-0.68] and 0.41 [95% CI, 0.24-0.71]). Adjusted 5-year incidence rates of distant metastasis were RP, 24% (95% CI, 19%-30%); EBRT, 24% (95% CI, 20%-28%); and EBRT+BT, 8% (95% CI, 5%-11%). EBRT+BT was associated with a significantly lower rate of distant metastasis (propensity-score-adjusted cause-specific HRs of 0.27 [95% CI, 0.17-0.43] for RP and 0.30 [95% CI, 0.19-0.47] for EBRT). Adjusted 7.5-year all-cause mortality rates were RP, 17% (95% CI, 11%-23%); EBRT, 18% (95% CI, 14%-24%); and EBRT+BT, 10% (95% CI, 7%-13%). Within the first 7.5 years of follow-up, EBRT+BT was associated with significantly lower all-cause mortality (cause-specific HRs of 0.66 [95% CI, 0.46-0.96] for RP and 0.61 [95% CI, 0.45-0.84] for EBRT). After the first 7.5 years, the corresponding HRs were 1.16 (95% CI, 0.70-1.92) and 0.87 (95% CI, 0.57-1.32). No significant differences in prostate cancer-specific mortality, distant metastasis, or all-cause mortality (≤7.5 and >7.5 years) were found between men treated with EBRT or RP (cause-specific HRs of 0.92 [95% CI, 0.67-1.26], 0.90 [95% CI, 0.70-1.14], 1.07 [95% CI, 0.80-1.44], and 1.34 [95% CI, 0.85-2.11]). Conclusions and Relevance: Among patients with Gleason score 9-10 prostate cancer, treatment with EBRT+BT with androgen deprivation therapy was associated with significantly better prostate cancer-specific mortality and longer time to distant metastasis compared with EBRT with androgen deprivation therapy or with RP.

A phase II randomized trial of Observation versus stereotactic ablative RadiatIon for OLigometastatic prostate CancEr (ORIOLE).

BACKGROUND: We describe a randomized, non-blinded Phase II interventional study to assess the safety and efficacy of stereotactic ablative radiotherapy (SABR) for hormone-sensitive oligometastatic prostate adenocarcinoma, and to describe the biology of the oligometastatic state using immunologic, cellular, molecular, and functional imaging correlates. 54 men with oligometastatic prostate adenocarcinoma will be accrued. The primary clinical endpoint will be progression at 6 months from randomization with the hypothesis that SABR to all metastases will forestall progression by disrupting the metastatic process. Secondary clinical endpoints will include local control at 6 months post-SABR, toxicity and quality of life, and androgen deprivation therapy (ADT)-free survival (ADT-FS). Further fundamental analysis of the oligometastatic state with be achieved through correlation with investigational 18F-DCFPyL PET/CT imaging and measurement of circulating tumor cells, circulating tumor DNA, and circulating T-cell receptor repertoires, facilitating an unprecedented opportunity to characterize, in isolation, the effects of SABR on the dynamics of and immunologic response to oligometastatic disease. METHODS/DESIGN: Patients will be randomized 2:1 to SABR or observation with minimization to balance assignment by primary intervention, prior hormonal therapy, and PSA doubling time. Progression after 6 months will be compared using Fisher's exact test. Hazard ratios and Kaplan-Meier estimates of progression free survival (PFS), ADT free survival (ADT-FS), time to locoregional progression (TTLP) and time to distant progression (TTDP) will be calculated based on an intention-to-treat. Local control will be assessed using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. Withdrawal from the study prior to 6 months will be counted as progression. Adverse events will be summarized by type and grade. Quality of life pre- and post- SABR will be measured by Brief Pain Inventory. DISCUSSION: The ORIOLE trial is the first randomized, non-blinded Phase II interventional study in the North America evaluating the safety and efficacy of SABR in oligometastatic hormone-sensitive prostate cancer. Leading-edge laboratory and imaging correlates will provide unique insight into the effects of SABR on the oligometastatic state. TRIAL REGISTRATIONS: ClinicalTrials.gov Identifier: NCT02680587. URL of Registry: https://clinicaltrials.gov/show/NCT02680587 Date of Registration: 02/08/2016. Date of First Participant Enrollment: 05/23/2016.

