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.
Project description:Patient-derived xenografts (PDXs) are widely recognised as a more physiologically relevant preclinical model than standard cell lines, but are expensive and low throughput, have low engraftment rate and take a long time to develop. Our newly developed conditional reprogramming (CR) technology addresses many PDX drawbacks, but lacks many in vivo factors. Here we determined whether PDXs and CRCs of the same cancer origin maintain the biological fidelity and complement each for translational research and drug development. Four CRC lines were generated from bladder cancer PDXs. Short tandem repeat (STR) analyses revealed that CRCs and their corresponding parental PDXs shared the same STRs, suggesting common cancer origins. CRCs and their corresponding parental PDXs contained the same genetic alterations. Importantly, CRCs retained the same drug sensitivity with the corresponding downstream signalling activity as their corresponding parental PDXs. This suggests that CRCs and PDXs can complement each other, and that CRCs can be used for in vitro fast, high throughput and low cost screening while PDXs can be used for in vivo validation and study of the in vivo factors during translational research and drug development.
Project description:BACKGROUND:The patient-derived xenograft (PDX) model is likely to reflect human tumor biology more accurately than cultured cell lines because human tumors are implanted directly into animals; maintained in an in vivo, three-dimensional environment; and never cultured on plastic. PDX models of head and neck squamous cell carcinoma (HNSCC) have been developed previously but were not well characterized at the molecular level. HNSCC is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies and the understanding of HNSCC biology both depend upon clinically relevant animal models. We developed and characterized the patient-derived xenograft (PDX) model because it is likely to recapitulate human tumor biology. METHODS:We transplanted 30 primary tumors directly into mice. The histology and stromal components were analyzed by immunohistochemistry. Gene expression analysis was conducted on patient tumors and on PDXs and cell lines derived from one PDX and from independent, human tumors. RESULTS:Five of 30 (17%) transplanted tumors could be serially passaged. Engraftment was more frequent among HNSCC with poor differentiation and nodal disease. The tumors maintained the histologic characteristics of the parent tumor, although human stromal components were lost upon engraftment. The degree of difference in gene expression between the PDX and its parent tumor varied widely but was stable up to the tenth generation in one PDX. For genes whose expression differed between parent tumors and cell lines in culture, the PDX expression pattern was very similar to that of the parent tumor. There were also significant expression differences between the human tumors that subsequently grew in mice and those that did not, suggesting that this model enriches for cancers with distinct biological features. The PDX model was used successfully to test targeted drugs in vivo. CONCLUSION:The PDX model for HNSCC is feasible, recapitulates the histology of the original tumor, and generates stable gene expression patterns. Gene expression patterns and histology suggested that the PDX more closely recapitulated the parental tumor than did cells in culture. Thus, the PDX is a robust model in which to evaluate tumor biology and novel therapeutics.
Project description:Head and neck squamous cell carcinoma (HNSCC) is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies for HNSCC and the understanding of its biology both depend upon clinically relevant animal models. An increasingly promising xenograft model, the patient derived xenograft (PDX), is developed by surgically implanting tumor tissue directly from a patient into an immunocompromised mouse. We transplanted 30 HNSCC primary tumors directly into mice. The histology and stromal components were analyzed using immunohistochemistry. Gene expression analysis with Affymetrix U133A-microarrays was conducted on patient tumors, including third generation and one tenth generation PDX; one PDX-derived cell line; and 2 established HNSCC cell lines. Five of 30 (17%) transplanted tumors could be serially passaged and used for therapeutic and mechanistic studies. One cell line has been established from a tongue primary. The tumors maintained the histologic appearance of the parent tumor although human stromal components were lost upon engraftment. One PDX model was derived from an HPV-positive tumor. From the >54,000 probes tested, there were widespread differences in gene expression between the tumors growing in mice vs. the corresponding human tumors from which they were derived. For genes differing between parent tumors and human cell lines in culture, the PDXs’ expression pattern was very similar to that of the parent tumors. There were also widespread expression differences between the human tumors that subsequently grew in mice vs. those that did not - suggesting that this model enriches for cancers with distinct biological features. Our results demonstrate the feasibility of a PDX model of HNSCC. Gene expression patterns suggest that the PDX more closely recapitulated the parental tumor than do cells in culture. The histology of the tumors in mice is similar to that of the same tumor in humans. Additionally, gene expression patterns and histology are stable over multiple generations. We transplanted HNSCC primary tumors directly into mice. Gene expression analysis with Affymetrix U133A-microarrays was conducted on patient tumors, including third generation and one tenth generation PDX; one PDX-derived cell line; and 2 established HNSCC cell lines.
Project description:Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR) amplicon length (ssPAL) differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS), an immunomagnetic mouse cell depletion (MCD) approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors.
