A novel cell-based transplantation method using a Rho kinase inhibitor and a specific catheter device for the treatment of salivary gland damage after head and neck radiotherapy

Abstract

Radiotherapy (RT) for head and neck cancer results in irreversible damage to salivary glands (SGs) and decreases saliva production, leading to a dry mouth. To date, there are no satisfactory therapies to solve this problem. We recently established a novel culturing method using a Rho kinase inhibitor (RI), Y-27632, that maintained cellular morphology and function for a prolonged period of time. In the present study, we investigated whether cell-based transplantation using our culturing method ameliorated the dysfunction of irradiated SGs. First, rat SG cells were cultured in a medium with RI. Cells were characterized by morphological findings and mRNA expression analysis. We also assessed features of SG cells in three-dimensional (3-D) culture by scanning electron microscopy and immunohistochemistry (IHC). The RI-containing medium led to higher cell proliferation of rat SG cells with preservation of cell morphology and higher alpha-amylase (AMY) expression in both 2-D and 3-D culture systems. To establish the atrophic-SG models, external RT at a dose of 15 Gy was delivered to the head and neck fields of nude rats. The SG cells derived from GFP-rats were cultured in medium with RI, after which they were transplanted into the submandibular glands of atrophic-SG rats using a catheter placed into Wharton's duct. IHC and salivary flow rate (SFR) analyses were measured 12 weeks after the transplantation. Following transplantation, donor cells (GFP-SG cells) were primarily located in the ductal region of the SG, and AMY expression in SGs and the SFR were increased in the SG cell transplantation group compared with the control. Those data indicated that cell-based therapy using RI-treated SG cells could restore salivary hypofunction of irradiated SGs by direct integration of the donor cells in the duct of SGs. We propose that these data support future clinical plans in which SG cells would be excised from the labial minor SGs of the patients with head and neck cancers prior to RT, cultured during RT, and auto-transplanted into SGs using a catheter into the Wharton's duct. We believe that our culturing and transplantation methods can be applied to SG cells, constituting a therapeutic approach for the treatment of patients with dry mouth after not only RT but also aging and Sjögren's syndrome.

Keywords: Auto-transplantation; Cell-based transplantation; Head and neck cancer; Radiotherapy; Rho kinase inhibitor; Salivary gland.

Fig. 1

Culture of rat SG cells with/without RI. A, Cell morphologies changed with continued growth in the absence of RI, but not in the presence of RI (passage-10). B, Cellular growth of rat SG cells with/without RI. The cellular proliferation assay demonstrated that RI-treated SG cells retained higher cell growth rates than did the non-RI culture condition (*, P < 0.05). C, Amy mRNA expression in rat SG cells with/without RI. Amy mRNA expression was significantly upregulated in the RI-treated SG cells compared with the cells cultured without RI (passage-10; *, P < 0.05; N = 3).

Fig. 2

Characterization of rat SG cells in a 3-D culture system. A, Rat SG cells were tightly connected and exhibited a typical cobblestone morphology in a 2-D culture system (2-D). Rat SG cells were cultured in Matrigel to permit formation of 3-D structures. These cells formed rounded clumps in certain areas of the Matrigel (3-D). B, SEM analysis of the 3-D structure of cultivated rat SG cells. The cell clumps were embedded in Matrigel (upper panel), and high magnification showed that the clumps were composed of closely associated cells (arrows in the upper panel). The hollow lumen structure and tightly connected cells aggregates were observed through a crack in the cell clump (lower panel). C, HE and PAS staining of rat SG cells. HE staining showed that the cell clumps that comprised two to three layers of SG cells, included luminal spaces and several cells were localized in those spaces (HE). The internal structure of the cell clumps stained positively for PAS, suggesting the presence of glycoproteins, carbohydrates, and mucins (PAS). D, IHC of rat SG cells. The cell clumps stained positively for AMY and mucin1, markers of mucopolysaccharides. Positive staining was also observed for AQP5 and NKCC1, but the localizations differed from normal SG tissues.

Fig. 3

Transplantation of GFP-rat SG cells to irradiated SG through the Wharton's duct. A, Establishment of irradiated SG rat models. Anesthetized rats were immobilized in a tube shielded with lead. Only the head and neck regions were exposed. The nude rats were locally irradiated in the head and neck regions with a single dose of 15 Gy. B, The catheter was inserted into Wharton's duct from the sublingual caruncle (red circle) to the submandibular gland. C, IHC of transplanted SGs was performed using anti-GFP antibody. GFP-positive cells were observed in the duct of the RT + SG cell group. D, AMY expression after RT in SGs. Low AMY expression was found in the RT group. AMY expression was recovered in the RT + SG cell group 12 weeks after the transplantation. E, SFR after the transplantation. SFR was significantly increased in the RT + SG cell group 12 weeks after the transplantation with SG cells (N = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Plans for SG auto-transplantation of human patients. A, Schematic diagram of human application shows that some labial minor SGs are extracted prior to RT and cultured during RT. Auto-transplantation of cultured SG cells through Wharton's duct into the atrophic submandibular gland after RT is performed. B, Morphology of human SG cells with/without RI. The human SG cells cultured with RI showed similar cell growth and cell morphology to those of rat SG cells. C, AMY expression in human SG cells with/without RI. AMY mRNA expression was significantly upregulated in RI-treated SG cells compared with non-RI culture condition (passage-10; *, P < 0.05; N = 3). The ICC data also showed strong immunoreactivity of AMY in the RI-treated SG cells, whereas SG cells cultured without RI showed weak immunoreactivity of AMY. D, Transplantation method was confirmed using a catheter device through Wharton's duct in healthy volunteers. Proper insertion of the catheter was confirmed by radiograph.