Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome.

BACKGROUND: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). METHODS: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and ß1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. RESULTS: Fibroblast growth factor receptor (FGFR 1-4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with ß1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. CONCLUSIONS: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.

Intraoperative MET-receptor targeted fluorescent imaging and spectroscopy for lymph node detection in papillary thyroid cancer: novel diagnostic tools for more selective central lymph node compartment dissection.

PURPOSE: Patients undergoing prophylactic central compartment dissection (PCLND) for papillary thyroid cancer (PTC) are often overtreated. This study aimed to determine if molecular fluorescence-guided imaging (MFGI) and spectroscopy can be useful for detecting PTC nodal metastases (NM) and to identify negative central compartments intraoperatively. METHODS: We used a data-driven prioritization strategy based on transcriptomic profiles of 97 primary PTCs and 80 normal thyroid tissues (NTT) to identify tumor-specific antigens for a clinically available near-infrared fluorescent tracer. Protein expression of the top prioritized antigen was immunohistochemically validated with a tissue microarray containing primary PTC (n = 741) and NTT (n = 108). Staining intensity was correlated with 10-year locoregional recurrence-free survival (LRFS). A phase 1 study (NCT03470259) with EMI-137, targeting MET, was conducted to evaluate safety, optimal dosage for detecting PTC NM with MFGI, feasibility of NM detection with quantitative fiber-optic spectroscopy, and selective binding of EMI-137 for MET. RESULTS: MET was selected as the most promising antigen. A worse LRFS was observed in patients with positive versus negative MET staining (81.9% versus 93.2%; p = 0.02). In 19 patients, no adverse events related to EMI-137 occurred. 0.13 mg/kg EMI-137 was selected as optimal dosage for differentiating NM from normal lymph nodes using MFGI (p < 0.0001) and spectroscopy (p < 0.0001). MFGI identified 5/19 levels (26.3%) without NM. EMI-137 binds selectively to MET. CONCLUSION: MET is overexpressed in PTC and associated with increased locoregional recurrence rates. Perioperative administration of EMI-137 is safe and facilitates NM detection using MFGI and spectroscopy, potentially reducing the number of negative PCLNDs with more than 25%. CLINICAL TRIAL REGISTRATION: NCT03470259.

Addition of HER2 and CD44 to 18F-FDG PET-based clinico-radiomic models enhances prediction of neoadjuvant chemoradiotherapy response in esophageal cancer.

OBJECTIVES: To assess the complementary value of human epidermal growth factor receptor 2 (HER2)-related biological tumor markers to clinico-radiomic models in predicting complete response to neoadjuvant chemoradiotherapy (NCRT) in esophageal cancer patients. METHODS: Expression of HER2 was assessed by immunohistochemistry in pre-treatment tumor biopsies of 96 patients with locally advanced esophageal cancer. Five other potentially active HER2-related biological tumor markers in esophageal cancer were examined in a sub-analysis on 43 patients. Patients received at least four of the five cycles of chemotherapy and full radiotherapy regimen followed by esophagectomy. Three reference clinico-radiomic models based on 18F-FDG PET were constructed to predict pathologic response, which was categorized into complete versus incomplete (Mandard tumor regression grade 1 vs. 2-5). The complementary value of the biological tumor markers was evaluated by internal validation through bootstrapping. RESULTS: Pathologic examination revealed 21 (22%) complete and 75 (78%) incomplete responders. HER2 and cluster of differentiation 44 (CD44), analyzed in the sub-analysis, were univariably associated with pathologic response. Incorporation of HER2 and CD44 into the reference models improved the overall performance (R2s of 0.221, 0.270, and 0.225) and discrimination AUCs of 0.759, 0.857, and 0.816. All models exhibited moderate to good calibration. The remaining studied biological tumor markers did not yield model improvement. CONCLUSIONS: Incorporation of HER2 and CD44 into clinico-radiomic prediction models improved NCRT response prediction in esophageal cancer. These biological tumor markers are promising in initial response evaluation. KEY POINTS: • A multimodality approach, integrating independent genomic and radiomic information, is promising to improve prediction of γpCR in patients with esophageal cancer. • HER2 and CD44 are potential biological tumor markers in the initial work-up of patients with esophageal cancer. • Prediction models combining 18F-FDG PET radiomic features with HER2 and CD44 may be useful in the decision to omit surgery after neoadjuvant chemoradiotherapy in patients with esophageal cancer.