Project description:Preclinical models, including patient-derived xenograft (PDX) and organoid and primary cell culture, are essential for studies of cancer cell biology and facilitate translational research and individualization of therapy. We explored the optimum preclinical model by modifying the conventional conditional reprogramming (CR) system followed by screening effective targeted drug combinations against colorectal cancer (CRC). By modifying the ingredients of the culture medium used in a conventional CR system, a novel individualized CR system (termed i-CR) was established. Tumor samples from CRC patients were collected and PDX models were derived followed by high-throughput i-CR drug screening and validation of the effective targeted drug combinations. The i-CR system selectively expanded tumor cells rather than normal epithelial cells and facilitated high-throughput drug screening when combined with high-content imaging and quantitative analysis of cell proliferation. Using inhibitors targeting multiple signaling pathways identified by high-throughput i-CR drug screening, we discovered that inhibition of the EGFR and MEK or CDK4/6 pathways exerted a synergistic inhibitory effect against CRC, and we noted super-synergistic effects when EGFR, MEK, and CDK4/6 inhibitors were used simultaneously. These data were validated using paired PDX models, which showed marked inhibition of tumor growth. The novel i-CR system combined with PDX models will enable individualization of therapy and drug discovery, and strategies combining EGFR, MEK, and CDK4/6 inhibitors warrant clinical validation.
Project description:Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.
Project description:Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer and disproportionately accounts for the majority of breast cancer related deaths. Effective treatment of this disease remains an unmet medical need. Over the past several decades, TNBC cell lines have been used as the foundation for drug development and disease modeling. However, ever-mounting research demonstrates striking differences between cell lines and clinical TNBC tumors, disconnecting bench research and actual clinical responses. In this review, we discuss the limitations of cell lines and the importance of using patients' tumors for translational research, and highlight the usage of patient-derived xenograft (PDXs) models that have emerged as a clinically relevant platform for preclinical studies. PDX tumors possess tumor heterogeneity with similar cellular, molecular, genetic and epigenetic properties akin to those found within patients' tumors. Moreover, PDX and clinical tumors possess abnormal vasculature with higher blood vessel permeability, a feature that is not always demonstrated in in vivo cell line xenografts. Development of clinically relevant, novel drug-nanoparticles capable of accumulating in PDX tumors through the enhanced permeability and retention effect in tumor vasculature may lead to new and effective TNBC treatments.
Project description:<h4>Purpose</h4>To generate a preclinical model of isocitrate dehydrogenase (IDH) mutant gliomas from glioma patients and design a MRS method to test the compatibility of 2-hydroxyglutarate (2HG) production between the preclinical model and patients.<h4>Methods</h4>Five patient-derived xenograft (PDX) mice were generated from two glioma patients with IDH1 R132H mutation. A PRESS sequence was tailored at 9.4 T, with computer simulation and phantom analyses, for improving 2HG detection in mice. 2HG and other metabolites in the PDX mice were measured using the optimized MRS at 9.4 T and compared with 3 T MRS measurements of the metabolites in the parental-tumor patients. Spectral fitting was performed with LCModel using in-house basis spectra. Metabolite levels were quantified with reference to water.<h4>Results</h4>The PRESS TE was optimized to be 96 ms, at which the 2HG 2.25 ppm signal was narrow and inverted, thereby leading to unequivocal separation of the 2HG resonance from adjacent signals from other metabolites. The optimized MRS provided precise detection of 2HG in mice compared to short-TE MRS at 9.4 T. The 2HG estimates in PDX mice were in excellent agreement with the 2HG measurements in the patients.<h4>Conclusion</h4>The similarity of 2HG production between PDX models and parental-tumor patients indicates that PDX tumors retain the parental IDH metabolic fingerprint and can serve as a preclinical model for improving our understanding of the IDH-mutation associated metabolic reprogramming.
Project description:<b>Background: </b>In vitro patient tumor models such as patient-derived organoids (PDO) and conditionally reprogrammed (CR) cell culture are important for translational research and pre-clinical drug testing. In this study we present a personalized drug sensitivity test for late stage, potentially operable colorectal cancer (CRC) using patient-derived primary tumor cells isolated with i-CR technology, an optimized CR method. We explored the clinical feasibility of using i-CR platform to guide CRC chemotherapy, and established the correlation between in vitro drug sensitivity and patient clinical response.<br><br><b>Methods: </b>Primary CRC tumor cells were isolated and cultured with the i-CR technology. NGS was performed and the WES and CNV results of i-CR cells were compared with that of the original patient tumor samples. In vitro drug screenings were done with guideline chemotherapy drugs for CRC. In vivo drug response was examined with paired PDX mouse models. A double-blind co-clinical cohort study was carried out and the clinical outcomes of the enrolled patients were compared with the i-CR results.<br><br><b>Results: </b>i-CR platform could be used to rapidly propagate primary colorectal tumor cells that represent individual patient tumors effectively by keeping the clonal heterogeneity and the genetic characteristics. Chemotherapy drug screenings with i-CR cells were comparable with that of PDX models. More importantly, i-CR results showed high accordance with the clinical outcomes of the enrolled CRC patients.<br><br><b>Conclusion: </b>i-CR platform was capable to test and optimize therapeutic regimens pre-clinically, study cancer cell biology, and model tumor re-emergence to identify new targeted therapeutics from an effective personalized medicine standpoint.