THE AMBULATORY AND EX-VIVO EFFECT OF INTRADETRUSOR IPSC-DERIVED HUMAN PROGENITOR SMOOTH MUSCLE CELLS IN A ROWETT NUDE RAT MODEL OF RADIATION CYSTITIS

Dobberfuhl A1, Briggs M2, Wallace S2, Wen Y2, Zhou Y2, Graves E3, Knox S3, Chen B2

Research Type

Basic Science / Translational

Abstract Category

Overactive Bladder

Abstract 580
E-Poster 3
Scientific Open Discussion ePoster Session 31
Friday 6th September 2019
13:35 - 13:40 (ePoster Station 3)
Exhibition Hall
Animal Study Basic Science Stem Cells / Tissue Engineering Biomechanics Voiding Dysfunction
1.Stanford University, Dept. of Urology, 2.Stanford University, Dept. of Obstetrics and Gynecology, 3.Stanford University, Dept. of Radiation Oncology
Presenter
A

Amy D Dobberfuhl

Links

Abstract

Hypothesis / aims of study
Pelvic malignancies account for one-third of all new cancer diagnoses and up to half of all patients receive XRT therapy. Adverse effects of pelvic XRT include radiation cystitis, urothelial hemorrhage and ischemic bladder fibrosis. Mesenchymal stem cells are known to promote tissue repair and inhibit inflammation and fibrosis. Our aim was to investigate the ambulatory and ex-vivo effect of intradetrusor iPSC-derived human progenitor smooth muscle cells (pSMC) in a rat model of radiation cystitis.
Study design, materials and methods
Twenty-four female Rowett nude rats were divided into 2 groups. After institutional animal protocol approval, bladders were identified by computed tomography and irradiated on day 0 [n=6 (0Gy); n=18 (20Gy)] using image-guided external beam radiotherapy (PXi X-Rad SmART). On day 53, randomized-blinded intradetrusor injection was performed [n=5 (20Gy-Vehicle); n=8 (20Gy-pSMC)]. Nocturnal ambulatory (overnight, 12-hours per cage cycle) micturition frequency and volume per void were recorded using a 12-channel 100-gram load cell sensor array (sensitivity 50 uL per void) and metabolic cages (Tecniplast, Model 3701M081) at baseline (day 0) and biweekly following radiation (day 0 to 81). Void frequency and volume were recorded in 24 hour intervals to allow animals to habituate to each cage cycle. At 4-weeks after intradetrusor injection (day 81), bladders were assessed by urethane anesthetized cystometry, organ bath myography and histology. Data were analyzed in SAS (Cary, North Carolina, USA). Metabolic cage data were analyzed using Pearson correlation, and univariate/multivariate linear regression models for outcome mean volume per void. Organ bath data were analyzed using T-test, analysis of variance (ANOVA) and generalized linear regression.
Results
On metabolic cage data analysis, there were 1,242 nocturnal voids (146 cage cycles, 7 time points). Volume per void was immediately reduced after irradiation. There was 28% (5/18) mortality from radiation proctitis (day 28-51). Following injection, survival of cells was confirmed by luciferase bioluminescence imaging. Nocturnal mean voided volume was partially improved in 20Gy-pSMC rats at 2-weeks after injection. After pSMC injection (day 51-81), there was normalization in number of voids (r=-0.032, p=0.892), total urine production (r=0.012, p=0.960) and stool output (r=-0.149, p=0.531). On multivariate linear regression (Table 1), after adjusting for covariates (water intake, total urine output, rat weight, stool output), there was a reduction in volume per void after vehicle injection (p<0.05, ref 0Gy), and partial improvement after pSMC injection (p=0.1864, ref 0Gy). Using a least squares (LS) estimate, mean voided volume was noted to partially normalize following pSMC treatment [LS-means: 0.50mL (0Gy), 0.42mL (20Gy-Vehicle), 0.45mL (20Gy-pSMC)].

On bladder cystometry data analysis, irradiation resulted in poor voiding efficiency (VE<15%) in 100% (4/4) of vehicle rats. The majority (67%, 4/6) of non-irradiated bladders had efficient voids (VE>20%). Improved voiding (VE>30%) was noted in 80% (4/5) of rats at 4-weeks after pSMC injection (day 81).

On organ bath myography analysis at day 81; there were 19 rats with 44 bladder strips (2-4 strips per rat), used to generate 660 maximum tension data points (total) as a result of 4 stimulation tests [electrical field stimulation (EFS) duration response (1 and 10 second), EFS frequency response (2, 8, 32 and 64 Hz), KCL dose response (100 and 200 mM), and carbachol dose response (0.01, 0.1, 1, 10, 20 µM]. On EFS of all rats, not excluding animals who died prior to day 81, the 1 and 10 second frequency response tension generated was increased at 32 and 64 Hz in pSMC treated animals (versus 0 Gy and 20Gy-Vehicle).

On organ bath myography analysis at 81+ days after radiation and 28+ days after injection, after excluding rats who did not survive to 81 days (3 pSMC rats died prior to 81 days due to radiation proctitis), our final group of rats subject to analysis were 0Gy (n=6), 20Gy-Vehicle (n=4), and 20Gy-pSMC (n=5). From this group, again an increase in tension generated (grams) is noted after pSMC treatment at 1 and 10 second EFS duration response. Sustained increase in contractility is noted at 64Hz [mean tension 1.21g (20Gy-pSMC) vs. 0.84g (0Gy), ANOVA p=0.12], however was not statistically significant as a result of the small number of animals per group and high morbidity as a result of radiation proctitis (Figure 1). Similarly pSMC partially improved potassium-chloride (KCL) response (100-200mM), however was not statistically significant. Radiotherapy weakened carbachol response in all tissues regardless of treatment, and anterior bladder contractility to carbachol was more impaired in 20Gy-Vehicle rats than pSMC rats.
Interpretation of results
Following pelvic irradiation, pSMC intradetrusor injection partially improved ambulatory mean voided volume, cystometric voiding efficiency, potassium-chloride response and electrical field stimulation contractility.
Concluding message
Further investigation is needed to assess the biochemical mechanisms of radiation induced bladder dysfunction on the ambulatory and ex-vivo physiologic response to pSMC treatment.
Figure 1 Table 1
Figure 2 Figure 1
References
  1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65(1):5-29. doi:10.3322/caac.21254
  2. Rajaganapathy BR, Jayabalan N, Tyagi P, Kaufman J, Chancellor MB. Advances in Therapeutic Development for Radiation Cystitis. Low Urin Tract Symptoms. 2014;6(1):1-10. doi:10.1111/luts.12045
  3. Wiafe B, Metcalfe PD, Adesida AB. Stem Cell Therapy: Current Applications and Potential for Urology. Curr Urol Rep. 2015;16(11):77. doi:10.1007/s11934-015-0551-5
Disclosures
Funding CIRM DISC1-08731 Clinical Trial No Subjects Animal Species Rat Ethics Committee Stanford University APLAC