high-throughput-in-vitro-assay-using-patient-derived-tumor-organoids

High-Throughput <em>In Vitro</em> Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Fukushima Medical University

Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel cell-based assay for evaluating the efficacy of anti-EGFR drugs against EGFR mutation.

Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation.

In recent years, human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been widely used to develop evaluation systems for drug cardiotoxicity, including the arrhythmia caused by QT prolongation. To accurately assess the arrhythmogenic potential of drugs, associated with QT prolongation, we developed an evaluation system using hiPS-CMs and gene expression analysis. hiPS-CMs were treated with 8 arrhythmogenic and 17 non-arrhythmogenic drugs at several concentrations for 24 hr to comprehensively analyze gene expression. The results showed that 19 genes were upregulated in the arrhythmogenic drug-treated cells compared with their expression levels in the non-treated and non-arrhythmogenic drug-treated cells. The arrhythmogenic risks of the drugs were evaluated by scoring gene expression levels. The results indicated that arrhythmogenic risks could be inferred when cells were treated at a concentration 100 times higher than the maximum blood concentration of the drug. Thus, we succeeded in developing a system for evaluation of the arrhythmogenic potential of drugs using gene expression analysis.

Evaluation system for arrhythmogenic potential of drugs using human-induced pluripotent stem cell-derived cardiomyocytes and gene expression analysis.

Patient-derived tumor xenograft models represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture; however, their use is low-throughput and costly. To overcome this limitation, patient-derived tumor organoids (PDOs) were established from human lung, ovarian and uterine tumor tissues, among others, to accurately and efficiently recapitulate the tissue architecture and function. PDOs were able to be cultured for >6 months, and formed cell clusters with similar morphologies to their source tumors. Comparative histological and comprehensive gene expression analyses proved that the characteristics of PDOs were similar to those of their source tumors, even following long-term expansion in culture. At present, 53 PDOs have been established by the Fukushima Translational Research Project, and were designated as Fukushima PDOs (F‑PDOs). In addition, the in vivo tumorigenesis of certain F‑PDOs was confirmed using a xenograft model. The present study represents a detailed analysis of three F‑PDOs (termed REME9, 11 and 16) established from endometrial cancer tissues. These were used for cell growth inhibition experiments using anticancer agents. A suitable high-throughput assay system, with 96- or 384‑well plates, was designed for each F‑PDO, and the efficacy of the anticancer agents was subsequently evaluated. REME9 and 11 exhibited distinct responses and increased resistance to the drugs, as compared with conventional cancer cell lines (AN3 CA and RL95-2). REME9 and 11, which were established from tumors that originated in patients who did not respond to paclitaxel and carboplatin (the standard chemotherapy for endometrial cancer), exhibited high resistance (half-maximal inhibitory concentration >10 µM) to the two agents. Therefore, assay systems using F‑PDOs may be utilized to evaluate anticancer agents using conditions that better reflect clinical conditions, compared with conventional methods using cancer cell lines, and to discover markers that identify the pharmacological effects of anticancer agents.

Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues.

Patient-derived tumor organoids (PDOs) represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture models. We have established PDOs from various human tumors to accurately and efficiently recapitulate the tissue architecture and function. Molecular targeted therapies with remarkable efficacy are currently in use against various tumors. Thus, there is a need for in vitro functional-potency assays that can be used to test the efficacy of molecular targeted drugs and model complex interactions between immune cells and tumor cells to evaluate the potential for cancer immunotherapy. This study represents an in vitro evaluation of different classes of molecular targeted drugs, including small-molecule inhibitors, monoclonal antibodies, and an antibody-drug conjugate, using lung PDOs. We evaluated epidermal growth factor receptor and human epidermal growth factor receptor 2 (HER2) inhibitors using a suitable high-throughput assay system. Next, the antibody-dependent cellular cytotoxicity (ADCC) activity of an anti-HER2 monoclonal antibody was evaluated to visualize the interactions of immune cells with PDOs during ADCC responses. Moreover, an evaluation system was developed for the immune checkpoint inhibitors, nivolumab and pembrolizumab, using PDOs. Our results demonstrate that the in vitro assay systems using PDOs were suitable for evaluating molecular targeted drugs under conditions that better reflect pathological conditions.

An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids.

An  assay system using patient-derived tumor models represents a promising preclinical cancer model that replicates the disease better than traditional cell culture models. Patient-derived tumor organoid (PDO) and patient-derived tumor xenograft (PDX) models have been previously established from different types of human tumors to recapitulate accurately and efficiently their tissue architecture and function. However, these models have low throughput and are challenging to construct. Thus, the present study aimed to establish a simple  high-throughput assay system using PDO and PDX models. Furthermore, the current study aimed to evaluate different classes of anticancer drugs, including chemotherapeutic, molecular targeted and antibody drugs, using PDO and PDX models. First, an  high-throughput assay system was constructed using PDO and PDX established from solid and hematopoietic tumors cultured in 384-well plates to evaluate anticancer agents. In addition, an  evaluation system of the immune response was developed using PDO and PDX. Novel cancer immunotherapeutic agents with marked efficacy have been used against various types of tumor. Thus, there is an urgent need for  functional potency assays that can simulate the complex interaction of immune cells with tumor cells and can rapidly test the efficacy of different immunotherapies or antibody drugs. An evaluation system for the antibody-dependent cellular cytotoxic activity of anti-epidermal growth factor receptor antibody and the cytotoxic activity of activated lymphocytes, such as cytotoxic T lymphocytes and natural killer cells, was constructed. Moreover, immune response assay systems with bispecific T-cell engagers were developed using effector cells. The present results demonstrated that  assay systems using PDO and PDX may be suitable for evaluating anticancer agents and immunotherapy potency with high reproducibility and simplicity.

Motoki Takagi

Medical-Industrial Translational Research Center

Fukushima Medical University

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids