A Novel Method To Procure Fresh Prostate Tissue for Viable Epithelial Cells.
Beth Palla, Hong Zhang, Jiaoti Huang. University of California at Los Angeles; Anhui Medical University, Hefei, China
Background: Cancer research depends on tumor models such as cell culture and mouse models but results from those studies need to be confirmed in human tissue. Archival human tissue is stored either as frozen tissue or in paraffin blocks, neither of which are suitable for studies that require live cells. We have designed a strategy to obtain fresh prostate tissue to prepare live prostate epithelial cells.
Design: 1. Prostatectomy specimens were sectioned into 3-4 mm thick slices from base to apex.
2. Levels 2 and 4 were divided into 4 quadrants and each quadrant was divided into superior and inferior halves. The superior half was submitted for frozen section to identify cancer versus benign tissue, while the inferior half was kept fresh on ice. The fresh tissue was then matched up with the frozen tissue section on the slide, and the fresh benign tissue was separated from cancer.
3. The fresh tissue was minced into small pieces, digested with collagenase and filtered to obtain a single cell suspension, which was incubated with antibodies against cell surface markers specific to each of the 3 prostate epithelial cell types, and sorted into distinct epithelial cell populations by fluorescence-activated cell sorting. The cell types were confirmed by RT-PCR and western assays.
Results: 1. Tissue from 80 cases was procured, 41% of which had cancer.
2. A total of 0.5-5 x 106 viable cells/case were obtained.
3. Using 3 antibodies against cell surface markers, distinct populations of epithelial cells were identified and sorted. Basal (Trop2highCD49fhigh), luminal (Trop2highCD49flow) and neuroendocrine (CD56high) cells were isolated from benign prostate while malignant lumina-type tumor cells and neuroendorcrine cells were isolated from tumor.
4. The benign and malignant cells contain high quality RNA as determined by ND8000 and Agilent 2100.
5. The benign epithelial cells were infected with lentivrisues expressing oncogenes, combined with supportive stroma, and transplanted into immunodeficient mice. High grade PIN occured at 12 weeks and invasive prostate cancer occured at 16 weeks, confirming tha viability of the isolated cells.
Conclusions: 1. We have developed a robust protocol to procure fresh human prostate tissue to obtain live benign and malignant epithelial cells
2. A combination of 3 antibodies separates prostate epithelial cells into distinct populations
3. The cells prepared are viable and can be used for biochemical and molecular studies as well as animal experiments, which will be invaluable in studying the molecular mechanisms directly applicable to human cancer.
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 246, Monday Morning