Despite considerable advances in radiation therapy, a growing number of head and neck cancer survivors, including survivors of oropharyngeal squamous carcinoma cancer (OPSCC), are living with treatment-related morbidity, the only remedies for which have minimal effectiveness [1,2]. During radiation therapy for OPSCC, healthy neighboring tissues are inevitably included in the radiation portal and the major salivary glands are highly radiation-sensitive despite a slow cell turnover. Radiation-induced salivary hypofunction and xerostomia (dry mouth) are among the most common and persistent side effects in previous head and neck cancer patients, predisposing these patients to problems with speech, swallowing, pain, dental decay, oral infections, sleep, and loss of quality of life. The effects of ionizing radiation on the major salivary glands are complex and not entirely understood, but they are characterized by a loss of acinar, progenitor, and stem cells as well as ongoing inflammation, reduced vascularization, and the formation of fibrosis. The present treatment for radiation-induced salivary gland hypofunction and xerostomia only addresses symptoms and provides extremely limited effects, which is why there is a great need for new treatments.
Growing evidence supports the potential of Mesenchymal stem/stromal cells (MSCs) to restore tissues damaged from irradiation. MSCs are somatic cells that are found in abundance in the vascularized tissue of the human body, where they are believed to facilitate homeostasis and regeneration. Preclinical studies indicate that rather than engrafting, MSCs possibly act by inducing anti-apoptosis, regeneration, immunomodulation, angiogenesis, anti-scarring, growth support, and differentiation of local stem and progenitor cells by releasing a concert of paracrine factors. However, human data regarding their mode of action are sparse.
For nearly a decade, our group at the Department of Otolaryngology, Head and Neck Surgery and Audiology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark has been working with adipose tissue-derived MSCs (AT-MSCs) for radiation-induced salivary gland damage. Our first trial was an investigator-initiated, randomized placebo-controlled trial that investigated autologous AT-MSC injection in the submandibular glands of previous OPSCC patients. The results of this trial showed encouraging signs of efficacy, with changes in unstimulated saliva flow rate and reduction of xerostomia-related complaints and no indications of safety concerns after two years of follow-up [3,4]. However, we found that autologous AT-MSCs were not applicable for clinical use, and our study revealed no information regarding the mechanisms that the AT-MSCs induced in the salivary glands. In collaboration with Copenhagen Cardiology Stem cell Centre (CSCC), we then designed a cryostored nonmatched allogeneic AT-MSCs from healthy donors to investigate the effects of their injection into both the parotid and submandibular glands in an open-label study, with dosage escalation of the cryostoring agent Dimethyl sulfoxide (DMSO). This study was designed to investigate the early safety, feasibility, efficacy, and mode of action of unmatched ex vivo expanded cryostored “off-the-shelf” AT-MSCs when injected (ultra-sound guided) in the submandibular and parotid glands in previous OPSCC patients with salivary gland damage after irradiation. The study took place in an out-patient clinic setting with a 120 days follow-up .
The present study, which was recently published in Communications Medicine, presents the results of changes in the human salivary proteome in eight patients irradiated for OPSCC compared to ten healthy age-, sex-, and education-matched control persons. The data were derived from an investigator-initiated, non-randomized, open-label, first-in-human, single-site study designed to investigate the early safety, feasibility, efficacy, and mode of action of unmatched ex vivo-expanded, cryostored “off-the-shelf” AT-MSC intraglandular treatment in previous OPSCC patients with salivary gland damage after irradiation in an out-patient clinic setting with a 120-day follow-up performed in collaboration with Department of Odontology, University of Copenhagen, Denmark. The patients all received ultrasound-guided injections of 25 x 106 AT-MSCs bilaterally in the submandibular glands and 50 x 106 AT-MSCs in each parotid gland.
The whole saliva proteome was analyzed using Mass spectrometry analysis, which provides detailed information on both the human proteome and microbiome (e.g. cohabiting microbiome in saliva), an approach known to reveal differences in patients with oral diseases compared to healthy control persons [6–9]. Our results showed that 140 human proteins were significantly differentially expressed in saliva from patients with radiation-induced hypofunction years after irradiation (baseline before AT-MSC treatment) compared to healthy controls. Five days after AT-MSC treatment, no changes were found in the whole saliva proteome. After 120 days, however, a significant impact occurred, as 99 proteins were differentially expressed at baseline compared to 120 days after treatment.
Interestingly, we found that AT-MSC treatment did not restore the proteome of whole saliva to a healthy condition, as 212 proteins were significantly differentially expressed in saliva 120 days after AT-MSC treatment, as compared to healthy controls. Among the differentially expressed proteins, several possess pleiotropic effects and could be involved in the regulation of cell growth and development, immune system functions, angiogenesis, or regeneration.
In conclusion, our study offers important insights into the effects induced by the injection of allogeneic AT-MSCs in radiation-damaged salivary glands. Intraglandular treatment with allogeneic AT-MSCs in irradiated patients stimulates an increase in proteins that may be related to tissue regeneration in stimulated whole saliva. This study also provides new data on how radiation therapy alters the proteome of whole saliva years after radiation treatment, which in the future may add significantly to a deeper understanding of the background biological mechanism responsible of the increased prevalence of oral diseases observed in this group of patients
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