Conditional Reprogramming Research Articles

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury.

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury (AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells (KPTECs) in 2D and 3D culture systems using conditional reprogramming (CR) and organoids techniques. These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters (SLC34A3 and cubilin). They also expressed angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3D organoids culture compared to that in 2D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike (S) protein was able to enter CR cells with luciferase reporter. This integrated 2D CR and 3D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00253-y) contains supplementary material, which is available to authorized users.

Conditional reprogramming: Modeling urological cancer and translation to clinics.

Patient‐derived models, including cell models (organoids and conditionally reprogrammed cells [CRCs]) and patient‐derived xenografts, are urgently needed for both basic and translational cancer research. Conditional reprogramming (CR) technique refers to a co‐culture system of primary human normal or tumor cells with irradiated murine fibroblasts in the presence of a Rho‐associated kinase inhibitor to allow the primary cells to acquire stem cell properties and the ability to proliferate indefinitely in vitro without any exogenous gene or viral transfection. Considering its robust features, the CR technique may facilitate cancer research in many aspects. Under in vitro culturing, malignant CRCs can share certain genetic aberrations and tumor phenotypes with their parental specimens. Thus, tumor CRCs can promisingly be utilized for the study of cancer biology, the discovery of novel therapies, and the promotion of precision medicine. For normal CRCs, the characteristics of normal karyotype maintenance and lineage commitment suggest their potential in toxicity testing and regenerative medicine. In this review, we discuss the applications, limitations, and future potential of CRCs in modeling urological cancer and translation to clinics.

Culture and application of conditionally reprogrammed primary tumor cells

Cancer is still a major public-health problem that threatens human life worldwide and further study needs to be carried out in the basic and preclinical areas. Although high-throughput sequencing technology and individualized precise therapy have made breakthroughs over the years, the high failure rate of clinical translational research has limited the innovation of antitumor drugs and triggered the urgent need for optimal cancer-research models. The development of cancerous cell lines, patient-derived xenograft (PDX) models, and organoid has strongly promoted the development of tumor-biology research, but the prediction values are limited. Conditional reprogramming (CR) is a novel cell-culture method for cancer research combining feeder cells with a Rho-associated coiled-coil kinase (ROCK) inhibitor, which enables the rapid and continuous proliferation of primary epithelial cells. In this review, we summarize the methodology to establish CR model and overview recent functions and applications of CR cell-culture models in cancer research with regard to the study of cancer-biology characterization, the exploration of therapeutic targets, individualized drug screening, the illumination of mechanisms about response to antitumor drugs, and the improvement of patient-derived animal models, and finally discuss in detail the major limitations of this cell-culture system.

Hybrid Stem Cell States: Insights Into the Relationship Between Mammary Development and Breast Cancer Using Single-Cell Transcriptomics.

Similarities between stem cells and cancer cells have implicated mammary stem cells in breast carcinogenesis. Recent evidence suggests that normal breast stem cells exist in multiple phenotypic states: epithelial, mesenchymal, and hybrid epithelial/mesenchymal (E/M). Hybrid E/M cells in particular have been implicated in breast cancer metastasis and poor prognosis. Mounting evidence also suggests that stem cell phenotypes change throughout the life course, for example, through embryonic development and pregnancy. The goal of this study was to use single cell RNA-sequencing to quantify cell state distributions of the normal mammary (NM) gland throughout developmental stages and when perturbed into a stem-like state in vitro using conditional reprogramming (CR). Using machine learning based dataset alignment, we integrate multiple mammary gland single cell RNA-seq datasets from human and mouse, along with bulk RNA-seq data from breast tumors in the Cancer Genome Atlas (TCGA), to interrogate hybrid stem cell states in the normal mammary gland and cancer. CR of human mammary cells induces an expanded stem cell state, characterized by increased expression of embryonic stem cell associated genes. Alignment to a mouse single-cell transcriptome atlas spanning mammary gland development from in utero to adulthood revealed that NM cells align to adult mouse cells and CR cells align across the pseudotime trajectory with a stem-like population aligning to the embryonic mouse cells. Three hybrid populations emerge after CR that are rare in NM: KRT18+/KRT14+ (hybrid luminal/basal), EPCAM+/VIM+ (hybrid E/M), and a quadruple positive population, expressing all four markers. Pseudotime analysis and alignment to the mouse developmental trajectory revealed that E/M hybrids are the most developmentally immature. Analyses of single cell mouse mammary RNA-seq throughout pregnancy show that during gestation, there is an enrichment of hybrid E/M cells, suggesting that these cells play an important role in mammary morphogenesis during lactation. Finally, pseudotime analysis and alignment of TCGA breast cancer expression data revealed that breast cancer subtypes express distinct developmental signatures, with basal tumors representing the most “developmentally immature” phenotype. These results highlight phenotypic plasticity of normal mammary stem cells and provide insight into the relationship between hybrid cell populations, stemness, and cancer.

