Purification and Ex Vivo Expansion of Fully Functional Salivary Gland Stem Cells

Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24hi/CD29hi). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes

Hedgehog Pathway as a Potential Intervention Target in Esophageal Cancer

Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog (HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures, fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures, as indicated by increased sphere formation after sorting for CD44+/CD24- EC cancer stem-like cell (CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH pathway could be an attractive approach to control CSCs.</p

Rescue of Salivary Gland Function after Stem Cell Transplantation in Irradiated Glands

Head and neck cancer is the fifth most common malignancy and accounts for 3% of all new cancer cases each year. Despite relatively high survival rates, the quality of life of these patients is severely compromised because of radiation-induced impairment of salivary gland function and consequential xerostomia (dry mouth syndrome). In this study, a clinically applicable method for the restoration of radiation-impaired salivary gland function using salivary gland stem cell transplantation was developed. Salivary gland cells were isolated from murine submandibular glands and cultured in vitro as salispheres, which contained cells expressing the stem cell markers Sca-1, c-Kit and Musashi-1. In vitro, the cells differentiated into salivary gland duct cells and mucin and amylase producing acinar cells. Stem cell enrichment was performed by flow cytrometric selection using c-Kit as a marker. In vitro, the cells differentiated into amylase producing acinar cells. In vivo, intra-glandular transplantation of a small number of c-Kit+ cells resulted in long-term restoration of salivary gland morphology and function. Moreover, donor-derived stem cells could be isolated from primary recipients, cultured as secondary spheres and after re-transplantation ameliorate radiation damage. Our approach is the first proof for the potential use of stem cell transplantation to functionally rescue salivary gland deficiency

ACE inhibition attenuates radiation-induced cardiopulmonary damage

BACKGROUND AND PURPOSE: In thoracic irradiation, the maximum radiation dose is restricted by the risk of radiation-induced cardiopulmonary damage and dysfunction limiting tumor control. We showed that radiation-induced sub-clinical cardiac damage and lung damage in rats mutually interact and that combined irradiation intensifies cardiopulmonary toxicity. Unfortunately, current clinical practice does not include preventative measures to attenuate radiation-induced lung or cardiac toxicity. Here, we investigate the effects of the ACE inhibitor captopril on radiation-induced cardiopulmonary damage.MATERIAL AND METHODS: After local irradiation of rat heart and/or lungs captopril was administered orally. Cardiopulmonary performance was assessed using biweekly breathing rate measurements. At 8weeks post-irradiation, cardiac hemodynamics were measured, CT scans and histopathology were analyzed.RESULTS: Captopril significantly improved breathing rate and cardiopulmonary density/structure, but only when the heart was included in the radiation field. Consistently, captopril reduced radiation-induced pleural and pericardial effusion and cardiac fibrosis, resulting in an improved left ventricular end-diastolic pressure only in the heart-irradiated groups.CONCLUSION: Captopril improves cardiopulmonary morphology and function by reducing acute cardiac damage, a risk factor in the development of radiation-induced cardiopulmonary toxicity. ACE inhibition should be evaluated as a strategy to reduce cardiopulmonary complications induced by radiotherapy to the thoracic area.</p

Generation and Differentiation of Adult Tissue-Derived Human Thyroid Organoids

Total thyroidectomy as part of thyroid cancer treatment results in hypothyroidism requiring lifelong daily thyroid hormone replacement. Unbalanced hormone levels result in persistent complaints such as fatigue, constipation, and weight increase. Therefore, we aimed to investigate a patient-derived thyroid organoid model with the potential to regenerate the thyroid gland. Murine and human thyroidderived cells were cultured as organoids capable of self-renewal and which expressed proliferation and putative stem cell and thyroid characteristics, without a change in the expression of thyroid tumor-related genes. These organoids formed thyroid-tissue-resembling structures in culture. (Xeno-)transplantation of 600,000 dispersed organoid cells underneath the kidney capsule of a hypothyroid mouse model resulted in the generation of hormone-producing thyroid-resembling follicles. This study provides evidence that thyroid-lineagespecific cells can form organoids that are able to self-renew and differentiate into functional thyroid tissue. Subsequent (xeno-)transplantation of these thyroid organoids demonstrates a proof of principle for functional miniature gland formation

