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

Personalized therapy for non‑small cell lung cancer (NSCLC), particularly lung adenocarcinoma, has recently been significantly improved by the discovery of various molecular targets. However, this has not been the case for lung squamous cell carcinoma (SCC). In the present study, we identified the family with sequence similarity 83, member B (FAM83B) as a candidate marker for SCC through a comprehensive gene expression analysis and examined its correlations with various clinicopathological factors. The subjects of this study consisted of 215 patients with NSCLC who underwent complete resection from 2005 to 2011 at the Fukushima Medical University Hospital (Fukushima, Japan). They included 102 patients with adenocarcinoma and 113 with SCC. FAM83B expression was first examined in some of the samples by gene expression analysis and western blotting, and then all clinical specimens were evaluated by immunohistochemistry (IHC). The relationship between the quantitative values for IHC and clinicopathological factors was statistically analyzed. The results showed that FAM83B mRNA expression was significantly higher in SCC than in normal lung or adenocarcinoma (P<0.0001). Immunoblot analysis also confirmed this trend. Specimens containing >10% positive area for FAM83B were judged as 'positive'; 94.3% (107/113) of SCC and 14.7% (15/102) of adenocarcinoma were positive. Patients were divided into two subgroups according to expression (54 high‑expression and 53 low‑expression patients); the high‑expression group was associated with a better disease‑free survival (DFS) rate (P=0.042, log‑rank test). In conclusion, FAM83B may be a reliable diagnostic and prognostic biomarker for SCC. Detailed analyses of FAM83B function in lung cancer are required to understand how its expression is associated with better prognosis in SCC.

FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma.

Basaloid squamous cell carcinoma of the esophagus (BSCE) is a rare variant of squamous cell carcinoma that is difficult to distinguish from other carcinomas by preoperative endoscopic biopsy because of its histological varieties. Accurate diagnosis is essential for adequate treatment, and the methods proposed so far (e.g., immunohistochemical staining) have limitations. In this study, we tried to identify the characteristic bundles of gene expression in BSCE using comprehensive gene expression analysis (CGEA). Subsequently, we constructed a gene expression scoring system for the proper diagnosis of BSCE. Fifty-seven surgical specimens, including seven BSCEs, obtained from 30 patients who underwent esophagectomy were used for constructing the scoring system. Three hundred and twelve biopsy specimens, including eight BSCEs, obtained from 80 patients and 20 commercially available formalin-fixed paraffin-embedded (FFPE) specimens diagnosed as esophageal cancer, including 13 BSCEs, were used for validation. After our original mathematical extraction algorithm, 75 genes were extracted to distinguish BSCE from non-BSCE. The cumulative converted values (gene expression score) of the respective 75 genes from each specimen were obtained and lined up in ascending order to assess the optimal gene expression cut-off score for a definitive diagnosis of BSCE. The validation of this scoring system showed high prediction of the biopsy specimens [area under the curve (AUC)=0.981; 95% confidence interval (CI): 0.952‑1.000] and the commercially available FFPE specimens (AUC=0.901; 95% CI: 0.750-1.000). In conclusion, using CGEA in a gene expression scoring system helps in differentiating BSCE from non-BSCE with high accuracy and may contribute in improving BSCE treatment.

A novel gene expression scoring system for accurate diagnosis of basaloid squamous cell carcinoma of the esophagus.

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor () gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required.  () is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for  expression using cDNA microarray analysis. The associations between  expression and clinicopathological parameters, including patient survival, were examined.  was highly expressed in tumors from males, smokers and in tumors with wild-type . Multivariate analyses further confirmed that wild-type  tumors were significantly positively associated with  expression. In survival analysis,  expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type . Furthermore,  knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by  RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines.  may be involved in lung ADC tumor proliferation and can be a predictor of poor survival.  is also a potential novel therapeutic target for ADC with wild-type .

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

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.

Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required.

Establishment and Characterization of a Novel Human Clear-cell Sarcoma of Soft-tissue Cell Line, RSAR001, Derived from Pleural Effusion of a Patient with Pleural Dissemination.

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.

In patients with branch-duct intraductal papillary mucinous neoplasms (BD-IPMN), we aimed to develop a novel blood-based biomarker utilizing a gene-expression profile for the detection of pancreatic malignancies, such as IPMN-derived carcinoma (IPMC) or pancreatic ductal adenocarcinoma (PDAC).

A Blood-based Gene-expression Scoring System for Cancer Screening in Patients With Branch-duct Intraductal Papillary Mucinous Neoplasms.

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.

Article	Year
FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma. International journal of oncology\| PubMed ID: 25586059	2015
A novel gene expression scoring system for accurate diagnosis of basaloid squamous cell carcinoma of the esophagus. International journal of oncology\| PubMed ID: 28731134	2017
Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor... Oncology letters\| PubMed ID: 29434849	2018
Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues. Oncology reports\| PubMed ID: 29917168	2018
Establishment and Characterization of a Novel Human Clear-cell Sarcoma of Soft-tissue Cell Line, RSAR001, Derived from Pleural Effusion of a Patient with Pleural Dissemination. Anticancer research\| PubMed ID: 30194147	2018
An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids. Cells\| PubMed ID: 31137590	2019
A Blood-based Gene-expression Scoring System for Cancer Screening in Patients With Branch-duct Intraductal Papillary Mucinous Neoplasms. Anticancer research\| PubMed ID: 33109597	2020
Construction of patient-derived tumor models to evaluate anticancer agents and cancer immunotherapy. Oncology letters\| PubMed ID: 33841567	2021

Hirosumi Tamura

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