To this day, most preclinical research and clinical use of chemo and radiation cancer therapies do not take sex-differences into consideration, despite women having higher risk of adverse treatment toxicities and making up a larger survivor population than men. Additionally, evolving advances in cancer treatments continue to increase clinical success and there remains a growing unmet need to characterize the long-term toxic effects of frontline therapies, used in both women and men, on cancer survivors’ quality of life. To address these needs, a series of experiments examined the sex-specific effects of paclitaxel, a commonly used taxane family chemotherapeutic, and thoracic radiotherapy treatments on rodents’ cognitive and/or cardiac function. Female and male Wild Type (WT) mice were treated with paclitaxel (150 and 300 mg/kg) administered weekly over 6 weeks or exposed to 19 Gy cardiac irradiation. Systemic, behavioral, and cardiovascular endpoints were examined to assess paclitaxel-induced toxicity and cardiac function and histology were used to assess thoracic radiotherapy-induced toxicity. Interestingly, female WT mice exhibited enhanced tolerance compared to male WT mice in both treatment regimens. To gain more understanding of the observed female-specific protection, RhoB-deficient and aged female WT mice (22 months) were used. RhoB GTPase deficiency was interrogated for the possible impact of sex-hormones in cancer therapy toxicity (RhoB proteins being associated with estrogen receptors in females and cardiac fibrosis in males). In female mice, RhoB deficiency and advanced age had no impact on paclitaxel-induced neurocognitive impairment, but RhoB deficiency did compromise survival after radiotherapy. Conversely, RhoB deficiency reduced paclitaxel-induced neurotoxicity in males and extended male survival after radiation. In summary, this study revealed novel sexually dimorphic toxicities for commonly used taxane and radiation treatments, mediated in-part through age and/or RhoB-dependent pathways, which are associated with estrogen function in females and immune function in both sexes. This study provides additional data to the growing preclinical and clinical body of literature revealing sex-differences in disease progression and the critical roles of sex-hormones in immune response mechanisms. Therefore, further research is justified to elucidate estrogen and androgen-dependent mechanisms involved in the sex- and organ-specific toxicities observed after taxane and radiation therapies in preclinical and clinical research.

Purpose of review

Recent findings

Summary

Purpose: FLASH radiotherapy's enhanced normal tissue protective effect without compromising cancer treatment efficacy has stimulated investigations exploring its mechanisms. To this date, mitochondrial endpoints after FLASH radiotherapy have not been explored. This master’s thesis aims to narrate the in vivo accounts of the normal tissue-sparing FLASH effect observed across organ systems and report the findings in ATP production and mitochondrial dynamics in the murine brain as possible explanations for the FLASH effect.

Methods: Two cohorts received 10 Gy whole-brain FLASH and conventional dose rate irradiations at two different facilities. Stanford FLASH dose rate per pulse was 5.3 × 10^5 Gy/s Gy/s, with mean dose rate of 225 Gy/s. CHUV FLASH dose rate was 5.6 × 10^6 Gy/s, where a single 1.8 microsecond pulse was used to administer the total dose. The conventional dose rate was 0.1 Gy/s at both locations. At 4 months post-irradiation, ATP concentration and OPA-1 mitochondrial fusion protein levels were measured from the hippocampus and frontal cortex tissues and compared across the treatment groups via one-way ANOVA and Pearson's correlation analyses.

Results: The hippocampus tissues from Stanford FLASH- and CHUV conventional-irradiated groups had significantly different associations between long and short OPA-1 isoforms compared to the control (p=0.005 and p=0.012, respectively). Although the strengths of associations in the hippocampal tissues were weaker than their cortex counterparts across the cohorts, only the Stanford CONV and FLASH irradiated groups showed significant and marginally significant differences between the brain regions (p=0.0496 and 0.057, respectively). These differences indicate that the mitochondrial dynamics may be dysregulated in the hippocampus at 4 months post-irradiation. The individual levels of ATP, L-OPA1, S-OPA1, and the ratios of isoforms did not significantly differ across the treatment groups.

Conclusion: Despite the low sample size (n≤12), there may be evidence for dysregulated mitochondrial dynamics in the hippocampus after CONV and FLASH irradiations at a late time point. Further study should confirm the radiation-induced effect on mitochondrial physiology through more refined tests.

