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Abstract Previous studies show increased sensitivity of older mice (28–29 months) compared with young adult mice (3 months, possessing a mature immune system) to radiation-induced GI lethality. Age-dependent lethality was associated with higher levels of apoptotic stem cells in small intestinal crypts that correlated with sphingomyelinase activity, a source of pro-apoptotic ceramide. The objective of this study is to determine whether the cycling crypt base columnar cells (CBCs) in aging animals are specifically more sensitive to radiation effects than the CBCs in young adult mice, and to identify factors that contribute to increased radiosensitivity. Mortality induced by subtotal body radiation was assessed at different doses (13 Gy, 14 Gy, and 15 Gy) in young adult mice versus older mice. Each dose was evaluated for the occurrence of lethal GI syndrome. A higher death rate due to radiation-induced GI syndrome was observed in older mice as compared with young adult mice: 30 vs. 0% at 13 Gy, 90 vs. 40% at 14 Gy, and 100 vs. 60% at 15 Gy. Radiation-induced damage to crypts was determined by measuring crypt regeneration (H&E staining, Ki67 expression), CBC biomarkers (lgr5 and ascl2), premature senescence (SA-β-gal activity), and apoptosis of CBCs. At all three doses, crypt microcolony survival assays showed that the older mice had fewer regenerating crypts at 3.5 days post-radiation treatment. Furthermore, in the older animals, baseline CBCs numbers per circumference were significantly decreased, correlating with an elevated apoptotic index. Analysis of tissue damage showed an increased number of senescent CBCs per crypt circumference in older mice relative to younger mice, where the latter was not significantly affected by radiation treatment. It is concluded that enhanced sensitivity to radiation-induced GI syndrome and higher mortality in older mice can be attributed to a decreased capacity to regenerate crypts, presumably due to increased apoptosis and senescence of CBCs.

PurposeTo explore whether prostate motion mitigation using the rectal distension-mediated technique is safe and effective in stereotactic ablative radiation therapy (SABR) salvage treatment of intraprostatic cancer recurrences following initial radiotherapy for primary prostate cancer.Materials and methodsBetween July 2013 and December 2020, 30 patients received salvage SABR for 68Ga- PSMA-11 PET/CT-detected intra-prostatic relapses. Median time from primary RT to salvage reirradiation was 70.2 (IQR, 51.3-116.0) months. Median PSA at retreatment was 3.6 ng/mL (IQR, 1.9-6.2). Rectal distension-mediated SABR was achieved with a 150-cm3 air-inflated endorectal balloon and a Foley catheter loaded with 3 beacon transponders was used for urethra visualization and on-line tracking. MRI-based planning employed a 2-mm expansion around the planned target volume (PTV), reduced to 0-mm at the interface with critical organs at risk (OARs). Volumetric Modulated Arc Therapy (VMAT) permitted a 20% dose reduction of the urethra. VMAT simultaneous integrated boost (SIB) of the dominant intraprostatic lesion was deployed when indicated. Median SABR dose was 35 Gy (7 Gy per fraction over 5 consecutive days; range 35-40 Gy). Toxicity assessment used CTCAE v.4 criteria.ResultsMedian follow-up was 44 months (IQR, 18-60). The actuarial 3- and 4-year biochemical relapse free survival was 53.4% and 47.5%, respectively. Intraprostatic post-salvage relapse by PSMA PET/CT was 53.3%. Acute grade 2 and 3 genitourinary (GU) toxicities were 20% and 0%, respectively. There were no instances of acute grade ≥2 rectal (GI) toxicity. Late grade 2 and 3 GU toxicities occurred in 13.3% and 0% of patients, respectively. There were no instances of grade ≥2 late rectal toxicity. Patient-reported QOL measures showed an acute transient deterioration in the urinary domain 1 month after treatment but returned to baseline values at 3 months. The median IPSS scores rose over baseline (≥5 points in 53% of patients) between month 6 and 12 post-treatment as a result of urinary symptoms flare, eventually receding at 18 months. The bowel domain metrics had no appreciable changes over time.ConclusionPursuit of local control in intraprostatic failures is feasible and can be achieved with an acceptably low toxicity profile associated with effective OAR sparing.

Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5–positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane–dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury–induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.