A functional screen identifies miRNAs that inhibit DNA repair and sensitize prostate cancer cells to ionizing radiation.

MicroRNAs (miRNAs) have been implicated in DNA repair pathways through transcriptional responses to DNA damaging agents or through predicted miRNA regulation of DNA repair genes. We hypothesized that additional DNA damage regulating miRNAs could be identified by screening a library of 810 miRNA mimetics for the ability to alter cellular sensitivity to ionizing radiation (IR). A prostate cancer Metridia luciferase cell model was applied to examine the effects of individual miRNAs on IR sensitivity. A large percentage of miRNA mimetics were found to increase cellular sensitivity to IR, while a smaller percentage were protective. Two of the most potent IR sensitizing miRNAs, miR-890 and miR-744-3p, significantly delayed IR induced DNA damage repair. Both miRNAs inhibited the expression of multiple components of DNA damage response and DNA repair. miR-890 directly targeted MAD2L2, as well as WEE1 and XPC, where miR-744-3p directly targeted RAD23B. Knock-down of individual miR-890 targets by siRNA was not sufficient to ablate miR-890 radiosensitization, signifying that miR-890 functions by regulating multiple DNA repair genes. Intratumoral delivery of miR-890 mimetics prior to IR therapy significantly enhanced IR therapeutic efficacy. These results reveal novel miRNA regulation of DNA repair and identify miR-890 as a potent IR sensitizing agent.

GSTP1 Loss results in accumulation of oxidative DNA base damage and promotes prostate cancer cell survival following exposure to protracted oxidative stress.

BACKGROUND: Epigenetic silencing of glutathione S-transferase π (GSTP1) is a hallmark of transformation from normal prostatic epithelium to adenocarcinoma of the prostate. The functional significance of this loss is incompletely understood. The present study explores the effects of restored GSTP1 expression on glutathione levels, accumulation of oxidative DNA damage, and prostate cancer cell survival following oxidative stress induced by protracted, low dose rate ionizing radiation (LDR). METHODS: GSTP1 protein expression was stably restored in LNCaP prostate cancer cells. The effect of GSTP1 restoration on protracted LDR-induced oxidative DNA damage was measured by GC-MS quantitation of modified bases. Reduced and oxidized glutathione levels were measured in control and GSTP1 expressing populations. Clonogenic survival studies of GSTP1- transfected LNCaP cells after exposure to protracted LDR were performed. Global gene expression profiling and pathway analysis were performed. RESULTS: GSTP1 expressing cells accumulated less oxidized DNA base damage and exhibited decreased survival compared to control LNCaP-Neo cells following oxidative injury induced by protracted LDR. Restoration of GSTP1 expression resulted in changes in modified glutathione levels that correlated with GSTP1 protein levels in response to protracted LDR-induced oxidative stress. Survival differences were not attributable to depletion of cellular glutathione stores. Gene expression profiling and pathway analysis following GSTP1 restoration suggests this protein plays a key role in regulating prostate cancer cell survival. CONCLUSIONS: The ubiquitous epigenetic silencing of GSTP1 in prostate cancer results in enhanced survival and accumulation of potentially promutagenic DNA adducts following exposure of cells to protracted oxidative injury suggesting a protective, anti-neoplastic function of GSTP1. The present work provides mechanistic backing to the tumor suppressor function of GSTP1 and its role in prostate carcinogenesis.

Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy.