Radiotherapy has long been used to control tumor growth by causing ionization of cellular macromolecules including DNA, RNA, lipids and proteins, leading to lethal or sublethal damage to a cell. Typically, convention radiotherapy (CONV-RT) is delivered at a dose rate between 0.07–0.1 Gy⋅s−1 and fractionated over an extended treatment plan (1 -2 months), allowing healthy normal tissue to mend sublethal damage while repair-compromised tumor tissue accumulates damage. Previous studies have identified that fractions under 2 Gy are required to reduce long-term neurocognitive damage1. These normal tissue dose tolerances limit the therapeutic benefit of radiotherapy. A recent breakthrough in radiation delivery modalities have shown that using ultra-high dose rates (>100 Gy⋅s−1), or ‘FLASH’ irradiation, can reduce cognitive damage, spare normal tissue injury, and minimize harm to critical systems like the blood brain barrier (BBB), all while maintaining isoefficient tumor control2–4. This new radiation modality has the potential to greatly extend the therapeutic benefit of radiotherapy while limiting the unintended neurological sequelae that are frequently caused by treatment.

At this point, our lab has produced substantial evidence that FLASH irradiation reduces damage done to the CNS normal tissue, protecting the homeostasis, and potentially reducing the onset and severity of late radiation-injury in brain tumor survivors subjected to cranial radiotherapy. Experiments performed by ourselves and collaborators have examined the FLASH effect using preclinical models of fish, rodents, cats, and mini-pigs2,5–8 and has successfully been used clinically in a single patient to treat multiresistant CD30+ T-cell cutaneous lymphoma4. While a plethora of data is available on the FLASH effect, the underlying mechanisms are still not fully understood. Our lab has shown that oxygenation levels of the tissue play a vital role in radiation induce tissue damage through mitigation of reactive oxygen species (ROS)2; however, there are likely other key mechanisms at play. The importance of tissue oxygenation and the role the BBB plays in maintaining CNS homeostasis are why we consider the vasculature to be a key element in deciphering the FLASH effect.

The Limoli lab has been publishing regularly on the benefits of FLASH irradiation in pre-clinical models for the last 4 years, highlighting the normal tissue sparing effects in the CNS. My work in particular has focused on the effect of both CONV and FLASH irradiation on the normal tissue and the BBB. I performed initial experiments utilizing FLASH and CONV radiation to determine effects on the BBB at early timepoints which led to a publication in Radiation Research9. Following this, I spent time exploring the literature behind CNS vasculature, radiotherapy, and new experimental therapeutic techniques which culminated into a published review in Free Radical Biology and Medicine10. I have used this rationale to examine the cognitive and vasculature effects of FLASH on juveniles11, a particularly radiation susceptible population. Results from the juvenile model have guided us to explore the neurological mechanistic basis of the effect we find in an adult model while simultaneously testing the threshold of dose tolerance. Further experiments on a similar cohort of adult animals to measure blood flow and tissue oxygenation levels using spatial frequency and domain imaging. Results from this publication are expected to be concluded soon. The objective of this dissertation is to, in part, determine if the normal tissue sparing effect of FLASH irradiation reduces radiation-induced pathology of the BBB, further explicate the neuro-mechanistic basis of cognitive protection and determine the effects on tumor vascularization. Results produced from this body of work will not only provide additional justification for clinical translation, but it may also help illuminate key mechanistic differences between cancer and normal tissue that may one day be used for therapeutic gain.

My field of focus has been the effects of ultra-high dose rate FLASH radiotherapy in pre-clinical models. In particular, my work has focused on both clinical and FLASH dose rates and their effect on the normal tissue, cognition, neuroinflammation, and the BBB. My graduate work led to numerous publications and is challenging the fundamental principles used in the field to improve .

ABSTRACT OF THE DISSERTATION

Radiation- and Chemotherapy-Induced Cognitive Deficits: Neural-Derived Extracellular Vesicles as Translational Therapiesby Sarah Mae Smith Doctor of Philosophy in Environmental Health Sciences University of California, Irvine, 2022 Professor Charles L. Limoli, Chair