Mouse parotid salivary gland organoids for the in vitro study of stem cell radiation response.

OBJECTIVE: Hyposalivation-related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro. MATERIALS AND METHODS: Single cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self-renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response. RESULTS: Single cells derived from parotid gland organoids were able to extensively self-renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid-derived stem cells displayed radiation dose-response curves similar to the submandibular gland. CONCLUSIONS: Murine parotid gland organoids harbor stem cells with long-term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.

Patient-derived tumor organoids for prediction of cancer treatment response.

Cancer treatment, in particular radiotherapy and chemotherapy, is often hindered by an inherent resistance of cancer cells. Cancer stem cells in particular have previously been shown to be more resistant than other cells within a tumor and are thought repopulate the tumour after therapies. Therefore, it is of utmost importance to develop tools and techniques that can be used to study mechanisms of resistance of cancer stem cells as potential treatment targets. Organoids (and cancer-derived organoids), are three-dimensional tissue-resembling cellular clusters derived from tissue or tumor specific stem cells that mimic the in vivo (tumor) characteristics, as well as (tumor) cell heterogeneity. Cancer organoids may further enhance the in vitro and in vivo models that are currently available, improve our understanding of cancer stem cell resistance and can be used to develop novel cancer treatments by improved targeting of cancer stem cells. In this review, we compare organoids with the more traditional laboratory models, such as cell lines and xenografts, and review the literature of the current role of cancer organoids in determining treatment responses.

Concise review: Adult salivary gland stem cells and a potential therapy for xerostomia.

The ability to speak, swallow, masticate, taste food, and maintain a healthy oral cavity is heavily reliant on the presence of saliva, the hugely important effect of which on our everyday lives is often unappreciated. Hyposalivation, frequently experienced by people receiving radiation therapy for head and neck cancers, results in a plethora of symptoms whose combined effect can drastically reduce quality of life. Although artificial lubricants and drugs stimulating residual function are available to ameliorate the consequences of hyposalivation, their effects are at best transient. Such management techniques do not address the source of the problem: a lack of functional saliva-producing acinar cells, resulting from radiation-induced stem cell sterilization. Post-radiotherapy stimulation of cell proliferation only results in improved saliva secretion when part of the tissue has been spared or when the dose to the salivary gland (SG) remains below a certain level. Therefore, stem cell replacement therapy may be a good option to treat radiation-induced hyposalivation. Substantial progress has been made lately in the understanding of cell turnover in the SG, and the recent identification of stem and progenitor cell populations in the SG provides a basis for studies toward development of a stem cell-based therapy for xerostomia. Here, we review the current state of knowledge of SG stem cells and their potential for use in a cell-based therapy that may provide a more durable cure for hyposalivation.

Radiation-induced Xerostomia is Related to Stem Cell Dose-dependent Reduction of Saliva Production.

