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Organoid Cultures As Preclinical Models of Non–Small Cell Lung

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Organoid Cultures As Preclinical Models of Non–Small Cell Lung Published OnlineFirst November 6, 2019; DOI: 10.1158/1078-0432.CCR-19-1376 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Organoid Cultures as Preclinical Models of Non–Small Cell Lung Cancer Ruoshi Shi1,2, Nikolina Radulovich1, Christine Ng1, Ni Liu1, Hirotsugu Notsuda1, Michael Cabanero1, Sebastiao N. Martins-Filho1, Vibha Raghavan1, Quan Li1, Arvind Singh Mer1, Joshua C. Rosen1,3, Ming Li1, Yu-Hui Wang1, Laura Tamblyn1, Nhu-An Pham1, Benjamin Haibe-Kains1,2,4,5,6, Geoffrey Liu1,2,7, Nadeem Moghal1,2, and Ming-Sound Tsao1,2,3 ABSTRACT ◥ Purpose: Non–small cell lung cancer (NSCLC) is the most Results: We have identified cell culture conditions favoring the common cause of cancer-related deaths worldwide. There is an establishment of short-term and long-term expansion of NSCLC unmet need to develop novel clinically relevant models of NSCLC to organoids derived from primary lung patient and PDX tumor tissue. accelerate identification of drug targets and our understanding of The NSCLC organoids recapitulated the histology of the patient and the disease. PDX tumor. They also retained tumorigenicity, as evidenced by Experimental Design: Thirty surgically resected NSCLC cytologic features of malignancy, xenograft formation, preservation primary patient tissue and 35 previously established of mutations, copy number aberrations, and gene expression pro- patient-derived xenograft (PDX) models were processed files between the organoid and matched parental tumor tissue by for organoid culture establishment. Organoids were histo- whole-exome and RNA sequencing. NSCLC organoid models also logically and molecularly characterized by cytology and preserved the sensitivity of the matched parental tumor to targeted histology, exome sequencing, and RNA-sequencing analysis. therapeutics, and could be used to validate or discover biomarker– Tumorigenicity was assessed through subcutaneous injection drug combinations. of organoids in NOD/SCID mice. Organoids were subjected Conclusions: Our panel of NSCLC organoids closely recapitu- to drug testing using EGFR, FGFR, and MEK-targeted lates the genomics and biology of patient tumors, and is a potential therapies. platform for drug testing and biomarker validation. Introduction tumors or drug sensitivity to targeted therapeutics of their patient tumors (5). In addition, although GEMMs and clinically relevant Non–small cell lung cancer (NSCLC) is the leading cause of cancer- PDXs may be closer to the ideal models to study drug response in related death worldwide with a 5-year overall survival rate of 15% (1). patients, studies using these models are labor intensive, costly, and Over the last decades, there has been tremendous effort in developing time consuming (6). Thus, research efforts are underway to develop preclinical models of NSCLC, including two-dimensional (2D) cell novel preclinical models derived from patient with NSCLC and PDX lines, air–liquid interface cultures, genetically engineered mouse mod- tissue that are economical, rapid to use, and accurately reflect the els (GEMM), and patient-derived xenografts (PDX; refs. 2–4). These biology of the disease. models have been used to accelerate our understanding of NSCLC Over the past few years, organoid cultures derived from primary biology and pathogenesis. Although cell lines are still widely used in patient tumors and PDXs of various cancers including the colon, preclinical studies, they often do not reflect the biology of their parental pancreas, prostate, liver, and breast have been described (7–16). These cancer organoids have been utilized for numerous applications, such as fi – 1University Health Network, Ontario Cancer Institute/Princess Margaret Cancer drug screening and biomarker identi cation (17 20). They have been Centre, Toronto, Ontario, Canada. 2Department of Medical Biophysics, Univer- proposed to be better in vitro models than 2D cell lines due to higher sity of Toronto, Toronto, Ontario, Canada. 3Department of Laboratory Medicine rates of preservation of key histologic and molecular traits of their 4 and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Depart- parental tumors (14, 15). In addition, drug screening in patient- ment of Computer Science, University of Toronto, Toronto, Ontario, Canada. derived organoids (POD) has shown high concordance with that of 5Ontario Institute for Cancer Research, Toronto, Ontario, Canada. 6Vector Institute for Artificial Intelligence, Toronto, Ontario, Canada. 