Enhanced proliferation of acinar and progenitor cells by prophylactic pilocarpine treatment underlies the observed amelioration of radiation injury to parotid glands

Background: Administration of pilocarpine before irradiation can ameliorate radiation-induced hyposalivation. Indirect evidence Suggests that this effect may be mediated through induction of a compensatory response. In this study, this hypothesis is tested directly, by assessing the proliferation of progenitor and secretory cells in irradiated and non-irradiated parotid gland tissue. Methods: In a rat model, parotid glands were unilaterally irradiated with a single dose of 15 Gy, 60 min after administration of pilocarpine (4.0 mg/kg). Rats were sacrificed for proliferating cell nuclear antigen (PCNA) labelling, assessing the number of proliferating progenitor and secretory cells, before, and 10 h, 1. 3, 7, 10, 20 and 30 days after irradiation. Results: A small radiation-induced increase in PCNA expressing cells was observed, both in the acinar (secretory cells) and intercalated duct cell (containing the progenitor cells) compartment. This increment was significantly enhanced in pilocarpine pre-treated glands. In fact, in this group of animals increased proliferation was observed both in the irradiated and the shielded gland. Conclusions: Amelioration of early loss of rat salivary gland function after radiation by pilocarpine pretreatment is, at least in part, due to compensatory mechanisms through increased proliferation of undamaged cells. (C) 2008 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 90 (2009) 253-25

BATH AND SHOWER EFFECTS IN THE RAT PAROTID GLAND EXPLAIN INCREASED RELATIVE RISK OF PAROTID GLAND DYSFUNCTION AFTER INTENSITY-MODULATED RADIOTHERAPY

Purpose: To assess in a rat model whether adding a subtolerance dose in a region adjacent to a high-dose irradiated subvolume of the parotid gland influences its response (bath-and-shower effect). Methods and Materials: Irradiation of the whole, cranial 50%, and/or the caudal 50% of the parotid glands of Wistar rats was performed using 150-MeV protons. To determine suitable (i.e., subtolerance) dose levels for a bath-dose, both whole parotid glands were irradiated with 5 to 25 Gy. Subsequently groups of Wistar rats received 30 Gy to the caudal 50% (shower) and 0 to 10 Gy to the cranial 50% (bath) of both parotid glands. Stimulated saliva flow rate (function) was measured before and up to 240 days after irradiation. Results : Irradiation of both glands up to a dose of 10 Gy did not result in late loss of function and is thus regarded subtolerance. Addition of a dose bath of I to 10 Gy to a high-dose in the caudal 50% of the glands resulted in enhanced function loss. Conclusion: Similar to the spinal cord, the parotid gland demonstrates a bath and shower effect, which may explain the less-than-expected sparing of function after IMRT. (C) 2009 Elsevier Inc

Optimum dose range for the amelioration of tong term radiation-induced hyposalivation using prophylactic pilocarpine treatment

Background: To determine dose and time dependency of pilocarpine pre-treatment protection from late damage after unilateral irradiation of the rat parotid gland. Methods and materials: The right parotid gland of saline (1 mg/ml) or pilocarpine (4 mg/kg) pre-treated rats was irradiated with 10, 15 and 20 Gy. Saliva was collected from the irradiated and shielded parotid before, 30, 60, 120 and 240 days after irradiation. The number of acinar cells/gland was determined 30, 120 and 240 days after irradiation by histological examination. Results: Pilocarpine pre-treated rats, protection of parotid gland function was seen in the early-intermediate phase (0-120 days) after 15 Gy and in the late phase (>120 days) after 10 and 15 Gy. Although no protection was observed after 20 Gy, a stimulatory effect of pilocarpine on the non-irradiated gland resulted in a significant increase in total saliva secretion. The increase in function after pilocarpine treatment was paralleled by a significant increase in the number of acinar cells in both the irradiated and shielded glands. Conclusions: Pre-irradiation treatment with pilocarpine induces compensatory response, at lower doses, in the irradiated and at higher doses in the non-irradiated gland reducing late damage, due to stimulation of unirradiated or surviving cells to divide. (c) 2007 Elsevier Ireland Ltd. All rights reserved

