Functionality of bone marrow mesenchymal stromal cells derived from head and neck cancer patients - A FDA-IND enabling study regarding MSC-based treatments for radiation-induced xerostomia

Abstract

Purpose: Salivary dysfunction is a significant side effect of radiation therapy for head and neck cancer (HNC). Preliminary data suggests that mesenchymal stromal cells (MSCs) can improve salivary function. Whether MSCs from HNC patients who have completed chemoradiation are functionally similar to those from healthy patients is unknown. We performed a pilot clinical study to determine whether bone marrow-derived MSCs [MSC(M)] from HNC patients could be used for the treatment of RT-induced salivary dysfunction.

Methods: An IRB-approved pilot clinical study was undertaken on HNC patients with xerostomia who had completed treatment two or more years prior. Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated and cultured. Culture-expanded MSC(M) were stimulated with IFNγ and cryopreserved prior to reanimation and profiling for functional markers by flow cytometry and ELISA. MSC(M) were additionally injected into mice with radiation-induced xerostomia and the changes in salivary gland histology and salivary production were examined.

Results: A total of six subjects were enrolled. MSC(M) from all subjects were culture expanded to > 20 million cells in a median of 15.5 days (range 8-20 days). Flow cytometry confirmed that cultured cells from HNC patients were MSC(M). Functional flow cytometry demonstrated that these IFNγ-stimulated MSC(M) acquired an immunosuppressive phenotype. IFNγ-stimulated MSC(M) from HNC patients were found to express GDNF, WNT1, and R-spondin 1 as well as pro-angiogenesis and immunomodulatory cytokines. In mice, IFNγ-stimulated MSC(M) injection after radiation decreased the loss of acinar cells, decreased the formation of fibrosis, and increased salivary production.

Conclusions: MSC (M) from previously treated HNC patients can be expanded for auto-transplantation and are functionally active. Furthermore IFNγ-stimulated MSC(M) express proteins implicated in salivary gland regeneration. This study provides preliminary data supporting the feasibility of using autologous MSC(M) from HNC patients to treat RT-induced salivary dysfunction.

Keywords: Cell therapy; Head and neck cancer; Mesenchymal Stromal Cells; Radiation; Xerostomia.

Figure 1:

Characterization of bone marrow derived mesenchymal stromal cells [MSC(M)] from patients with head and neck cancer (HNC). A) Graph of the normalized median fluorescent intensity of classical markers of HNC MSC(M). There was a significant increase of CD90, CD105, CD73 as compared to isotype control (*p=<0.01), with no significant difference in the median fluorescent intensity between MSCs and isotype control for the non-MSC markers (CD14, CD20, CD34, and CD45). B) Histograms of the 6 HNC MSC(M) showing the expression of classical markers (CD90, CD105, and CD73) and no expression of non-MSC Markers (CD14, CD20, CD34, and CD45) (MSC(M) shown as black outline, isotype control shown as gray curve). C) Differentiation of the MSC(M) into adipocytes and osteocytes was achieved for all 6 patients, with healthy control shown for comparison. D) Growth curve of the 6 HNC MSCs after IFNγ stimulation, cryopreservation, and thawing, showing variable speeds of continued growth over two weeks. E) Median fluorescent intensity (MFI) of immunosuppressive functional markers of head and neck cancer patients’ MSCs with and without IFNγ stimulation. There is a significant increase in MHC I, MHC II, IDO, and ICAM-1, with a trend in increased PD-L1 expression without statistical significance, all MFIs are median with standard error bars, *p<0.05.

Figure 2:

Cytokine expression of IFNγ stimulated bone marrow derived mesenchymal stromal cells [MSC(M)] from patients with treated head and neck cancers (HNC) and healthy donors after cryopreservation and culture rescue. A) GDNF expression, mean GDNF for HNC MSCs was 117.5 ± 15.9 pg/mL (standard deviation 15.9 pg/mL) mean GDNF for healthy donors was 155.7 ng/mL (standard deviation 21.6 ng/mL). B) WNT1 expression, mean WNT1 was 9.4 ng/mL (standard deviation 0.8 ng/mL, mean WNT1 for healthy donors was 9.3 ng/mL (standard deviation 1.6 ng/mL). C) R-spondin 1 expression, mean R-spondin 1 was 65.8 pg/mL (standard deviation 29.7 pg/mL), mean R-spondin 1 for healthy donors was 87.8 pg/mL (standard deviation 48.1 pg/mL).

Figure 3:

Further analysis of secretome. A) Heat map of the correlation between each pairing of cytokines examined in the secretome, with darker colors demonstrating a stronger correlation. B) Pairwise scatter plot of the relationship between FGF and VEGF-A with each patient represented as an empty circle, showing the clustering of head and neck cancer (HNC) patients and control patients, with each group’s mean shown in a solid circle. C) Pairwise scatter plot of the relationship between FGF and IL-8 (CXCL8) with each patient represented as an empty circle, showing the clustering of HNC patients and control patients, with the group’s mean shown in a solid circle. D) Concentrations of cytokines that may be used for discriminating between HNC mesenchymal stromal cells (MSC) and healthy control MSC, means reported for all MSC: mean IL8 2464.0 pg/mL, mean FGF 24.8 pg/mL, and mean VEGF 19.7 pg/mL.

Figure 4:

Treatment of irradiated mice with MSC(M) allows for salivary gland recovery. A) Murine submandibular glands at 20x magnification show changes after radiation and MSC(M) injection. Radiation (RT) resulted in histopathologic changes including decreased mucin, decreased amylase, and increased fibrosis (collagen). In irradiated mice injected with IFNγ-stimulated MSC(M) there was restoration of amylase and mucin production and decreased fibrosis, similar to unirradiated controls. ALB – Alician blue stains mucin, MTC – Massons trichrome stains collage fibers. B) Bar graph of the ratio of salivary production (in grams) to mouse body weight (in grams) showing MSC(M) injection allows for recovery of salivary production. Graph shows mean and SEM error bars. **p=0.0015