Checkpoint blockade immunotherapy has received mainstream attention as a result of striking and durable clinical responses in some patients with metastatic disease and a reasonable response rate in many tumour types. The activity of checkpoint blockade immunotherapy is not restricted to melanoma or lung cancer, and additional indications are expected in the future, with responses already reported in renal cancer, bladder cancer, and Hodgkin's lymphoma among many others. Additionally, the interactions between radiation and the immune system have been investigated, with several studies describing the synergistic effects on local and distant tumour control when radiation therapy is combined with immunotherapy. Clinical enthusiasm for this approach is strengthened by the many ongoing trials combining immunotherapy with definitive and palliative radiation. Herein, we discuss the biological and mechanistic rationale behind combining radiation with checkpoint blockade immunotherapy, with a focus on the preclinical data supporting this potentially synergistic combination. We explore potential hypotheses and important considerations for clinical trial designs. Finally, we reintroduce the notion of radiosensitising immunotherapy, akin to radiosensitising chemotherapy, as a potential definitive therapeutic modality.

Bromopyridone Nucleotide Analogues, Anoxic Selective Radiosensitizing Agents That Are Incorporated in DNA by Polymerases.

Ionizing radiation is frequently used to kill tumor cells. However, hypoxic solid tumor cells are more resistant to this treatment, providing the impetus to develop molecules that sensitize cells to ionizing radiation. 5-Bromo-2'-deoxyuridine (BrdU) has been investigated as a radiosensitizing agent in the lab and clinic for almost 5 decades. Recent reports that BrdU yields DNA interstrand cross-links (ICLs) in non-base-paired regions motivated us to develop radiosensitizing agents that generate cross-links in duplex DNA selectively under anoxic conditions. 4-Bromo- and 5-bromopyridone analogues of BrdU were synthesized and incorporated into oligonucleotides via solid-phase synthesis. Upon irradiation, these molecules yield DNA interstrand cross-links under anaerobic conditions. The respective nucleotide triphosphates are substrates for some DNA polymerases. ICLs are produced upon irradiation under anoxic conditions when the 4-bromopyridone is present in a PCR product. Because the nucleoside analogue is a poor phosphorylation substrate for human deoxycytidine kinase, a pro-nucleotide form of the 4-bromopyridone was used to incorporate this analogue into cellular DNA. Despite these efforts, the 4-bromopyridone nucleotide was not detected in cellular DNA. Although these molecules are improvements over previously reported nucleotide analogues designed to be hypoxic radiosensitizing agents, additional advances are needed to create molecules that function in cells.

Stereotactic radiation therapy combined with immunotherapy: augmenting the role of radiation in local and systemic treatment.

Stereotactic radiosurgery and stereotactic body radiation therapy are two contemporary radiation modalities that can treat tumors in any area of the body using highly focused radiation. Recently, immunotherapy has established itself as a viable and powerful anticancer treatment. In this review we detail the rationale supporting a combination of immunotherapy and stereotactic radiation. Additionally, we discuss the evidence for the immune stimulatory effects of focused radiation and the role that radiation may play in enhancing the systemic treatment effects of immunotherapy.

Magnetic nanoparticle hyperthermia enhances radiation therapy: A study in mouse models of human prostate cancer.

