Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer

Abstract

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

Expansion of airway basal epithelial cells from primary human non‐small cell lung cancer tumors

“…Relevant biological differences might also exist between mitotic inactivation using irradiation4, 9 rather than mitomycin C, as used here. Alternatively, different tissue acquisition methods,9 feeder cell‐conditioned medium,10, 27 low oxygen,10 harsh tissue digestion protocols26 and/or initially serum‐starving cells6 might help to select tumor cells by inducing differentiation or apoptosis in normal epithelial cells.…”

“…Traditionally, the primary culture of human cancer cells has been challenging, with few tumors amenable to culture on plastic, so this protocol, known as “conditional reprogramming” or “3T3 + Y,” has naturally attracted attention in the cancer community. To date, variants of this protocol have allowed cancer cell cultures to be established across multiple cancer types including lung, prostate, pancreas and colon 4, 5, 6…”

“…To date, variants of this protocol have allowed cancer cell cultures to be established across multiple cancer types including lung, prostate, pancreas and colon. [4][5][6] In non-small cell lung cancer (NSCLC), a number of reports demonstrate successful primary tumor cell culture using fibroblast co-culture and ROCK inhibition. [7][8][9][10] However, others have found that normal epithelial cells are preferentially expanded in these conditions.…”

Establishment of pancreatic cancer cell lines with endoscopic ultrasound‐guided biopsy via conditionally reprogrammed cell culture

“…However, PDX takes many months to establish and is difficult to use for high‐throughput screening in cancer patients . Because of these limitations, a recent patient‐derived cancer cell line model, CRCs, are extensively used in cancer research . CRCs induce the propagation and immortalization of human tumor epithelial cells and generate patient‐specific cell lines within 2 or 4 weeks .…”

“…3 Because of these limitations, a recent patient-derived cancer cell line model, CRCs, are extensively used in cancer research. 11,15,17,18 CRCs induce the propagation and immortalization of human tumor epithelial cells and generate patient-specific cell lines within 2 or 4 weeks. 10,11 Considering that small tissues from biopsy can be a source of primary cell lines using the CRC technique, EUS-FNB should also be evaluated as a source of primary cancer cell lines.…”