Proton radiotherapy has been implemented into the standard-of-care for cancer patients within recent years. However, experimental studies investigating cellular and molecular mechanisms are lacking, and prognostic biomarkers are needed. Cancer stem cell (CSC)-related biomarkers, such as aldehyde dehydrogenase (ALDH), are known to influence cellular radiosensitivity through inactivation of reactive oxygen species, DNA damage repair, and cell death. In a previous study, we found that ionizing radiation itself enriches for ALDH-positive CSCs. In this study, we analyze CSC marker dynamics in prostate cancer, head and neck cancer, and glioblastoma cells upon proton beam irradiation. We find that proton irradiation has a higher potential to target CSCs through induction of complex DNA damages, lower rates of cellular senescence, and minor alteration in histone methylation pattern compared with conventional photon irradiation. Mathematical modeling indicates differences in plasticity rates among ALDH-positive CSCs and ALDH-negative cancer cells between the two irradiation types.

Tumor heterogeneity and cellular plasticity are key determinants of tumor progression, metastatic spread, and therapy response driven by the cancer stem cell (CSC) population. Within the current study, we analyzed irradiation-induced plasticity within the aldehyde dehydrogenase (ALDH)-positive (ALDH+) population in prostate cancer. The radiosensitivity of xenograft tumors derived from ALDH+ and ALDH-negative (ALDH−) cells was determined with local tumor control analyses and demonstrated different dose–response profiles, time to relapse, and focal adhesion signaling. The transcriptional heterogeneity was analyzed in pools of 10 DU145 and PC3 cells with multiplex gene expression analyses and illustrated a higher degree of heterogeneity within the ALDH+ population that even increases upon irradiation in comparison with ALDH− cells. Phenotypic conversion and clonal competition were analyzed with fluorescence protein-labeled cells to distinguish cellular origins in competitive three-dimensional cultures and xenograft tumors. We found that the ALDH+ population outcompetes ALDH− cells and drives tumor growth, in particular upon irradiation. The observed dynamics of the cellular state compositions between ALDH+ and ALDH− cells in vivo before and after tumor irradiation was reproduced by a probabilistic Markov compartment model that incorporates cellular plasticity, clonal competition, and phenotype-specific radiosensitivities. Transcriptional analyses indicate that the cellular conversion from ALDH− into ALDH+ cells within xenograft tumors under therapeutic pressure was partially mediated through induction of the transcriptional repressor SNAI2. In summary, irradiation-induced cellular conversion events are present in xenograft tumors derived from prostate cancer cells and may be responsible for radiotherapy failure. Implications: The increase of ALDH+ cells with stem-like features in prostate xenograft tumors after local irradiation represents a putative cellular escape mechanism inducing tumor radioresistance.

(1) Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features. These cellular characteristics are dynamically regulated in response to treatment and may lead to an enrichment of radioresistant cells with a cancer stem cell (CSC) phenotype. Epigenetic mechanisms, particularly DNA and histone methylation, are key regulators of gene-specific transcription and cellular plasticity. Therefore, we hypothesized that specific epigenetic targeting may prevent irradiation-induced plasticity and may sensitize HNSCC cells to radiotherapy. (2) Methods: We compared the DNA methylome and intracellular concentrations of tricarboxylic acid cycle metabolites in radioresistant FaDu and Cal33 cell lines with their parental controls, as well as aldehyde dehydrogenase (ALDH)-positive CSCs with negative controls. Moreover, we conducted a screen of a chemical library targeting enzymes involved in epigenetic regulation in combination with irradiation and analyzed the clonogenic potential, sphere formation, and DNA repair capacity to identify compounds with both radiosensitizing and CSC-targeting potential. (3) Results: We identified the histone demethylase inhibitor GSK-J1, which targets UTX (KDM6A) and JMJD3 (KDM6B), leading to increased H3K27 trimethylation, heterochromatin formation, and gene silencing. The clonogenic survival assay after siRNA-mediated knock-down of both genes radiosensitized Cal33 and SAS cell lines. Moreover, high KDM6A expression in tissue sections of patients with HNSCC was associated with improved locoregional control after primary (n = 137) and post-operative (n = 187) radio/chemotherapy. Conversely, high KDM6B expression was a prognostic factor for reduced overall survival. (4) Conclusions: Within this study, we investigated cellular and molecular mechanisms underlying irradiation-induced cellular plasticity, a key inducer of radioresistance, with a focus on epigenetic alterations. We identified UTX (KDM6A) as a putative prognostic and therapeutic target for HNSCC patients treated with radiotherapy.