PURPOSE: We aimed to characterise magnetic nanoparticle hyperthermia (mNPH) with radiation therapy (RT) for prostate cancer. METHODS: Human prostate cancer subcutaneous tumours, PC3 and LAPC-4, were grown in nude male mice. When tumours measured 150 mm3 magnetic iron oxide nanoparticles (MIONPs) were injected into tumours to a target dose of 5.5 mg Fe/cm3 tumour, and treated 24 h later by exposure to alternating magnetic field (AMF). Mice were randomly assigned to one of four cohorts to characterise (1) intratumour MIONP distribution, (2) effects of variable thermal dose mNPH (fixed AMF peak amplitude 24 kA/m at 160 ± 5 kHz) with/without RT (5 Gy), (3) effects of RT (RT5: 5 Gy; RT8: 8 Gy), and (4) fixed thermal dose mNPH (43 °C for 20 min) with/without RT (5 Gy). MIONP concentration and distribution were assessed following sacrifice and tissue harvest using inductively coupled plasma mass spectrometry (ICP-MS) and Prussian blue staining, respectively. Tumour growth was monitored and compared among treated groups. RESULTS: LAPC-4 tumours retained higher MIONP concentration and more uniform distribution than did PC3 tumours. AMF power modulation provided similar thermal dose for mNPH and combination therapy groups (CEM43: LAPC-4: 33.6 ± 3.4 versus 25.9 ± 0.8, and PC3: 27.19 ± 0.7 versus 27.50 ± 0.6), thereby overcoming limitations of MIONP distribution and yielding statistically significant tumour growth delay. CONCLUSION: PC3 and LAPC-4 tumours represent two biological models that demonstrate different patterns of nanoparticle retention and distribution, offering a model to make comparisons of these effects for mNPH. Modulating power for mNPH offers potential to overcome limitations of MIONP distribution to enhance mNPH.

Irreversible inhibition of DNA polymerase ß by small-molecule mimics of a DNA lesion.

Abasic sites are ubiquitous DNA lesions that are mutagenic and cytotoxic but are removed by the base excision repair pathway. DNA polymerase ß carries out two of the four steps during base excision repair, including a lyase reaction that removes the abasic site from DNA following incision of its 5'-phosphate. DNA polymerase ß is overexpressed in cancer cells and is a potential anticancer target. Recently, DNA oxidized abasic sites that are produced by potent antitumor agents were shown to inactivate DNA polymerase ß. A library of small molecules whose structures were inspired by the oxidized abasic sites was synthesized and screened for the ability to irreversibly inhibit DNA polymerase ß. One candidate (3a) was examined more thoroughly, and modification of its phosphate backbone led to a molecule that irreversibly inactivates DNA polymerase ß in solution (IC50 ≈ 21 µM), and inhibits the enzyme's lyase activity in cell lysates. A bisacetate analogue is converted in cell lysates to 3a. The bisacetate is more effective in cell lysates, more cytotoxic in prostate cancer cells than 3a and potentiates the cytotoxicity of methyl methanesulfonate between 2- and 5-fold. This is the first example of an irreversible inhibitor of the lyase activity of DNA polymerase ß that works synergistically with a DNA damaging agent.

Androgen withdrawal fails to induce detectable tissue hypoxia in the rat prostate.

BACKGROUND: It has been reported that significant hypoxia may occur in the rat prostate following androgen deprivation (AD). It is well known that hypoxia substantially reduces radiation sensitivity of cells both in vitro and in vivo. Given that contemporary management of men with intermediate and high-risk prostate cancer includes the use of neoadjuvant androgen suppression and radiation, AD-induced hypoxia in the prostate could result in suboptimal therapeutic results. Given this concern, we fully investigate possible AD-induced hypoxia in the ventral prostate (VP) of adult rats by two independent methods. METHODS: Tissue pO2 levels in the VP of adult Spraque-Dawley rats were evaluated prior to and at various time points following castration by two independent techniques. First, an Oxylab tissue oxygen monitor with a 240 µm probe was used for quantitative monitoring of global VP oxygenation. Second, fluorescence immunohistochemistry using the hypoxia marker EF5, known to be metabolically activated by hypoxic cells, was used to evaluate cell-to-cell variation in hypoxia at various days post-castration. RESULTS: Neither the oxygen probe nor EF5 method demonstrate any substantive change in pO2 levels in the rat VP at any time point post-castration. CONCLUSIONS: We find no evidence that the rat VP becomes hypoxic at any point following castration using an animal model that closely mimics the human prostate. These data are in contrast to previous reports suggesting prostatic hypoxia occurs following AD and provide assurance that our present therapeutic strategy of neoadjuvant AD followed by radiation is not compromised by AD-induced tissue hypoxia.