PURPOSE: Previous studies have shown that the mean dose to the parotid gland stem cell rich regions (Dmean,SCR) is the strongest dosimetric predictor for the risk of patient-reported daytime xerostomia. This study aimed to test whether the relationship between patient-reported xerostomia and Dmean,SCR is explained by a dose-dependent reduction of saliva production. METHODS AND MATERIALS: In 570 patients with head and neck cancer treated with definitive radiation therapy (RT), flow from the parotid (FLOWPAR) and submandibular/sublingual (FLOWSMSL) glands, and patient-reported daytime (XERDAY) and nighttime (XERNIGHT) xerostomia were prospectively measured before, at 6 months, and 12 months after RT. Using linear mixed effect models, the relationship of the mean dose to the parotid glands (Dmean,par), Dmean,SCR, non-SCR parotid gland tissue (Dmean,non-SCR), submandibular glands (Dmean,sub), and oral cavity (Dmean,oral) with salivary flow and xerostomia was analyzed while correcting for known confounders. RESULTS: Dmean,SCR proved to be responsible for the effect of Dmean,par on FLOWPAR (P ≤ .03), while Dmean,non-SCR did not affect FLOWPAR (P ≥ .11). To illustrate, increasing Dmean,SCR by 10 Gy at a fixed Dmean,non-SCR reduced FLOWPAR by 0.02 mL/min (25%) after RT. However, if the opposite happened, no change in FLOWPAR was observed (0.00 mL/min [4%]). As expected, Dmean,sub was significantly associated with FLOWSMSL (P < .001). For example, increasing Dmean,sub by 10 Gy reduced FLOWSMSL by 0.07 mL/min (26%) after RT. Xerostomia scores were also affected by dose to the salivary glands. Dmean,SCR and Dmean,oral were associated with higher XERDAY scores (P ≤ .05), while Dmean,sub increased XERNIGHT scores (P = .01). For example, an increase of 10 Gy in Dmean,SCR raised XERDAY scores by 2.13 points (5%) after RT, while an additional 10 Gy in Dmean,subs increased XERNIGHT scores by 2.20 points (6%) after RT. Salivary flow was not only associated with radiation dose, but also with xerostomia scores in line with the salivary glands' functions; ie, FLOWPAR only influenced XERDAY (P < .001, 10.92 points lower XERDAY per 1 mL/min saliva), while FLOWSMSL affected XERDAY and XERNIGHT (P ≤ .004, 6.69 and 5.74 points lower XERDAY and XERNIGHT, respectively, per 1 mL/min saliva). Therefore, the observed relationships between dose and xerostomia were corrected for salivary flow. As hypothesized, Dmean,SCR only increased XERDAY scores via reducing FLOWPAR, whereas the effects of Dmean,oral on XERDAY and Dmean,sub on XERNIGHT were independent of salivary flow. CONCLUSIONS: Higher SCR region dose reduced parotid gland saliva production, subsequently resulting in higher daytime xerostomia scores. Consequently, this study supports the clinical implementation of stem cell sparing RT to preserve salivary flow with the aim of reducing the risk of xerostomia.

Small Molecule MIF Modulation Enhances Ferroptosis by Impairing DNA Repair Mechanisms.

Ferroptosis is a form of regulated cell death that can be modulated by small molecules and has the potential for the development of therapeutics for oncology. Although excessive lipid peroxidation is the defining hallmark of ferroptosis, DNA damage may also play a significant role. In this study, a potential mechanistic role for MIF in homologous recombination (HR) DNA repair is identified. The inhibition or genetic depletion of MIF or other HR proteins, such as breast cancer type 1 susceptibility protein (BRCA1), is demonstrated to significantly enhance the sensitivity of cells to ferroptosis. The interference with HR results in the translocation of the tumor suppressor protein p53 to the mitochondria, which in turn stimulates the production of reactive oxygen species. Taken together, the findings demonstrate that MIF-directed small molecules enhance ferroptosis via a putative MIF-BRCA1-RAD51 axis in HR, which causes resistance to ferroptosis. This suggests a potential novel druggable route to enhance ferroptosis by targeted anticancer therapeutics in the future.

Role of immediate early genes in the development of salivary gland organoids in polyisocyanopeptide hydrogels.

Human salivary gland organoids have opened tremendous possibilities for regenerative medicine in patients undergoing radiotherapy for the treatment of head and neck cancer. However, their clinical translation is greatly limited by the current use of Matrigel for organoid derivation and expansion. Here, we envisage that the use of a fully, synthetic hydrogel based on the oligo (-ethylene glycol) functionalized polymer polyisocyanopeptides (PICs) can provide an environment suitable for the generation and expansion of salivary gland organoids (SGOs) after optimization of PIC polymer properties. We demonstrate that PIC hydrogels decorated with the cell-binding peptide RGD allow SGO formation from salivary gland (SG)-derived stem cells. This self-renewal potential is preserved for only 4 passages. It was found that SGOs differentiated prematurely in PIC hydrogels affecting their self-renewal capacity. Similarly, SGOs show decreased expression of immediate early genes (IEGs) after culture in PIC hydrogels. Activation of multiple signalling pathways involved in IEG expression by ß-adrenergic agonist isoproterenol, led to increased stem cell self-renewal capacity as measured by organoid forming efficiency (OFE). These results indicate that PIC hydrogels are promising 3D matrices for SGOs, with the option to be used clinically, after further optimization of the hydrogel and culture conditions.