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

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.

Clinical implications of drug-screening assay for recurrent metastatic hormone receptor-positive, human epidermal receptor 2-negative breast cancer using conditionally reprogrammed cells

Various new drugs have been developed for treating recurrent hormone receptor-positive (HR+)/human epidermal receptor 2-negative (HER2−) breast cancer. However, directly identifying effective drugs remains difficult. In this study, we elucidated the clinical relevance of cultured cells derived from patients with recurrent HR+/HER2− metastatic breast cancer. The recently established conditionally reprogrammed (CR) cell system enables us to examine heterogeneity, drug sensitivity and cell function using patient-derived tumour samples. The results of microarray analysis, DNA target sequencing and xenograft experiments indicated that the mutation status and pathological features were preserved in CR cells, whereas RNA expression was different from that in the primary tumour cells, especially with respect to cell adhesion-associated pathways. The results of drug sensitivity assays involving the use of primary breast cancer CR cells were consistent with gene expression profiling test data. We performed drug-screening assays using liver metastases, which were sensitive to 66 drugs. Importantly, the result reflected the actual clinical course of this patient. These results supported the use of CR cells obtained from the metastatic lesions of patients with HR+/HER2− breast cancer for predicting the clinical drug efficacy.

Establishment of ornithine transcarbamylase deficiency-derived primary human hepatocyte with hepatic functions

A long-term hepatocyte culture maintaining liver-specific functions is very essential for both basic research and the development of bioartificial liver devices in clinical application. However, primary hepatocytes rapidly lose their proliferation and hepatic functions over a few days in culture. This work is to establish an ornithine transcarbamylase deficiency (OTCD) patient-derived primary human hepatocyte (OTCD-PHH) culture with hepatic functions for providing an in vitro cell model. Liver tissue from an infant with OTCD was dispersed into single cells. The cells were cultured using conditional reprogramming. To characterize the cells, we assessed activities and mRNA expression of CYP3A4, 1A1, 2C9, as well as albumin and urea secretion. We found that the OTCD-PHH can be subpassaged for more than 15 passages. The cells do not express mRNA of fibroblast-specific maker, whereas they highly express markers of epithelial cells and hepatocytes. In addition, the OTCD-PHH retain native CYP3A4, 1A1, 2C9 activities and albumin secretion function at early passages. The OTCD-PHH at passages 2, 6, 9 and 13 have identical DNA fingerprint as the original tissue. Furthermore, under 3D culture environment, low urea production and hepatocyte marker staining of the OTCD-PHH were detected. The established OTCD-PHH maintain liver-specific functions at early passages and can be long-term cultured in vitro. We believe the established long-term OTCD-PHH culture is highly relevant to study liver diseases, particularly in infants with OTCD.

