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Generation of stable PDX derived cell lines using conditional reprogramming.

ABSTRACT: Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

SUBMITTER: Borodovsky A

PROVIDER: S-EPMC5719579 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Generation of stable PDX derived cell lines using conditional reprogramming.

Borodovsky Alexandra A McQuiston Travis J TJ Stetson Daniel D Ahmed Ambar A Whitston David D Zhang Jingwen J Grondine Michael M Lawson Deborah D Challberg Sharon S SS Zinda Michael M Pollok Brian A BA Dougherty Brian A BA D'Cruz Celina M CM

Molecular cancer 20171206 1

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from p ...[more]

PMID: 29212548

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Project description: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.

Project description:BackgroundBreast cancer is a highly heterogeneous disease characterized by multiple histologic and molecular subtypes. While a myriad of breast cancer cell lines have been developed over the past 60 years, estrogen receptor alpha (ER)+ disease and some mutations associated with this subtype remain underrepresented. Here we describe six breast cancer cell lines derived from patient-derived xenografts (PDX) and their general characteristics.MethodsEstablished breast cancer PDX were processed into cell suspensions and placed into standard 2D cell culture; six emerged into long-term passageable cell lines. Cell lines were assessed for protein expression of common luminal, basal, and mesenchymal markers, growth assessed in response to estrogens and endocrine therapies, and RNA-seq and oncogenomics testing performed to compare relative transcript levels and identify putative oncogenic drivers.ResultsThree cell lines express ER and two are also progesterone receptor (PR) positive; PAM50 subtyping identified one line as luminal A. One of the ER+PR+ lines harbors a D538G mutation in the gene for ER (ESR1), providing a natural model that contains this endocrine-resistant genotype. The third ER+PR-/low cell line has mucinous features, a rare histologic type of breast cancer. The three other lines are ER- and represent two basal-like and a mixed ductal/lobular breast cancer. The cell lines show varied responses to tamoxifen and fulvestrant, and three were demonstrated to regrow tumors in vivo. RNA sequencing confirms all cell lines are human and epithelial. Targeted oncogenomics testing confirmed the noted ESR1 mutation in addition to other mutations (i.e., PIK3CA, BRCA2, CCND1, NF1, TP53, MYC) and amplifications (i.e., FGFR1, FGFR3) frequently found in breast cancers.ConclusionsThese new generation breast cancer cell lines add to the existing repository of breast cancer models, increase the number of ER+ lines, and provide a resource that can be genetically modified for studying several important clinical breast cancer features.

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Generation of stable PDX derived cell lines using conditional reprogramming.

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Generation of stable PDX derived cell lines using conditional reprogramming.

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