Radiation-induced cardiac side-effects: The lung as target for interacting damage and intervention.

Radiotherapy is part of the treatment for many thoracic cancers. During this treatment heart and lung tissue can often receive considerable doses of radiation. Doses to the heart can potentially lead to cardiac effects such as pericarditis and myocardial fibrosis. Common side effects after lung irradiation are pneumonitis and pulmonary fibrosis. It has also been shown that lung irradiation has effects on cardiac function. In a rat model lung irradiation caused remodeling of the pulmonary vasculature increasing resistance of the pulmonary vascular bed, leading to enhanced pulmonary artery pressure, right ventricle hypertrophy and reduced right ventricle performance. Even more pronounced effects are observed when both, lung and heart are irradiated. The effects observed after lung irradiation show striking similarities with symptoms of pulmonary arterial hypertension. In particular, the vascular remodeling in lung tissue seems to have similar underlying features. Here, we discuss the similarities and differences of vascular remodeling observed after thoracic irradiation compared to those in pulmonary arterial hypertension patients and research models. We will also assess how this knowledge of similarities could potentially be translated into interventions which would be beneficial for patients treated for thoracic tumors, where dose to lung tissue is often unavoidable.

Role of mTOR through Autophagy in Esophageal Cancer Stemness.

Esophageal cancer (EC) is a highly aggressive disease with a poor prognosis. Therapy resistance and early recurrences are major obstacles in reaching a better outcome. Esophageal cancer stem-like cells (CSCs) seem tightly related with chemoradiation resistance, initiating new tumors and metastases. Several oncogenic pathways seem to be involved in the regulation of esophageal CSCs and might harbor novel therapeutic targets to eliminate CSCs. Previously, we identified a subpopulation of EC cells that express high levels of CD44 and low levels of CD24 (CD44+/CD24-), show CSC characteristics and reside in hypoxic niches. Here, we aim to clarify the role of the hypoxia-responding mammalian target of the rapamycin (mTOR) pathway in esophageal CSCs. We showed that under a low-oxygen culture condition and nutrient deprivation, the CD44+/CD24- population is enriched. Since both low oxygen and nutrient deprivation may inhibit the mTOR pathway, we next chemically inhibited the mTOR pathway using Torin-1. Torin-1 upregulated SOX2 resulted in an enrichment of the CD44+/CD24- population and increased sphere formation potential. In contrast, stimulation of the mTOR pathway using MHY1485 induced the opposite effects. In addition, Torin-1 increased autophagic activity, while MHY1485 suppressed autophagy. Torin-1-mediated CSCs upregulation was significantly reduced in cells treated with autophagy inhibitor, hydroxychloroquine (HCQ). Finally, a clearly defined CD44+/CD24- CSC population was detected in EC patients-derived organoids (ec-PDOs) and here, MHY1485 also reduced this population. These data suggest that autophagy may play a crucial role in mTOR-mediated CSCs repression. Stimulation of the mTOR pathway might aid in the elimination of putative esophageal CSCs.

Role of quiescent cells in the homeostatic maintenance of the adult submandibular salivary gland.

Stem/progenitor cells are required for maintenance of salivary gland (SG) function and serve as untapped reservoirs to create functional cells. Despite recent advancements in the identification of stem/progenitor pools, in the submandibular gland (SMG), a knowledge gap remains. Furthermore, the contribution to adult SMG homeostasis of stem/progenitor cells originating from embryonic development is unclear. Here, we employ an H2B-GFP embryonic and adult pulse-and-chase system to characterize potential SMG stem/progenitor cells (SGSCs) based on quiescence at different stages. Phenotypical profiling of quiescent cells in the SMG revealed that label-retaining cells (LRCs) of embryonic or adult origin co-localized with CK8+ ductal or vimentin + mesenchymal, but not with CK5+ or CK14 + stem/progenitor cells. These SMG LRCs failed to self-renew in vitro while non-label retaining cells displayed differentiation and long-term expansion potential as organoids. Collectively, our data suggest that an active cycling population of cells is responsible for SMG homeostasis with organoid forming potential.

