Abstract CN03-02: Conditionally reprogrammed cells for personalized medicine.

Primary epithelial and mesenchymal cells derived from human tissue have limited ability to proliferate in standard tissue culture conditions. These limits to proliferation, often referred to as the Hayflick limit, consist of two complex restrictive phases of growth referred to as M1 (senescence) and M2 (agonescence). Until recently, normal human cells could not be passaged indefinitely unless these restriction points were inactivated by cellular or viral oncogenes. A simple example is provided by studies of cell immortalization by the high-risk human papillomaviruses. These viruses contain two oncogenes, E6 and E7, which singly bypass the M1 phase of cell growth and together can bypass both M1 and M2, effecting perpetual proliferation or immortalization. However, such genetic manipulation disrupts critical cell regulatory pathways, including the Rb and p53 tumor suppressor functions, and induces altered growth and differentiation phenotypes. Recently we demonstrated that the combination of feeder cells and a Rho kinase inhibitor (Y-27632) induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro and this process occurs without the need for transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in matrigel. This stem-like state is reminiscent of an adult stem-like phenotype since the cells exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44 and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of these stem-like cells is rapid (occurs within two days) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. Interestingly, these cultures (conditionally reprogrammed cells, CRCs) do not over-express the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g. Sox2, Oct4, Nanog, or Klf4). In contrast to the selection of rare stem-like cells, the described growth conditions can generate millions of cells from needle biopsies within a week and can generate cultures from cryopreserved tissue and from fewer than 4 viable cells. Continued cell proliferation is dependent upon the continued presence of both feeder cells and Y-27632, and the CRCs retain a normal karyotype and remain non-tumorigenic. More important, these CRCs retain their lineage commitment and establish normal epithelial architecture when returned to appropriate environmental stimuli. Thus, the induction of CRCs is reversible (conditional) and the removal of Y-27632 and feeders allows the cells to differentiate normally. When CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well-differentiated stratified squamous epithelium, while the tracheal cells form a ciliated airway epithelium. While our initial studies revealed that co-culturing of epithelial cells with JS Swiss 3T3 cells was essential for the induction of CRCs, we have now shown with transwell culture plates that physical contact between feeders and epithelial cells is not required for inducing CRCs and, more important, that irradiation of the feeder cells is required for this induction. Consistent with the transwell experiments, conditioned medium is shown to induce and maintain CRCs, which is accompanied by a concomitant increase of cellular telomerase activity. The activity of the conditioned medium correlated directly with radiation-induced apoptosis of the feeder cells. Thus, conditional reprogramming of epithelial cells is mediated by a combination of Y-27632 and a soluble factor(s) released by apoptotic feeder cells. CRCs can be established from many vertebrate species, including mouse, rat, ferret, pig, cow, and horse. CRCs can also be established efficiently from mammalian tumors. CRCs established from a human prostate adenocarcinoma display instability of chromosome 13, proliferate abnormally in matrigel, and form tumors in SCID mice. CRCs established from a human squamous cell carcinoma of the tongue retain the genetic phenotype of the primary tumor and induce tumors in nude mice. Finally, breast tumors from transgenic mice or DMBA-treated rats can be established readily into CRCs to allow for molecular analysis. However, the most important use of CRCs in cancer biology is their potential for predicting or monitoring clinical responses to therapy. A recent successful application of CRCs for evaluating human disease and developing appropriate therapeutics is exemplified by a clinical case of recurrent respiratory papillomatosis (RRP). A male patient with a twenty-year history of RRP developed progressive, bilateral tumor invasion of the lung parenchyma. CRCs were generated from the patient's normal and tumor lung tissue and analysis revealed that the laryngeal tumors contained a wild-type 8 kb HPV-11 genome whereas the metastatic pulmonary tumor cells contained a mutant 10.4 kb genome with amplification of the promoter/oncogene region, giving an apparent explanation for the aggressive behavior of this normally benign disease. More important, chemosensitivity studies on the tumor cell cultures identified Zolinza (vorinostat) as a potential therapeutic and the patient exhibited regression/stabilization of tumor growth after 3 months of treatment, with durable effects for two years. Clearly other human tumors need to be examined for the ability of CRCs to enhance the clinical choice of therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN03-02. Citation Format: Richard Schlegel, Nancy Pelacor-Ceron, Frank Suprynowicz, Christopher Albanese, Hang Yuan, Xuefeng Liu. Conditionally reprogrammed cells for personalized medicine. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN03-02.