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Review
. 2019 Oct 27;8(11):1327.
doi: 10.3390/cells8111327.

Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks

Nancy Palechor-Ceron  1 , Ewa Krawczyk  1 , Aleksandra Dakic  1 , Vera Simic  1 , Hang Yuan  1 , Jan Blancato  2 , Weisheng Wang  2 , Fleesie Hubbard  3 , Yun-Ling Zheng  2 , Hancai Dan  3 , Scott Strome  3 , Kevin Cullen  3 , Bruce Davidson  4 , John F Deeken  5 , Sujata Choudhury  1 , Peter H Ahn  6 , Seema Agarwal  1 , Xuexun Zhou  7 , Richard Schlegel  1 , Priscilla A Furth  2 , Chong-Xian Pan  8 , Xuefeng Liu  1   2
Affiliations
Review

Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks

Nancy Palechor-Ceron et al. Cells. .

Abstract

Traditional cancer models including cell lines and animal models have limited applications in both basic and clinical cancer research. Genomics-based precision oncology only help 2-20% patients with solid cancer. Functional diagnostics and patient-derived cancer models are needed for precision cancer biology. In this review, we will summarize applications of conditional cell reprogramming (CR) in cancer research and next generation living biobanks (NGLB). Together with organoids, CR has been cited in two NCI (National Cancer Institute, USA) programs (PDMR: patient-derived cancer model repository; HCMI: human cancer model initiatives. HCMI will be distributed through ATCC). Briefly, the CR method is a simple co-culture technology with a Rho kinase inhibitor, Y-27632, in combination with fibroblast feeder cells, which allows us to rapidly expand both normal and malignant epithelial cells from diverse anatomic sites and mammalian species and does not require transfection with exogenous viral or cellular genes. Establishment of CR cells from both normal and tumor tissue is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 × 106 cells in five days from a core biopsy of tumor tissue. Normal CR cell cultures retain a normal karyotype and differentiation potential and CR cells derived from tumors retain their tumorigenic phenotype. CR also allows us to enrich cancer cells from urine (for bladder cancer), blood (for prostate cancer), and pleural effusion (for non-small cell lung carcinoma). The ability to produce inexhaustible cell populations using CR technology from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (NGLB: next-generation living biobank) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool for precision cancer medicine. We discussed analyses of patient-derived matched normal and tumor models using a case with tongue squamous cell carcinoma as an example. Last, we summarized applications in cancer research, disease modeling, drug discovery, and regenerative medicine of CR-based NGLB.

Keywords: conditionally reprogrammed cells; living biobanks; organoids; patient-derived cancer models.

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Conflict of interest statement

Several patents for conditional reprogramming technology has been awarded to Georgetown University by the United States Patent Office. The license for this technology has been given to Propagenix for commercialization. The inventors, R.S., X.L., and Georgetown University receive potential royalties and payments from Propagenix.

Figures

Figure 1
Figure 1
In vitro culture of matched normal and tumor conditionally reprogrammed cell cultures (CRCs). (A) Cell morphology: under light microscope the non-malignant cells appeared to grow in tight colonies (inner square) surrounded by feeder cells, a characteristic of cells cultured in CRC, whereas malignant cells grew as individual big cells (inner square) displaying prominent intercellular junctions. Non-malignant cells exhibited a heterogeneous population composed of small, dark, hexagonal cells and some big and flat, while tumor cells displayed a highly homogenous population of big, dark, hexagonal cells (outer squares). Bars: 50 and 200 µm. (B) Karyotype: cytogenetic analysis of normal and tumor CRCs were performed at early passage. The non-malignant line (46, XX) displayed a diploid karyotype with no apparent chromosomal alterations (left) whereas the malignant line consisted of a near-triploid clone karyotype (74, XXX, +1, +1, +1, +2, +2, +3, +3, +4, +5, +6, +6, +7, +7, +10, +10, +11, +11, +12, +16, +16, +17, +17, +18, +19, +19, +20, +21, +22) (right).
Figure 2
Figure 2
Organoid cultures of normal and tumor CRCs in matrigel. Morphogenesis: normal CRC (A) formed small spheres of approximately 100 µm characterized by displaying only one lobule with a mass-like shape and central growth, while tumor CRC (C) formed more prominent spheres of more than 200 µm consisting of multiple lobules with a grape-like shape and defined edges. Bars 100 µm. Unlike normal CRC that grew as independent spheres (B), tumor CRC developed invasive processes (D) which communicated adjacent spheres. Bars 400 µm. Immunohistochemistry: spheres from on-malignant cells depicted a polarized central growth with a structure similar to an arrested acinus (E) whereas the spheres from malignant cells were formed by actively proliferating cells (I) correlated with Ki-67 where the non-malignant displayed a negative staining (G) but stained moderately positive to P63 (H) while a high percentage of malignant cells stained strongly positive for Ki-67 (K) and the remaining for P63 (L). All the spheres from both lines stained strongly and diffusely positive for CK (cytokeratin) 14. Bars: 100 µm.
Figure 3
Figure 3
Tumorigenicity Assays. (A) Soft agar colony formation assay: in an anchorage-independent growth assay only tumor CR cells formed visible spheres after two weeks in soft agar culture. Scale bars: 500 µm. (B) Xenografts: tumorigenic properties of tumor CR cells were defined by an in vivo assay. Six-week-old athymic mice were inoculated with 1 × 106 normal or tumor conditional cell reprogramming (CR) cells (left). The resulting tumors were resected after 3–3.5 months of injection and stained with Hematoxylin and eosin (H&E) (middle) staining (right). (C) Tumor growth: tumor CR cells formed palpable tumors three weeks after injection in both flank and mammary sites displaying similar growth rates and patterns.
Figure 4
Figure 4
In vitro chemosensitivity of normal and tumor CRCs. Differential toxicity of SAHA and cisplatin was established in the matched normal and tumor CR cells via ATP bioluminescence. Malignant cells showed more sensitivity to both compounds by displaying a median curative dose of 1.09 µM for Vorinostat (SAHA) and 10.47 µM for cisplatin compared to 5.83 µM and 39.91 µM respectively for the normal line (p values: 0.011 and 0.014).
Figure 5
Figure 5
Top 20 active pathways (A) and their distributions of molecular (B) and biological (C) functions in tumor CRC. RNA sequencing analysis showed that the angiogenesis, Wnt, integrin, inflammation mediated by chemokine and cytokine as well as the gonadotropin releasing hormone receptor pathways were the most highly active in tumor CRCs.
Figure 6
Figure 6
Workflow of next generation living biobanks (NGLB). Specimens including surgical specimens, core biopsies, needle biopsies, brushed cells, or cells from liquid biopsies (blood, urine, etc.) from patients with disease and/or different stages of disease (for example, before and after treatment, primary or metastatic, etc.) will be collected, cryopreserved, and stored together with their corresponding -omics (genomic, transcriptomic, proteomic, metabolomic, etc.) and clinical information. CR technology will be used to generate an unexhausted cell cultures for living cell banks for future demands in drug discovery, disease modeling, regenerative medicine. CR cells can be used to generate other types of living biobanks such organoids or patient-derived xenografts (PDX) (for tumor).
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