Approximately 24,000 patients are diagnosed with brain tumors each year, which represents an estimated 166,000 people living with brain cancer as of 2015. Radiotherapy constitutes one of the principle therapies for primary and metastatic brain tumors along with chemotherapy and surgery, with roughly 200,000 patients receiving brain radiation treatment each year in the United States. One of the most common and most damaging iatrogenic effects of cancer treatment is cognitive dysfunction, including impairments in working memory, learning ability, executive function, and attention. The deleterious effects of cranial ionizing radiation exposure are exacerbated by concomitant use of chemotherapeutic agents that elevate neurotoxicity. While the mechanisms underlying radiation-induced cognitive dysfunction have not yet been elucidated, much of the underlying pathology believed to be contributory is related to decreased hippocampal neurogenesis, increased neuroinflammation, microvascular injury and alterations in neuronal structure that disrupts dendritic morphology, spine density and synaptic proteins. Stem cell-based transplantation strategies have also shown considerable promise in ameliorating the negative effects of cranial irradiation. My initial work in the Limoli lab sought to examine the impact of bilateral and unilateral hemisphere transplantations of human neural stem cell (hNSC) and hNSC-derived extracellular vesicles on the structural integrity of hippocampal neurons in the irradiated rodent brain, in order to ascertain the extent and range of the beneficial effects of cell-based therapies as a function of distance from the transplant site (which has important therapeutic implications for dosing and administration). We found that stem cell transplantation decreases the number of activated microglia at one month post-irradiation, signifying a reduction in neuroinflammation. In addition, grafted stem cells preserved host neuronal structure one month and four months after irradiation. Stem cell transplantation also allayed the increased expression of the major synaptic scaffolding protein PSD-95 caused by irradiation. Further, as clinical utility of stem cell transplantation strategies to offset normal tissue damage caused by irradiation may be limited by the downstream potential for teratoma formation and immune rejection, we also attempted to circumvent such caveats by evaluating the promise of using transplanted human neural stem cell-derived extracellular vesicles in place of stem cells. We showed that unilateral transplantation of both stem cells and extracellular vesicles 48 hours post-irradiation signals for the renormalization of synaptic proteins such as PSD-95 in the contralateral hippocampus, and found that unilateral transplantation has the same protective effects on neuroinflammation, dendritic morphology, and spine density as bilateral transplantation. My later studies evaluate the impact of GABAergic neuron-derived extracellular vesicles on irradiation- and chemotherapy-associated neurocognitive deficits associated with clinically-relevant treatments, including fractionated radiation and the chemotherapeutic agent temozolomide (TMZ). We first examined whether retro-orbital injection of extracellular vesicles (EV) derived from GABAergic neurons can alleviate radiation-associated deficits in cognition at one month following treatment. GABAergic neuron-derived EVs are proposed to improve cognition after cancer therapy through providing an inhibitory impetus to compensate for irradiation- and chemotherapy-induced hippocampal hyperexcitation, as is apparent in preliminary electrophysiology studies in irradiated animals. Our behavioral data suggests that fractionated irradiation has deleterious neurological effects as determined by performance on hippocampus-, prefrontal cortex- and amygdala-dependent behavioral tasks, and that retro-orbital injection of GABAergic EVs attenuates some of these effects and rescues cognition. Intriguingly, these benefits were not replicated with the application of glutamatergic neuron-derived EVs, which did not reverse the observed decrements in cognitive function after irradiation. In exploring the neurobiochemical basis for this rescue in cognition on neuronal morphology, synaptic integrity, and neurotrophic factors, we found that dendritic spine density in the dentate gyrus of the hippocampus is significantly reduced following irradiation, but statistically similar to controls following the application of retro-orbital GABAergic EVs. We also determined that irradiation reduced levels of neurotrophins in the hippocampus, but retro-orbital injections of GABAergic EVs ameliorated this decrease. We built upon these studies by next exploring whether retro-orbital injection of GABAergic neuron-derived EVs can ameliorate the adverse effects associated with a systemic insult to the brain, intraperitoneal injections of the chemotherapeutic agent temozolomide (TMZ), in the context of a clinically-relevant treatment protocol also including fractionated radiation. Temozolomide is an alkylating agent that crosses the blood-brain barrier and is used primarily to treat glioblastoma. Again, several of the behavioral tasks show neurocognitive decrements following the TMZ+IRR protocol, which were improved with the GABAergic EV injections. Likewise, tissue from the hippocampi of these animals was analyzed for neurotrophic factor levels, which revealed that glial cell-derived neurotrophic factor was reduced after irradiation but improved in the GABAergic EV cohort. Finally, we have been evaluating the optimal timecourse for delivery of GABAergic neuron-derived EVs being employed to rescue cognitive decrements associated with irradiation by examining whether retro-orbitally injecting GABAergic neuron-derived EVs the week before the first fractionated radiation dose could result in a preemptive protective effect. We have identified that the protective paradigm of GABAergic EV application mitigates radiation-induced cognitive injury and enhances the quantity of neurotrophins in the hippocampus relative to irradiated animals.