Brain volume and microglial density changes are correlated in a juvenile mouse model of cranial radiation and CSF1R inhibitor treatment.

Microglia have been shown to proliferate and become activated following cranial radiotherapy (CRT), resulting in a chronic inflammatory response. We investigated the role of microglia in contributing to widespread volume losses observed in the brain following CRT in juvenile mice. To manipulate microglia, we used low-dose treatment with a highly selective CSF1R inhibitor called PLX5622 (PLX). We hypothesized that alteration of the post-CRT microglia population would lead to changes in brain development outcomes, as evaluated by structural MRI. Wild-type C57BL/6J mice were provided with daily intraperitoneal injections of PLX (25 mg/kg) or vehicle from postnatal day (P)14 to P19. Mice also received whole-brain irradiation (7 Gy) or sham irradiation (0 Gy) at 16 days of age. In one cohort of mice, immunohistochemical assessment in tissue sections was conducted to assess the impact of the selected PLX and CRT doses as well as their combination. In a separate cohort, mice were imaged using MRI at P14 (pretreatment), P19, P23, P42 and P63 in order to assess induced volume changes, which were measured based on structures from a predefined atlas. We observed that PLX and radiation treatments led to sex-specific changes in the microglial cell population. Across treatment groups, MRI-detected anatomical volumes at P19 and P63 were associated with microglia and proliferating microglia densities, respectively. Overall, our study demonstrates that low-dose PLX treatment produces a sex-dependent response in juvenile mice, that manipulation of microglia alters CRT-induced volume changes and that microglia density and MRI-derived volume changes are correlated in this model.

Chromatin- and nucleosome-associated features in liquid biopsy: implications for cancer biomarker discovery.

Cell-free DNA (cfDNA) from the bloodstream has been studied for cancer biomarker discovery, and chromatin-derived epigenetic features have come into the spotlight for their potential to expand clinical applications. Methylation, fragmentation, and nucleosome positioning patterns of cfDNA have previously been shown to reveal epigenomic and inferred transcriptomic information. More recently, histone modifications have emerged as a tool to further identify tumor-specific chromatin variants in plasma. A number of sequencing methods have been developed to analyze these epigenetic markers, offering new insights into tumor biology. Features within cfDNA allow for cancer detection, subtype and tissue of origin classification, and inference of gene expression. These methods provide a window into the complexity of cancer and the dynamic nature of its progression. In this review, we highlight the array of epigenetic features in cfDNA that can be extracted from chromatin- and nucleosome-associated organization and outline potential use cases in cancer management.

Isolation of salivary cell-free DNA for cancer detection.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

Cell-free DNA topology depends on its subcellular and cellular origins in cancer.

Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in nonvesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane protected and disproportionately derived from nontumor cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.

The Utility of Liquid Biopsies in Radiation Oncology.

The use of therapeutic radiation is primarily guided by clinicopathologic factors and medical imaging, whereas molecular biomarkers currently play a comparatively minor role in most settings. Liquid biopsies provide a rich source of noninvasive tumor-specific biomarkers and are amenable to repeated and noninvasive assessment. Here, we review the current status of liquid biopsies and their potential impact on the field of radiation oncology. We focus on established and emerging approaches to analyze circulating tumor DNA and circulating tumor cells from peripheral blood. These promising classes of biomarkers could have an outsized impact on cancer management by meaningfully stratifying patients into risk groups, tracking radiation therapy efficacy during and after treatment, and identifying patients with radiosensitive or radioresistant disease. Finally, we highlight opportunities for future investigation including the need for prospective interventional studies employing liquid biopsies to guide the management of radiation therapy-treated patients.