The response of phyllodes tumor of the breast to anticancer therapy: An in vitro and ex vivo study.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

The Sustained Induction of c-MYC Drives Nab-Paclitaxel Resistance in Primary Pancreatic Ductal Carcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

High-throughput screening identifies candidate drugs for the treatment of recurrent respiratory papillomatosis

Recurrent respiratory papillomatosis (RRP) is a benign neoplasm of the larynx caused mainly by human papillomavirus type 6 or 11 and its standard treatment involves repeated surgical debulking of the laryngeal tumors. However, significant morbidity and occasional mortality due to multiple recurrences occur. Conditional reprogramming (CR) was used to establish a HPV-6 positive culture from an RRP patient, named GUMC-403. High-throughput screening was performed at the National Center for Advanced Technology (NCATS) to identify potential drugs to treat this rare but morbid disease. GUMC-403 cells were screened against the NPC library of >2800 approved drugs and the MIPE library of >1900 investigational drugs to identify new uses for FDA-approved drugs or drugs that have undergone significant research and development. From the two libraries, we identified a total of 13 drugs that induced significant cytotoxicity in RRP cells at IC50 values that were clinically achievable. We validated the efficacy of the drugs in vitro using CR 2D and 3D models and further refined our list of drugs to panobinostat, dinaciclib and forskolin as potential therapies for RRP patients.

Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors.

The response to acute and long-term arginine starvation results in a conditional adaptive metabolic reprogramming that can be harnessed for therapeutic opportunities in ASS1-negative tumors. Here, we investigate the underlying biology of priming ASS1- tumors with arginine deiminase (ADI-PEG20) before treatment with gemcitabine (GEM) and docetaxel (DTX) in sarcoma, pancreatic cancer, and melanoma cell lines. ASS1- tumor cell lines were treated to create LTAT (long-term ADI treated) cell lines (ASS1+) and used for drug combination studies. Protein expression of ASS1, dCK, RRM2, E2F1, c-MYC, and hENT1 was measured. c-MYC activity was determined, live-cell immunofluorescent studies for hENT1, uptake assays of FITC-cytosine probe, and rescue studies with a c-MYC inhibitor were all determined in the presence or absence of the ADI-PEG20:GEM:DTX. In examining modulations within the pyrimidine pathway, we identified that the addition of DTX to cells treated with ADI-PEG20 resulted in translocation of stabilized c-Myc to the nucleus. This resulted in an increase of hENT1 cell-surface expression and rendered the cells susceptible to GEM. In vivo studies demonstrate that the combination of ADI-PEG20:GEM:DTX was optimal for tumor growth inhibition, providing the preclinical mechanism and justification for the ongoing clinical trial of ADI-PEG20, GEM, and DTX in sarcoma. The priming of tumors with ADI-PEG20 and DTX results in the stabilization of c-MYC potentiating the effect of GEM treatment via an increase in hENT1 expression. This finding is applicable to ASS1-deficient cancers that are currently treated with GEM.

Fidelity of a PDX-CR model for bladder cancer

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.

