Hypothesis / aims of study
Radiation therapy (RT) is commonly used for treating malignancies of pelvis including those arising from genitourinary, anorectal, and gynecologic organs. Radiation poses therapeutic effects on target diseases by direct tissue injury and interference on cell proliferation. However, damage to normal tissues involved in radiation field is inevitable that researchers focused on reducing this unnecessary damage by patient immobilization, intensity modulation, three-dimensional conformation, and improved image guidance. Development of technology enabled more precise focusing on target while minimizing damage to normal tissues in some cancers, but percentage of cancer survivors with RT history who develop related late complications should not be overlooked.
RT-related late effects are collectively termed pelvic radiation disease and main concern for urologists is radiation induced hemorrhagic cystitis. Two main issues of RT induced cystitis are unpredictability and untreatable nature of the disease. Radiation cystitis might occur in the acute phase after RT in minority of patients, but its onset is usually delayed - several years or decades later. The prevalence for radiation cystitis varies among studies and not all patients who undergo RT suffer from RT induced cystitis. In addition, present treatment strategies focus only on improvement in symptoms and are not able to definitely cure the disease. Radiation cystitis deteriorates patients’ quality of life, restrict active socio-economic activities and increase medical costs.
We have previously demonstrated therapeutic efficacy of mesenchymal stem cells in bladder dysfunction preclinical models. In this present study, we aimed to develop a radiation cystitis animal model and investigate therapeutic effect of stem cells with enhanced function in created model.
Study design, materials and methods
Stem Cell Preparation
Human umbilical cords(UCs) were collected and mesenchymal stem cells (MSCs) (UC-MSCs) were isolated as previously described. Human UC-MSCs were grown in low-glucose DMEM containing 10% heat-inactivated fetal bovine serum (FBS; HyClone, Pittsburgh, PA), 5 ng/mL human epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL basic fibroblast growth factor (bFGF), and 50 ng/mL long-R3 insulin-like growth factor-1 (IGF-1; ProSpec, Rehovot, Israel), as described. All MSCs used in this study were expanded for fewer than seven passages to ensure their functionality and were maintained at 37°C in a humidified atmosphere containing 5% CO2.
To enhance stem cell viability and engraftment, a technique named PFO (Primed Fresh Oct4) was used. For the PFO procedure, MSCs were plated at a density of 7 × 10^4 cells/mL and maintained in culture medium supplemented with 0.74 mM ascorbic acid 2-glucoside (AA2G; Sigma-Aldrich) for the indicated number of days. One day before functional evaluation, 50 nM sphingosine 1-phosphate (S1P; Sigma-Aldrich) and 0.5 mM valproic acid (VPA; Sigma-Aldrich) were added to the culture medium containing 0.74 mM AA2G to improve in vivo engraftment of MSCs, as previously described.
Study Design
In first part of the study, we determined optimal dose of radiation and optimal timing for bladder evaluation. Six-week old female C57BL/6J mice were divided into three groups - sham (n=15), 20Gy group (n =15), and 30Gy group (n = 15). Radiation was performed once in accordance with designated dosage. Mice in each group were sacrificed 4, 8, and 12 weeks after radiation to evaluate voiding habit and bladder histology (n=5, respectively).
Next, additional mice were divided into three groups - sham (n=5), RT (n=5), and RT+PFO (n=5) to evaluate the therapeutic efficacy of functionally improved stem cells. RT and RT+PFO group received pre-determined dosage of radiation and 100k PFO-MSCs were directly injected to the bladder of RT+PFO mice after fixed period of time, while others were injected phosphate buffer solution (PBS). Voiding profiles were assessed with voiding spot assay and bladders were harvested one week after injection for histological analysis. To investigate long-term therapeutic efficacy, voiding spot assay was weekly performed up to 8 weeks after stem cell injection in RT+PFO group (n=5).
Results
Optimal radiation dose for RC model was 20Gy, as 30Gy radiation presented high mortality rate. Radiated mice presented frequent urination and defecation compared to sham group, and it was more evident at 8-week after RT.
For further study, RC models received 20Gy radiation to pelvis, and phosphate buffer solution or 100k PFO-MSCs were directly injected to the bladder 8 weeks after radiation. Bladder tissue of RT group was characterized with edema and ischemic necrosis of urothelium with increased inflammation and fibrosis of detrusor while injection of PFO-MSCs alleviated histological changes (Figure 1). In addition, therapeutic efficacy of PFO-MSCs lasted up to 8 weeks after injection that improved urinary frequency was steadily maintained (Figure 2).
Interpretation of results
In previous studies, we have utilized MSCs originating from human embryonic stem cell -derived mesenchymal stem cells (hESC-MSCs). However, one of the major concerns with hESC-MSCs is possibility of carcinogenesis. All of the patients with radiation cystitis received RT due to pelvic malignancies that using hESC-MSCs would be more challenging as RT itself could increase the risk of malignancies in the affected field (ex; bladder cancer risk in prostate cancer patients with RT). Therefore, we decided to use UC-MSCs and enhance the stem cell function with PFO technique instead.