Tumor heterogeneity and cellular plasticity are key determinants of tumor progression, metastatic spread, and therapy response driven by the cancer stem cell (CSC) population. Within the current study, we analyzed irradiation-induced plasticity within the aldehyde dehydrogenase (ALDH)-positive (ALDH+) population in prostate cancer. The radiosensitivity of xenograft tumors derived from ALDH+ and ALDH-negative (ALDH−) cells was determined with local tumor control analyses and demonstrated different dose–response profiles, time to relapse, and focal adhesion signaling. The transcriptional heterogeneity was analyzed in pools of 10 DU145 and PC3 cells with multiplex gene expression analyses and illustrated a higher degree of heterogeneity within the ALDH+ population that even increases upon irradiation in comparison with ALDH− cells. Phenotypic conversion and clonal competition were analyzed with fluorescence protein-labeled cells to distinguish cellular origins in competitive three-dimensional cultures and xenograft tumors. We found that the ALDH+ population outcompetes ALDH− cells and drives tumor growth, in particular upon irradiation. The observed dynamics of the cellular state compositions between ALDH+ and ALDH− cells in vivo before and after tumor irradiation was reproduced by a probabilistic Markov compartment model that incorporates cellular plasticity, clonal competition, and phenotype-specific radiosensitivities. Transcriptional analyses indicate that the cellular conversion from ALDH− into ALDH+ cells within xenograft tumors under therapeutic pressure was partially mediated through induction of the transcriptional repressor SNAI2. In summary, irradiation-induced cellular conversion events are present in xenograft tumors derived from prostate cancer cells and may be responsible for radiotherapy failure.Implications:The increase of ALDH+ cells with stem-like features in prostate xenograft tumors after local irradiation represents a putative cellular escape mechanism inducing tumor radioresistance.

Background: Lewy Body pathology can be detected by alpha-synuclein-Seed Amplification Assay (αSyn-SAA) in cerebrospinal fluid (CSF) with high sensitivity and specificity in cohort studies. Yet, little is known about the results that can be expected from αSyn-SAA in CSF in samples outside cohort studies as obtained in clinical routine. Objective: This study analyzed the concordance of αSyn-SAA findings in CSF with clinical diagnosis in patients from clinical routine with diverse neurologic and psychiatric conditions. Methods: In this cross-sectional study, CSF from patients who underwent lumbar puncture for therapeutic or diagnostic purposes were tested in αSyn-SAA. Analysis included binary αSyn-SAA findings, data collected during neurological examination, and structured medical history. Results: All 356 participants (Mean Age 67.1 years, SD = 16.2; 55.9% male) were included in the primary analysis, including 90 patients with Parkinsonian syndromes, 139 with predominant cognitive disorders, 25 with other movement disorders, 35 with inflammatory or (para)neoplastic syndromes, and 67 with further diseases. αSyn-SAA was positive in all samples from patients with Parkinson's disease (41), dementia with Lewy bodies (30), pure autonomic failure (4), and in a subset of patients with Alzheimer's disease (13/46), normal pressure hydrocephalus (7/14) and others. Conclusions: αSyn-SAA findings show high concordance with a clinical diagnosis of PD and DLB. Findings are comparable to results from well-characterized cohort studies, supporting potential diagnostic value in future clinical routine. Challenges may result from the fact that αSyn-SAA detect LB co-pathology that is known from neuropathological studies for several neurodegenerative diseases.

Supplementary Figure from Plasticity within Aldehyde Dehydrogenase–Positive Cells Determines Prostate Cancer Radiosensitivity