1. Mechanically-induced molecular signaling in prostate development
Elizabeth Marcin
Department of Biomedical Engineering: Gleghorn Lab
University of Delaware Summer Fellows 2018

2. Background
The prevalence of both benign prostate hyperplasia (BPH) and prostate cancer increases with age: Over 55% of men over 50 exhibit BPH symptoms, and 12% of men are diagnosed with prostate cancer.
Though, they differ in cellular specific gene expression, both pathologies involve abnormal regulation of growth signaling pathways similar to that of embryonic phenotypes – so it’s possible that factors involved in prostate development are crucial to understanding BPH and cancer progression.
Interestingly, both pathologies are defined by varying degrees of abnormal stromal and epithelial growth: prostatic intraepithelial neoplasia (PIN) structures.
During development, epithelial cells begin to bud off the urethra and canalize to form hollow lumen (where the prostate can synthesize and secrete seminal fluid) surrounded by a layer of smooth muscle and then mesenchymal cells.
The smooth muscle is contractile throughout development, creating mechanical forces and pressure changes around the lumen to initiate fluid movement.
Research Questions
Are these mechanical forces necessary for epithelial growth?
Could mechanical forces like hydrostatic pressure and stretch influence adult epithelial function and growth?
Image Adapted from Roxanne Toivanen and Michael M. Shen, Development, 2017

3. Hypothesis

4. Experimental Design
Prostate epithelium was manually isolated from adult and adolescent mice and cultured in a Matrigel-media mixture enhanced with various growth factors and hormones to form hollow organoids. This smaller model is ideal for measurements specific to pressure-epithelial relationships as opposed to effects on the entire organ
Whole mount murine prostate explants were cultured on a membrane in two conditions: one in which the system was sutured closed at both the urethra and at the vas deferens allowing for fluidic pressure to be established in the lumen; the other in which the system remained open, preventing any luminal pressure establishment.
*culture method used for preliminary experiment*
Prostate Explants were sutured as described above, cultured upright in 2.5mL of media, and tethered to the base of the well by urethral-suture threads. This system is used to easily access the ventral lumen for pressure measurements of explants at varying time points and conditions

5. Preliminary Experiment Results
Murine Prostate, Open System Cultured Model.
Explanted at post natal day 5, cultured for 48 hours.
ECAD stain (white) marker for epithelium
Murine Prostate, Open System Cultured Model.
Postnatal day 5 for 48 hours
EdU stain (green) marker of cell replication
Murine Prostate, Closed System Cultured Model.
Postnatal day 5 for 48 hours
ECAD stain (white)
Interestingly enough, the closed system prostates
exhibited more PROLIFERATION
specifically EPITHELIAL cells
in the VENTRAL Lobe
Murine Prostate, Closed System Cultured Model.
Postnatal day 5 for 48 hours
EdU stain (gree

6. Pressure Measurement Examples
Explants were suspended and cultured in media for 24 hours before measurements were taken
A needle was inserted into lumen of the ventral lobe of the prostates and a baseline intralumenal pressure over time was established at various ages using a device engineered in Dr. Gleghorn’s lab.
These baselines for changes in luminal pressure throughout development will provide insight to role or pressure organogenesis and how growth is altered in response to pressure changes
Pressure inside the lumen (234 Pa)
Sensor removed, initiating return to system base of 0 Pa
Sensor inserted into pressurized lumen
Initial system pressure (0 Pa)
* Peaks at t=0 excluded as consistent erroneous initial measurement

7. Current/Continuing Experiments
Take pressure measurements of individual acini to compare to physiological base line
Chemically manipulate luminal pressure of lobe explants by altering ion channel function (using Forskolin and CFTR inhibitor)
Use immunofluorescent microscopy to determine membrane localization and concentration of CFTR channels in response to various pressures
Run qPCR after altering pressures in order to identify cell polarity and quantify expression of mechanosensitive Calcium channels such as TRPC6, TRPC1, TRPA1 and possible down stream protein targets involved in epithelial growth such as NFAT and EGF.
Example of epithelial acini after canalization: image taken with an epi-flourescent microscope in bright field with green stain for ECAD expression to confirm epithelial cell identity.

8. Acknowledgments