Tyrosine Phosphatase PTPRO Deficiency in ERBB2-Positive Breast Cancer Contributes to Poor Prognosis and Lapatinib Resistance.

Despite the initial benefit from treating ERBB2-positive breast cancer with tyrosine kinase inhibitor lapatinib, resistance develops inevitably. Since the expression of protein tyrosine phosphatase receptor-type O (PTPRO), a member of the R3 subfamily of receptor protein tyrosine phosphatases (PTPs), is inversely correlated with the aggressiveness of multiple malignancies, we decided to explore the correlation between PTPRO and lapatinib resistance in ERBB2-positive breast cancer. Results of immunohistochemical (IHC) staining and the correlation analysis between the expression levels of PTPRO and the clinicopathological parameters indicate that PTPRO is downregulated in cancer tissues as compared with normal tissues and negatively associated with differentiation, tumor size, tumor depth, as well as the expression of ERBB2 and Ki67. Results from Kaplan-Meier analyses indicate that lower expression of PTPRO is correlated with shorter relapse-free survival for patients with ERBB2-positive breast cancer, and multivariable Cox regression analysis found that PTPRO can potentially serve as an independent prognostic indicator for ERBB2-positive breast cancer. Results from both human breast cancer cells with PTPRO knockdown or overexpression and mouse embryonic fibroblasts (MEFs) which derived from Ptpro +/+ and Ptpro -/- mice with then stably transfected plasmid FUGW-Erbb2 consistently demonstrated the essentiality of PTPRO in the lapatinib-mediated anticancer process. Our findings suggest that PTPRO is not only able to serve as an independent prognostic indicator, but upregulating PTPRO can also reverse the lapatinib resistance of ERBB2-positive breast cancer.

Generation and Application of Inducible Chimeric RNA ASTN2-PAPPAas Knockin Mouse Model.

Chimeric RNAs (chiRNAs) play many previously unrecognized roles in different diseases including cancer. They can not only be used as biomarkers for diagnosis and prognosis of various diseases but also serve as potential therapeutic targets. In order to better understand the roles of chiRNAs in pathogenesis, we inserted human sequences into mouse genome and established a knockin mouse model of the tamoxifen-inducible expression of ASTN2-PAPPA antisense chimeric RNA (A-PaschiRNA). Mice carrying the A-PaschiRNA knockin gene do not display any apparent abnormalities in growth, fertility, histological, hematopoietic, and biochemical indices. Using this model, we dissected the role of A-PaschiRNA in chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis of esophageal squamous cell carcinoma (ESCC). To our knowledge, we are the first to generate a chiRNA knockin mouse model using the Cre-loxP system. The model could be used to explore the roles of chiRNA in pathogenesis and potential targeted therapies.

Patient-derived parathyroid organoids as a tracer and drug-screening application model.

Parathyroid diseases are characterized by dysregulation of calcium homeostasis and alterations in parathyroid hormone (PTH) excretion. The development of parathyroid-targeted treatment and imaging tracers could benefit from in vitro models. Therefore, we aim to establish a patient-derived parathyroid organoid model representing human parathyroid tissue. Hyperplastic parathyroid tissue was dispersed, and parathyroid organoids (PTOs) were cultured and characterized. PTO-derived cells exhibited self-renewal over several passages, indicative of the presence of putative stem cells. Immunofluorescence and RNA sequencing confirmed that PTOs phenocopy hyperplastic parathyroid tissue. Exposure of PTOs to increasing calcium concentrations and PTH-lowering drugs resulted in significantly reduced PTH excretion. PTOs showed specific binding of the imaging tracers 11C-methionine and 99mTc-sestamibi. These data show the functionality of PTOs resembling the parathyroid. This PTO model recapitulates the originating tissue on gene and protein expression and functionality, paving the way for future physiology studies and therapeutic target and tracer discovery.

Parotid Gland Stem Cell Sparing Radiation Therapy for Patients With Head and Neck Cancer: A Double-Blind Randomized Controlled Trial.