Abstract 1283: Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance

Abstract Background: Acquired resistance to systemic chemotherapy is the main complication in pancreatic ductal adenocarcinoma (PDAC) treatment. Although there are studies focused on gemcitabine resistance mechanisms, our understanding of the mechanisms of nab-paclitaxel (n-PTX) treatment failure remains extremely limited. To enhance the use of properly powered patient-derived platforms, we adopted the conditionally reprogrammed (CR) cell culture technique in order to develop both parental and nab-PTX-resistant cells. The CR approach allowed us to identify the critical role of c-MYC and ERK in the PDAC drug response. Small molecule activators of PP2A (SMAPS) have showed activity in inhibiting lung KRAS-mutant tumor growth. We used SMAPS as new therapeutic agents in PDAC, for its ability to alter c-MYC activity through PP2A dysregulation and enhance PDAC sensitivity to n-PTX. Methods: Long-term cultures of PDAC CRs were established from treatment-naive PDAC patients’ biopsies, and used to generate drug-resistant cells. Zebrafish and mouse model were used to test the cells’ ability to form tumors and to verify the drug resistance in vivo. Molecular analyses were used to characterize the drug-resistant cells and to identify key pathways involved in the drug resistance evolution. Genomic and chemical alterations of the key proteins were used to confirm the involvement in the drug resistance mechanism. We regulated the expression of c-MYC and ERK using SMAPS as a new targeting agent and trametenib to verify the direct correlation between c-MYC and ERK and the drug resistance mechanism. Results: Using the credentialed KRAS-mutant CR cultures, we generated n-PTX-resistant cell lines. The parental and nab-PTX resistant cells were subjected to subcutaneous injections in nude mice, and formed tumors in 2-3 weeks. Histological evaluation showed that the CRs self-assembled into ductal structures, surrounded by a desmoplastic stromal microenvironment that faithfully recapitulates human PDAC. Resistant profiles were verified both in mouse and Zebrafish model. RNA microarrays identified a sustained induction of a pro-inflammatory pathway leading to c-MYC overexpression. c-MYC silencing and overexpression confirmed the role of c-MYCin the evolution of nab-PTX resistance. Treatment of the resistant CRs with either trametenib or with SMAPS resulted in enhanced sensitivity to nab-PTX. We furtherverified that the enhanced sensitivity was commensurate with a reduction in p-Erk and c-Myc. Conclusion: The CR methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of n-PTX resistance is a significant advancement in the field. Our data showed that SMAPs or trametanib overcome a significant component of the n-PTX resistance providing new hope for refractory PDAC. Citation Format: Erika Maria Parasido, George S. Avetian, Jonathan Brody, Jordan Winter, Eric Londin, Michael Pishvaian, Eric Glasgow, Stephen Byers, Goutham Narla, Christopher Albanese. Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1283.

Abstract 3716: Conditional reprogramming technology for Next Generation Living Biobanks (NGLB)

Abstract As one of the next generation biobanks, living biobanks (iPSC, organoids, CRC, PDX, etc.) and related functional analyses are rapidly growing in basic research, clinics, and industry (especially immune-oncology and targeting therapies) as well. Most recently NCI launched PDMR (patient-derived model repository) using PDX, CRC and organoid cultures, NCI/ATCC/Broad/Sanger launched HCMI (Human Cancer Models Initiative) using Organoids and CRC cultures. In this study, we established a conditional reprogramming (CR) protocol which allows to generate cell cultures from cryopreserve fresh tissue specimens from surgery, core biopsies, needle biopsies, and brushed cells.It has been extremely difficult to generate primary cultures from tumor specimens, it has not been possible to expand and indefinitely propagate cells derived from adult tissues while retaining lineage-commitment, normal growth control and differentiation potential. The CR method rapidly expands 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 cell cultures from both normal and tumor tissue (many types) is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 x 106cells in 5 days from a core biopsy of breast tumor. Normal cultures retain a normal karyotype and differentiation potential and cell lines derived from tumors retain their tumorigenic phenotype. We were also able to generate their corresponding fibroblast cultures from the same specimens. These fibroblasts can be immortalized by exogenously expressed hTERT. This approach allows to establish in vitro "epithelial-stroma interactions" to study normal cell differentiation and tumor microenvironment. The ability to produce inexhaustible cell populations from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (termed as NGLB - next generation living biobanks) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool (or a living biomarker) for precision cancer medicine. CR cells can be also an inexhaustible resource to generate CR cells derived iPSC, organoids, and xenografts (CDXs). Citation Format: Xuefeng Liu. Conditional reprogramming technology for Next Generation Living Biobanks (NGLB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3716.

