Optimization of the Production Process of Clinical-Grade Human Salivary Gland Organoid-Derived Cell Therapy for the Treatment of Radiation-Induced Xerostomia in Head and Neck Cancer.

Head and neck cancer is a common cancer worldwide. Radiotherapy has an essential role in the treatment of head and neck cancers. After irradiation, early effects of reduced saliva flow and hampered water secretion are seen, along with cell loss and a decline in amylase production. Currently, there is no curative treatment for radiation-induced hyposalivation/xerostomia. This study aimed to develop and optimize a validated manufacturing process for salivary gland organoid cells containing stem/progenitor cells using salivary gland patient biopsies as a starting material. The manufacturing process should comply with GMP requirements to ensure clinical applicability. A laboratory-scale process was further developed into a good manufacturing practice (GMP) process. Clinical-grade batches complying with set acceptance and stability criteria were manufactured. The results showed that the manufactured salivary gland-derived cells were able to self-renew, differentiate, and show functionality. This study describes the optimization of an innovative and promising novel cell-based therapy.

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.

Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal.

Relatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand, replace and regenerate. Resident stem/progenitor cells are key in this process because, upon activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating in vivo. Accordingly, autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.

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.

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 thyroid-derived 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-lineage-specific 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.

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.

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.

Lack of DNA Damage Response at Low Radiation Doses in Adult Stem Cells Contributes to Organ Dysfunction.

PURPOSE: Radiotherapy for head and neck cancer may result in serious side effects, such as hyposalivation, impairing the patient's quality of life. Modern radiotherapy techniques attempt to reduce the dose to salivary glands, which, however, results in low-dose irradiation of the tissue stem cells. Here we assess the low-dose sensitivity of tissue stem cells and the consequences for tissue function. EXPERIMENTAL DESIGN: Postirradiation rat salivary gland secretory function was determined after pilocarpine induction. Murine and patient-derived salivary gland and thyroid gland organoids were irradiated and clonogenic survival was assessed. The DNA damage response (DDR) was analyzed in organoids and modulated using different radiation modalities, chemical inhibition, and genetic modification. RESULTS: Relative low-dose irradiation to the high-density stem cell region of rat salivary gland disproportionally impaired function. Hyper-radiosensitivity at doses <1 Gy, followed by relative radioresistance at doses ≥1 Gy, was observed in salivary gland and thyroid gland organoid cultures. DDR modulation resulted in diminished, or even abrogated, relative radioresistance. Furthermore, inhibition of the DDR protein ATM impaired DNA repair after 1 Gy, but not 0.25 Gy. Irradiation of patient-derived salivary gland organoid cells showed similar responses, whereas a single 1 Gy dose to salivary gland-derived stem cells resulted in greater survival than clinically relevant fractionated doses of 4 × 0.25 Gy. CONCLUSIONS: We show that murine and human glandular tissue stem cells exhibit a dose threshold in DDR activation, resulting in low-dose hyper-radiosensitivity, with clinical implications in radiotherapy treatment planning. Furthermore, our results from patient-derived organoids highlight the potential of organoids to study normal tissue responses to radiation.

Sparing the region of the salivary gland containing stem cells preserves saliva production after radiotherapy for head and neck cancer.

Each year, 500,000 patients are treated with radiotherapy for head and neck cancer, resulting in relatively high survival rates. However, in 40% of patients, quality of life is severely compromised because of radiation-induced impairment of salivary gland function and consequent xerostomia (dry mouth). New radiation treatment technologies enable sparing of parts of the salivary glands. We have determined the parts of the major salivary gland, the parotid gland, that need to be spared to ensure that the gland continues to produce saliva after irradiation treatment. In mice, rats, and humans, we showed that stem and progenitor cells reside in the region of the parotid gland containing the major ducts. We demonstrated in rats that inclusion of the ducts in the radiation field led to loss of regenerative capacity, resulting in long-term gland dysfunction with reduced saliva production. Then we showed in a cohort of patients with head and neck cancer that the radiation dose to the region of the salivary gland containing the stem/progenitor cells predicted the function of the salivary glands one year after radiotherapy. Finally, we showed that this region of the salivary gland could be spared during radiotherapy, thus reducing the risk of post-radiotherapy xerostomia.

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 (CD24(hi)/CD29(hi)). 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.

Prediction of response to radiotherapy in the treatment of esophageal cancer using stem cell markers.

BACKGROUND AND PURPOSE: In this study, we investigated whether cancer stem cell marker expressing cells can be identified that predict for the response of esophageal cancer (EC) to CRT. MATERIALS AND METHODS: EC cell-lines OE-33 and OE-21 were used to assess in vitro, stem cell activity, proliferative capacity and radiation response. Xenograft tumors were generated using NOD/SCID mice to assess in vivo proliferative capacity and tumor hypoxia. Archival and fresh EC biopsy tissue was used to confirm our in vitro and in vivo results. RESULTS: We showed that the CD44+/CD24- subpopulation of EC cells exerts a higher proliferation rate and sphere forming potential and is more radioresistant in vitro, when compared to unselected or CD44+/CD24+ cells. Moreover, CD44+/CD24- cells formed xenograft tumors faster and were often located in hypoxic tumor areas. In a study of archival pre-neoadjuvant CRT biopsy material from EC adenocarcinoma patients (N=27), this population could only be identified in 50% (9/18) of reduced-responders to neoadjuvant CRT, but never (0/9) in the complete responders (P=0.009). CONCLUSION: These results warrant further investigation into the possible clinical benefit of CD44+/CD24- as a predictive marker in EC patients for the response to chemoradiation.

Bone marrow stromal cell interaction reduces syndecan-1 expression and induces kinomic changes in myeloma cells.

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.