PURPOSE: Radiation therapy for head and neck cancer frequently leads to salivary gland damage and subsequent xerostomia. The radiation response of the parotid glands of rats, mice, and patients critically depends on dose to parotid gland stem cells, mainly located in the gland's main ducts (stem cell rich [SCR] region). Therefore, this double-blind randomized controlled trial aimed to test the hypothesis that parotid gland stem cell sparing radiation therapy preserves parotid gland function better than currently used whole parotid gland sparing radiation therapy. METHODS AND MATERIALS: Patients with head and neck cancer (n = 102) treated with definitive radiation therapy were randomized between standard parotid-sparing and stem cell sparing (SCS) techniques. The primary endpoint was >75% reduction in parotid gland saliva production compared with pretreatment production (FLOW12M). Secondary endpoints were several aspects of xerostomia 12 months after treatment. RESULTS: Fifty-four patients were assigned to the standard arm and 48 to the SCS arm. Only dose to the SCR regions (contralateral 16 and 11 Gy [P = .004] and ipsilateral 26 and 16 Gy [P = .001] in the standard and SCS arm, respectively) and pretreatment patient-rated daytime xerostomia (35% and 13% [P = .01] in the standard and SCS arm, respectively) differed significantly between the arms. In the SCS arm, 1 patient (2.8%) experienced FLOW12M compared with 2 (4.9%) in the standard arm (P = 1.00). However, a trend toward better relative parotid gland salivary function in favor of SCS radiation therapy was shown. Moreover, multivariable analysis showed that mean contralateral SCR region dose was the strongest dosimetric predictor for moderate-to-severe patient-rated daytime xerostomia and grade ≥2 physician-rated xerostomia, the latter including reported alteration in diet. CONCLUSIONS: No significantly better parotid function was observed in SCS radiation therapy. However, additional multivariable analysis showed that dose to the SCR region was more predictive of the development of parotid gland function-related xerostomia endpoints than dose to the entire parotid gland.

Development of a facility for high-precision irradiation of cells with carbon ions.

PURPOSE: Compared to photons, using particle radiation in radiotherapy reduces the dose and irradiated volume of normal tissues, potentially reducing side effects. The biological effect of dose deposited by particles such as carbon ions, however, differs from that of dose deposited by photons. The inaccuracy in models to estimate the biological effects of particle radiation remains the most important source of uncertainties in particle therapy. Improving this requires high-precision studies on biological effects of particle radiation. Therefore, the authors aimed to develop a facility for reproducible and high-precision carbon-ion irradiation of cells in culture. The combined dose nonuniformity in the lateral and longitudinal direction should not exceed +/-1.5%. Dose to the cells from particles than other carbon ions should not exceed 5%. METHODS: A uniform lateral dose distribution was realized using a single scatter foil and quadrupole magnets. A modulator wheel was used to create a uniform longitudinal dose distribution. The choice of beam energy and the optimal design of these components was determined using GEANT4 and SRIM Monte Carlo simulations. Verification of the uniformity of the dose distribution was performed using a scintillating screen (lateral) and a water phantom (longitudinal). The reproducibility of dose delivery between experiments was assessed by repeated measurements of the spatial dose distribution. Moreover, the reproducibility of dose-response measurements was tested by measuring the survival of irradiated HEK293 cells in three independent experiments. RESULTS: The relative contribution of dose from nuclear reaction fragments to the sample was found to be <5% when using 90 MeV/u carbon ions. This energy still allows accurate dosimetry conforming to the IAEA Report TRS-398, facilitating comparison to dose-effect data obtained with other radiation qualities. A 1.3 mm long spread-out Bragg peak with a diameter of 30 mm was created, allowing the irradiation of cell samples with the specified accuracy. Measurements of the transverse and longitudinal dose distribution showed that the dose variation over the sample volume was +/-0.8% and +/-0.7% in the lateral and longitudinal directions, respectively. The track-averaged LET of 132 +/- 10 keV/microm and dose-averaged LET of 189 +/- 15 keV/microm at the position of the sample were obtained from a GEANT4 simulation, which was validated experimentally. Three separately measured cell-survival curves yielded nearly identical results. CONCLUSIONS: With the new facility, high-precision carbon-ion irradiations of biological samples can be performed with highly reproducible results.