Abstract 3698: Conditionally reprogrammed cells from patient-derived xenograft to model neuroendocrine prostate cancer development

Abstract Treatment-emergent neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of advanced prostate cancer that occurs via NE transdifferentiation of prostate adenocarcinomas in response to androgen receptor (AR)-inhibition therapy. Study of t-NEPC has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we established conditionally reprogrammed (CR) cells from the adenocarcinoma PDX tumor line LTL331. These LTL331-derived CR (LTL331-CR) cells retained the same genomic mutations of the parental tumor and, can be genetically manipulated and continuously propagated in vitro. Further androgen deprivation treatment on LTL331-CR cells showed no effect on cell proliferation. Transcriptomic analyses of the LTL331-CR cells revealed profound downregulation of androgen response pathway, and enrichment of stem/progenitor-like marker genes, compared with the parental tumor LTL331. Notably, when grafted back into the subrenal capsule of male NOD/SCID mice, these LTL331-CR cells gave rise to NEPC tumors directly as manifested by histological expression of NE markers. Transcriptomic analyses of the newly developed NEPC tumors also demonstrated marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel strategy to investigate the mechanisms underlying t-NEPC development with a unique PDX by enabling gene manipulation ex vivo and subsequent functional evaluation in vivo. Citation Format: Xinpei Ci, Jun Hao, Xin Dong, Hui Xue, Rebecca Wu, Anne M. Haegert, Colin C. Collins, Dong Lin, Yuzhuo Wang. Conditionally reprogrammed cells from patient-derived xenograft to model neuroendocrine prostate cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3698.

Personalized Drug Sensitivity Screening for Bladder Cancer Using Conditionally Reprogrammed Patient-derived Cells

Many patients with muscle-invasive bladder cancer (BC) are either ineligible for or do not benefit from cisplatin-based chemotherapy, and there is an unmet need to estimate individuals’ drug sensitivities. We investigated the suitability of conditionally reprogrammed (CR) cells for the characterization of BC properties and their feasibility for personalized drug sensitivity screening. The CR cultures were established from six BC tumors with varying histology and stage. Four cultures were successfully propagated for genomic, transcriptomic, and protein expression profiling and compared to the parental tumors. Two out of four CR cultures (urothelial carcinoma and small cell neuroendocrine carcinoma [SmCC]) corresponded well to their parental tumors and underwent drug sensitivity screening to identify novel drugs for the respective tumors. Both cultures were sensitive to standard BC chemotherapy agents (eg cisplatin and gemcitabine) and to conventional drugs such as taxanes and inhibitors of topoisomerase and proteasome. The SmCC cells were also sensitive to statins (eg, atorvastatin and pitavastatin). In summary, after confirming their representativeness and origin, we conclude that CR cells are a feasible platform for personalized drug sensitivity testing and might thus add to the approaches used to personalize BC treatment strategies. Patient summaryWe investigated the conditional reprogramming method for generating patient-derived bladder cancer cell cultures and studied their feasibility for planning personalized treatment strategies.

Long-term culture and characterization of patient-derived primary hepatocytes using conditional reprogramming.