7Division of Medical the matched patient tumor (14, 18). Some reports have demonstrated Oncology and Hematology, Princess Margaret Cancer Centre, University of the ability to generate normal lung organoids composed of airway cell Toronto, Toronto, Ontario, Canada. lineages (21, 22). These models were primarily generated from normal Note: Supplementary data for this article are available at Clinical Cancer mouse and human airways to understand normal lung development Research Online (http://clincancerres.aacrjournals.org/). and function. In addition, methods to generate lung organoids from pluripotent stem cells have been reported to aid in the study of genetic R. Shi and N. Radulovich contributed equally to this article. pulmonary diseases such as cystic fibrosis (21, 23). A major advance Corresponding Author: Ming-Sound Tsao, University Health Network, 101 was outlined in recent reports describing protocols for the develop- College Street 11-314, Toronto, Ontario M5G 1L7, Canada. Phone: 416-634- – 8721; E-mail: [email protected] ment of NSCLC organoids (24 26). However, although many of the models reported in these studies were cultured short-term and were – Clin Cancer Res 2020;XX:XX XX useful for acute studies, lack of systematic documentation of doi: 10.1158/1078-0432.CCR-19-1376 organoid tumor cell purity was a significant issue and specific details Ó2019 American Association for Cancer Research. regarding long-term growth of the models were not provided (25, 26). AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 6, 2019; DOI: 10.1158/1078-0432.CCR-19-1376 Shi et al. resuspended in 100% growth factor–reduced Matrigel (VWR), plated Translational Relevance in 24-well tissue culture plates as Matrigel domes and maintained in Currently, there is an urgent need for clinically relevant pre- 37 C5%CO2 with media overlaying the Matrigel dome. Refer to clinical models of non–small cell lung cancer (NSCLC) for bio- Supplementary Materials and Methods for a list of media components. marker and drug discovery due to the lack of preclinical models Organoid growth was monitored weekly for the detection of initiated that recapitulate the biology of the patient tumor. Three- organoids, and organoids were kept in the same passage for no longer dimensional (3D) organoids have become valuable preclinical than four weeks. The identity of PDX and organoids were authenti- models to study disease pathogenesis and identify novel drug cated by short tandem repeat (STR) analysis and matched to patient targets. We have established a protocol for the development tissue. Organoid cultures were tested routinely for Mycoplasma. of NSCLC organoids from patient tumor and patient-derived Additional methods can be found in Supplementary Materials and xenograft models. This protocol allowed for the efficient generation Methods. of organoids for multiple potential applications. Importantly, we showed that these organoids retained the histologic and molecular IHC features of their parental tumors and demonstrated their utility for Fresh tumor tissue was fixed in 10% formalin for 24 to 48 hours, drug testing. Our organoid platform provides additional preclinical followed by fixation in 70% ethanol prior to paraffin embedding. models of NSCLC and may be useful for future drug screening Organoids were fixed with 10% formalin for 24 to 48 hours and 70% biomarker identification. ethanol with eosin and embedded in Histolgel (Thermo Fisher Sci- entific) before processing for H&E and IHC. Formalin-fixed paraffin- embedded tumor tissues and organoids were cut into 4-mm-thick slices and allowed to dry overnight at 60C. Prepared tissue sections were Furthermore, there still remains a great need to develop a NSCLC stained with appropriate antibodies using BenchMark XT autostainer organoid platform suitable for drug screening and biomarker identi- (Ventana Medical Systems). Primary antibody specific to CK5/6 fication in lung cancer. (Ventana), TP63, TTF-1, and CK7 (Dako) were used for IHC analysis. Here, we describe a culturing protocol that enables generation of The slides were scanned and imaged using Aperio Scanscope XT short-term (1–3 months, 1–9 passages) and long-term (>3 months, (Leica). >10 passages) NSCLC organoids from most and a subset of primary lung patient tumors and PDXs, respectively. These models were able to DNA extraction and WES analysis initiate from tumor tissues with 88% (57/65) success rate. Specifically, Snap-frozen tumor tissues and fresh organoid pellets were lysed in 72% (47/65) of the organoids were maintained in culture short-term, tris-buffered saline solution with 10% SDS and proteinase K (1 mg/mL) whereas 15% (10/65) were maintained in culture long-term. We overnight at 55C. DNA was isolated and eluted on spin columns using demonstrated that both short-term and long-term established NSCLC proprietary solutions provided by a DNA Extraction Kit (Norgen organoids grown in vitro and as xenografts recapitulated
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