DNA Damage-Induced Inflammatory Microenvironment and Adult Stem Cell Response.

Adult stem cells ensure tissue homeostasis and regeneration after injury. Due to their longevity and functional requirements, throughout their life stem cells are subject to a significant amount of DNA damage. Genotoxic stress has recently been shown to trigger a cascade of cell- and non-cell autonomous inflammatory signaling pathways, leading to the release of pro-inflammatory factors and an increase in the amount of infiltrating immune cells. In this review, we discuss recent evidence of how DNA damage by affecting the microenvironment of stem cells present in adult tissues and neoplasms can affect their maintenance and long-term function. We first focus on the importance of self-DNA sensing in immunity activation, inflammation and secretion of pro-inflammatory factors mediated by activation of the cGAS-STING pathway, the ZBP1 pathogen sensor, the AIM2 and NLRP3 inflammasomes. Alongside cytosolic DNA, the emerging roles of cytosolic double-stranded RNA and mitochondrial DNA are discussed. The DNA damage response can also initiate mechanisms to limit division of damaged stem/progenitor cells by inducing a permanent state of cell cycle arrest, known as senescence. Persistent DNA damage triggers senescent cells to secrete senescence-associated secretory phenotype (SASP) factors, which can act as strong immune modulators. Altogether these DNA damage-mediated immunomodulatory responses have been shown to affect the homeostasis of tissue-specific stem cells leading to degenerative conditions. Conversely, the release of specific cytokines can also positively impact tissue-specific stem cell plasticity and regeneration in addition to enhancing the activity of cancer stem cells thereby driving tumor progression. Further mechanistic understanding of the DNA damage-induced immunomodulatory response on the stem cell microenvironment might shed light on age-related diseases and cancer, and potentially inform novel treatment strategies.

The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways.

Radiotherapy is involved in the treatment of many cancers, but damage induced to the surrounding normal tissue is often inevitable. Evidence suggests that the maintenance of homeostasis and regeneration of the normal tissue is driven by specific adult tissue stem/progenitor cells. These tasks involve the input from several signaling pathways. Irradiation also targets these stem/progenitor cells, triggering a cellular response aimed at achieving tissue regeneration. Here we discuss the currently used in vitro and in vivo models and the involved specific tissue stem/progenitor cell signaling pathways to study the response to irradiation. The combination of the use of complex in vitro models that offer high in vivo resemblance and lineage tracing models, which address organ complexity constitute potential tools for the study of the stem/progenitor cellular response post-irradiation. The Notch, Wnt, Hippo, Hedgehog, and autophagy signaling pathways have been found as crucial for driving stem/progenitor radiation-induced tissue regeneration. We review how these signaling pathways drive the response of solid tissue-specific stem/progenitor cells to radiotherapy and the used models to address this.

The Hippo signaling pathway effector YAP promotes salivary gland regeneration after injury.

Salivary glands are damaged by radiotherapy for head and neck cancers, which often culminates in radiation-induced hyposalivation and xerostomia that may be permanent. Here, we identified a central role for YAP in the regenerative response of the salivary gland. Activation of the Hippo signaling pathway inhibits the phosphorylation of YAP, leading to its nuclear translocation and transcriptional activity. Using mice with salivary gland injury induced by surgical ligation and salivary gland­derived organoids, we found that YAP nuclear localization in the salivary gland epithelium changed dynamically between homeostasis and regeneration. Whereas local injury had no effect on nuclear YAP localization in saliva-producing acinar cells, it triggered nuclear accumulation of YAP in saliva-transporting ductal cells. Injury also stimulated the proliferation of ductal cells, which were mainly quiescent under homeostatic conditions and in nonregenerating areas distal to the injury site, thus enabling salivary gland regeneration. Overexpressing YAP or driving YAP nuclear translocation by inhibiting upstream Hippo pathway kinases increased the capacity of mouse and human salivary gland cells, including human cells that had been irradiated, to form lobed organoids in vitro. Our results identify a YAP-driven regeneration program in salivary gland ductal cells that could be used to promote salivary gland regeneration after irradiation-induced damage.