Cultivation of primary human hepatocytes (PHHs) often faces obstacles including failure of long-term in vitro culture, weak proliferation ability, rapid loss of liver-specific function and morphology, and tendency of fibrosis. Previous research focused on immortalization methods, such as telomerase and viral, to culture immortalized primary human hepatocytes, which may lose some of the normal properties. However, non-immortalized PHHs often fail to maintain long-term viability and functionality. These highlight the urgent need for developing new culture strategy for PHHs. In the present study, we isolated PHHs from fresh human liver tissues representing different liver diseases and age groups. We used conditional reprogramming, without permanent immortalization, for long-term in vitro primary human hepatocytes cultivation and characterization. For functional characterization, we assessed CYP3A4, 1A1 and 2C9 activities and measured the mRNA expression of albumin, s100a4, krt8, krt18, cyp1a1, cyp3a4, cyp2b6, cyp2c8, cyp2c9, and cyp2d6. Additionally, we compared the DNA fingerprint of the cells against their original liver tissues using short tandem repeat (STR) analysis. We found that PHHs-derived from young patients can survive for more than three months, while the lifespan of primary human hepatocytes derived from adult patients ranges from two to three months, which is longer than most commercial primary hepatocytes. Importantly, the cells at early passages retain strong CYP3A4, 1A1 and 2C9 activities and the DNA fingerprints are identical with their original tissues. Through conditional programming, we achieved, for the first time, a high level of success rate in the long-term in vitro cultivation of primary human hepatocytes-derived patients representing diverse liver disease. Moreover, the conditional programming cell culture technology reported in this paper requires neither co-culture with additive cells, nor complex and expensive components, such as collagen sandwich or spheroid culture. We thus believe that the patient-derived PHHs cultivation using conditional programming may provide a viable and valuable cell model to study liver disease-related mechanisms. Impact statement Commercially available primary human hepatocytes rapidly lose their proliferative ability and liver-specific functions over a few cultivation days. The demand for pharmaceutical toxicity screening and liver disease research requires the development of long-term primary hepatocyte culture methods. This manuscript addresses this challenge by introducing for the first time successful long-term in vitro cultivation of primary human hepatocytes from a range of liver transplantation patients using conditional reprogramming technique. The beauty of this technique is that it is not a permanent immortalization and does not require co-culture with additive cells. The primary human hepatocytes retain proliferative capacity, genetic stability, and hepatocyte-specific functions at early passages. In view of these, we believe that scientists and researchers will benefit from using these highly valuable cell models to study diverse liver diseases.

Proliferation of human hepatocellular carcinoma cells from surgically resected specimens under conditionally reprogrammed culture.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality worldwide, which is partially due to the lack of appropriate therapeutic options. The development of HCC is accompanied with unique and continuous genomic and epigenetic modifications. Therefore, the absence of a personalized and reproducible human model reduces the ability to determine the potential of candidate treatments. Conditional reprogramming (CR) culture has been used to establish and indefinitely grow patient-derived tumor cell lines in a rapid and efficient manner. In the present study, primary HCC cells were isolated from tumor specimens and cultured under CR conditions. The proliferative potential and capacity of cells to undergo continuous regeneration were evaluated by cell viability and proliferation assays, and the expression of tumor-specific markers was determined by western blotting and immunofluorescence to determine the prospects for use in clinical settings. It was demonstrated that ~55% of tumor samples were able to generate HCC cells that could be continuously expanded and passaged under CR conditions; this ability was associated with the source and composition of the tumor tissues. Furthermore, the expression of the tumor-specific marker α-fetoprotein and the proliferative ability of cells were maintained following cycles of cryopreservation and resuscitation. In conclusion, with further optimization, the CR system may be a useful tool for the precise therapeutic treatment of patients with HCC.

African-American Prostate Normal and Cancer Cells for Health Disparities Research.

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.

Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer deaths among American men. Unfortunately, there is no cure once the tumor is established within the bone niche. Although osteocytes are master regulators of bone homeostasis and remodeling, their role in supporting PCa metastases remains poorly defined. This is largely due to a lack of suitable ex vivo models capable of recapitulating the physiological behavior of primary osteocytes. To address this need, we integrated an engineered bone tissue model formed by 3D-networked primary human osteocytes, with conditionally reprogrammed (CR) primary human PCa cells. CR PCa cells induced a significant increase in the expression of fibroblast growth factor 23 (FGF23) by osteocytes. The expression of the Wnt inhibitors sclerostin and dickkopf-1 (Dkk-1), exhibited contrasting trends, where sclerostin decreased while Dkk-1 increased. Furthermore, alkaline phosphatase (ALP) was induced with a concomitant increase in mineralization, consistent with the predominantly osteoblastic PCa-bone metastasis niche seen in patients. Lastly, we confirmed that traditional 2D culture failed to reproduce these key responses, making the use of our ex vivo engineered human 3D bone tissue an ideal platform for modeling PCa-bone interactions.