Decreasing Irradiated Rat Lung Volume Changes Dose-Limiting Toxicity From Early to Late Effects

Purpose: Technological developments in radiation therapy result in smaller irradiated volumes of normal tissue. Because the risk of radiation therapy-induced toxicity generally depends on irradiated volume, changing volume could change the doselimiting toxicity of a treatment. Recently, in our rat model, we found that early radiation-induced lung dysfunction (RILD) was closely related to irradiated volume dependent vascular remodeling besides inflammation. The exact relationship between early and late RILD is still unknown. Therefore, in this preclinical study we investigated the dose-volume relationship of late RILD, assessed its dependence on early and late pathologies and studied if decreasing irradiated volume changed the doselimiting toxicity. Methods and Materials: A volume of 25%, 32%, 50%, 63%, 88%, or 100% of the rat lung was irradiated using protons. Until 26 weeks after irradiation, respiratory rates were measured. Macrovascular remodeling, pulmonary inflammation, and fibrosis were assessed at 26 weeks after irradiation. For all endpoints dose-volume response curves were made. These results were compared to our previously published early lung effects. Results: Early vascular remodeling and inflammation correlated significantly with early RILD. Late RILD correlated with inflammation and fibrosis, but not with vascular remodeling. In contrast to the early effects, late vascular remodeling, inflammation and fibrosis showed a primarily dose but not volume dependence. Comparison of respiratory rate increases early and late after irradiation for the different dose-distributions indicated that with decreasing irradiated volumes, the dose-limiting toxicity changed from early to late RILD. Conclusions: In our rat model, different pathologies underlie early and late RILD with different dose-volume dependencies. Consequently, the dose-limiting toxicity changed from early to late dysfunction when the irradiated volume was reduced. In patients, early and late RILD are also due to different pathologies. As such, new radiation techniques reducing irradiated volume might change the dose-limiting toxicity of the radiation therapy treatment. (C) 2016 Elsevier Inc. All rights reserved

The impact of heart irradiation on dose-volume effects in the rat lung

Purpose: To test the hypothesis that heart irradiation increases the risk of a symptomatic radiation-induced loss of lung function (SRILF) and that this can be well-described as a modulation of the functional reserve of the lung. Methods and Materials: Rats were irradiated with 150-MeV protons. Dose-response curves were obtained for a significant increase in breathing frequency after irradiation of 100%, 75%, 50%, or 25% of the total lung volume, either including or excluding the heart from the irradiation field. A significant increase in the mean respiratory rate after 6-12 weeks compared with 0-4 weeks was defined as SRILF, based on biweekly measurements of the respiratory rate. The critical volume (CV) model was used to describe the risk of SRILF. Fits were done using a maximum likelihood method. Consistency between model and data was tested using a previously developed goodness-of-fit test. Results: The CV model could be fitted consistently to the data for lung irradiation only. However, this fitted model failed to predict the data that also included heart irradiation. Even refitting the model to all data resulted in a significant difference between model and data. These results imply that, although the CV model describes the risk of SRILF when the heart is spared, the model needs to be modified to account for the impact of dose to the heart on the risk of SRILE Finally, a modified CV model is described that is consistent to all data. Conclusions: The detrimental effect of dose to the heart on the incidence of SRILF can be described by a dose dependent decrease in functional reserve of the lung. (c) 2007 Elsevier Inc