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In this study, we sought to investigate the protocols, expansion capacity, cryopreservation ability, genetic stability, and feasibility of larger-scale bioproduction of a subset of the models generated by the Human Cancer Models Initiative (HCMI), an international collaborative effort. We cultured organoid models derived from human colon, pancreas, esophagus, and mammary tissues developed by laboratories contributing to the HCMI. While organoid culture represents a significant divergence from typical two-dimensional monolayer culture of continuous cell lines, our results show that these next-generation in vitro models are suitable for larger-scale bioproduction. This is vital to ensure the widespread availability of these models within the research community to facilitate applications like pre-clinical drug discovery and basic cancer research.

In this study, we used CRISPR/Cas9 genome-editing technology to generate a drug resistant MEK1Q56P mutation within the A375 melanoma cell line which naturally harbors the BRAFV600E mutation. We validated this new isogenic cell model using both molecular and biofunctional approaches. Drug responses to BRAF- and MEK1-specific inhibitors and non-specific chemotherapy drugs were compared between the A375 MEK1Q56P cell line and the parental cell line in 2D and 3D culture environments. Results demonstrated that the isogenic MEK1Q56P cell line showed significant and specific resistance to BRAF inhibitors in comparison to the A375 line. This new approach to cell line development provides direct, in vitro, bio-functional evidence of a drug-resistant gene that drives tumor cell survival under targeted anti-cancer treatments. Furthermore, the A375 MEK1Q56P cell line represents a new type of drug resistance model that contains a defined genetic resistance mechanism.

In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblast (NAFs), and normal prostate epithelial (PrE) cells; all three lines were immortalized by human telomerase reverse transcriptase (hTERT) alone and then were cryopreserved, thawed, and continuously passaged without any indication of a decrease in growth rate. Cell proliferation was measured under the influence of CAFs and NAFs for various prostate-derived epithelial cells. The effects of stromal cells on prostate cell proliferation was cell line-dependent. CAF cells promoted the growth of PrE, DU145, and A549, while NAF cells inhibited their growth. Meanwhile, CAF cells inhibited the growth of the viral transduced RWPE1 cell line, while NAF cells promoted the growth of the prostate epithelial cell LNCap. This study demonstrates that these hTERT-immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.

In this study, we use the CRISPR/Cas9 system to generate isogenic drug-resistant 2D or 3D cancer models, or used in studies designed to further our understanding of the mechanisms of acquired drug resistance. Two different models were generated from the BRAF inhibitor sensitive A375 melanoma cell line. We introduced either the NRAS Q61K or the KRAS G13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF resistant tumor samples. We then assessed the susceptibility of these new isogenic cell lines to traditional BRAF inhibitors in 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of programmed death-ligand 1 (PD-L1).

Mutations in Isocitrate dehydrogenase (IDH) have been linked to human cancers such as glioma and acute myeloid leukemia (AML). We sought to use CRISPR/Cas9 gene-editing technology to create two in vitro disease models that harbor either the IDH1 or IDH2 mutations. An IDH1R132H mutation was introduced in the U-87 MG cell line, and an IDH2R140Q mutation was introduced in the TF-1 cell line. The IDH1R132H mutant U-87 MG Isogenic Cell Line showed an increase in D-2HG and elevated level of histone methylation. In the IDH2R140Q mutant TF-1 Isogenic Cell Line, an increase in D-2HG was also observed. In response to IDH2-specific inhibitors, we demonstrated that IDH2R140Q TF-1 cells exhibited decreases in both D-2HG and histone methylation levels. These data demonstrate that isogenic in vitro models are valuable tools for elucidating mechanisms involved in tumorigenesis and use in screening anti-cancer compounds for drug discovery.

In lung cancer, vimentin (VIM) is associated with epithelial to mesenchymal transition (EMT) and the metastatic spread of cancer. VIM expression is generally upregulated when epithelial cells transition to the mesenchymal phenotype. We used CRISPR/Cas9 gene editing to generate a VIM-red fluorescent protein (RFP) fusion reporter cell line in the A549 cell line, enabling end-point or real-time tracking of the EMT status as the cells transition from epithelial to mesenchymal phenotype under defined conditions. Bio-functional evaluation of the A549 VIM RFP cell line shows sensitivity to metastatic NSCLC drugs. These results provide the foundation for the use of this cell line in high-throughput screening applications, including the identification of new anti-EMT drugs for metastatic NSCLC.

In vitro angiogenesis models, such as the analysis of tubule formation, provide useful tools to study normal and disease state processes. We established an in vitro co-culture model system consisting of an assay-ready mixture of TeloHAEC-GFP and an hTERT-immortalized, adipose-derived mesenchymal stem cell line in a specially formulated medium containing VEGF supplement (Angio-Ready™ Angiogenesis Assay System). Here we report further optimization of the system into a 1,536-well, high-throughput format and a shortening of the assay time frame to. Using this format, we evaluated 2,816 drugs from NCATS Pharmaceutical Collection, and 35 potent inhibitors were identified. Many known and novel angiogenesis inhibitors were identified from this study. These results demonstrate that the co-culture model described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

Human induced pluripotent stem cells (iPSC)-derived neural progenitor cells (NPCs) and neurons are an attractive in vitro model to study neurological development and neurotoxicity and to model diseases. We investigated the expression of genes associated with the differentiation of NPCs during three weeks in dopaminergic differentiation media. To validate that our NPCs and dopaminergic neuron differentiation media are suitable for drug screening, we conducted neurotoxicity screenings in three types of NPCs and NPCs-derived neurons using Reliablue™ cell viability reagent assay and high content imaging analysis. This study demonstrates that ATCC NPCs and dopaminergic differentiation media are suitable for studying neurological development and neurotoxicity screening.

Kidney transporter cell models using well-characterized hTERT-immortalized primary renal proximal tubule epithelial cells (RPTEC) that stably overexpress either the OAT1, OCT2, or OAT3 gene were generated. We verified that the overexpressed transporters have normal transport activities using 6-CF and EAM-1 uptake assays in a high throughput format. Uptake of these compounds is blocked in a dose dependent manner by well-known SLC inhibitors, indicating that the overexpressed kidney transporters functioned as expected. These data demonstrate that modified RPTEC maintain kidney transporter expression over time, and provide physiologically relevant, highly sensitive models of human kidney transporter functions.

ATCC Corporate Workshop

Association of Molecular Pathology (AMP) Annual Meeting

11/16/2017 — 11/18/2017

The complexities involved in 16S rRNA and shotgun metagenomic analysis methods pose significant challenges for microbiome research and frequently result in the introduction of biases. One of the primary obstacles in assay standardization is the limited availability of reference materials and robust analytical tools. To support this need, ATCC has developed mock microbial communities from fully sequenced and characterized ATCC strains, selected based on their phenotypic and genotypic attributes or relevance in disease-specific research. These mock communities mimic mixed metagenomics samples and offer a universal control for microbiome analyses and assay development.