Purpose: Early positron emission tomography–derived metrics post–oligometastasis radioablation may predict impending local relapses (LRs), providing a basis for a timely ablation. Methods and Materials: Positron emission tomography data of 623 lesions treated with either 24 Gy single-dose radiation therapy (SDRT) (n = 475) or 3 × 9 Gy stereotactic body radiation therapy (SBRT) (n = 148) were analyzed in a training data set (n = 246) to obtain optimal cutoffs for pretreatment maximum standardized uptake value (SUVmax) and its 3-month posttreatment decline (ΔSUVmax) in predicting LR risk, validated in a data set unseen to testing (n = 377). Results: At a median of 21.7 months, 91 lesions developed LRs: 39 of 475 (8.2%) after SDRT and 52 of 148 (35.1%) after SBRT. The optimal cutoff values were 12 for SUVmax and –75% for ΔSUVmax. Bivariate SUVmax/ΔSUVmax permutations rendered a 3-tiered LR risk stratification of dual-favorable (low risk), 1 adverse (intermediate risk) and dual-adverse (high risk). Actuarial 5-year local relapse-free survival rates were 93.9% versus 89.6% versus 57.1% (P < .0001) and 76.1% versus 48.3% versus 8.2% (P < .0001) for SDRT and SBRT, respectively. The SBRT area under the ROC curve was 0.71 (95% CI, 0.61-0.79) and the high-risk subgroup yielded a 76.5% true positive LR prediction rate. Conclusions: The SBRT dual-adverse SUVmax/ΔSUVmax category LR prediction power provides a basis for prospective studies testing whether a timely ablation of impending LRs affects oligometastasis outcomes.

PurposeTo explore whether the rectal distension-mediated technique, harnessing human physiology to achieve intrafractional prostate motion mitigation, enables urethra sparing by inverse dose painting, thus promoting dose escalation with extreme hypofractionated stereotactic ablative radiotherapy (SABR) in prostate cancer.Materials and MethodsBetween June 2013 and December 2018, 444 patients received 5 × 9 Gy SABR over 5 consecutive days. Rectal distension-mediated SABR was employed via insertion of a 150-cm3 air-inflated endorectal balloon. A Foley catheter loaded with 3 beacon transponders was used for urethra visualization and online tracking. MRI-based planning using Volumetric Modulated Arc Therapy - Image Guided Radiotherapy (VMAT-IGRT) with inverse dose painting was employed in delivering the planning target volume (PTV) dose and in sculpting exposure of organs at risk (OARs). A 2-mm margin was used for PTV expansion, reduced to 0 mm at the interface with critical OARs. All plans fulfilled Dmean ≥45 Gy. Target motion ≥2 mm/5 s motions mandated treatment interruption and target realignment prior to completion of the planned dose delivery.ResultsPatient compliance to the rectal distension-mediated immobilization protocol was excellent, achieving reproducible daily prostate localization at a patient-specific retropubic niche. Online tracking recorded ≤1-mm intrafractional target deviations in 95% of treatment sessions, while target realignment in ≥2-mm deviations enabled treatment completion as scheduled in all cases. The cumulative incidence rates of late grade ≥2 genitourinary (GU) and gastrointestinal (GI) toxicities were 5.3% and 1.1%, respectively. The favorable toxicity profile was corroborated by patient-reported quality of life (QOL) outcomes. Median prostate-specific antigen (PSA) nadir by 5 years was 0.19 ng/ml. The cumulative incidence rate of biochemical failure using the Phoenix definition was 2%, 16.6%, and 27.2% for the combined low/favorable–intermediate, unfavorable intermediate, and high-risk categories, respectively. Patients with a PSA failure underwent a 68Ga-labeled prostate-specific membrane antigen (68Ga-PSMA) scan showing a 20.2% cumulative incidence of intraprostatic relapses in biopsy International Society of Urological Pathology (ISUP) grade ≥3.ConclusionThe rectal distension-mediated technique is feasible and well tolerated. Dose escalation to 45 Gy with urethra-sparing results in excellent toxicity profiles and PSA relapse rates similar to those reported by other dose-escalated regimens. The existence of intraprostatic recurrences in patients with high-risk features confirms the notion of a high α/β ratio in these phenotypes resulting in diminished effectiveness with hypofractionated dose escalation.

After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.

These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.

BACKGROUND:Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates. METHODOLOGY/PRINCIPAL FINDINGS:MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax. CONCLUSIONS/SIGNIFICANCE:Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either “normalize” dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C16-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase+/+ mice or asmase−/− littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase−/− mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either "normalize" dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C(16)-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase(+/+) mice or asmase(-/-) littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase(-/-) mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.