Use of Ex Vivo Patient-Derived Tumor Organotypic Spheroids to Identify
Total Page：16
File Type：pdf, Size：1020Kb
Published OnlineFirst February 7, 2020; DOI: 10.1158/1078-0432.CCR-19-1844 CLINICAL CANCER RESEARCH | TRANSLATIONALCANCER MECHANISMS AND THERAPY Use of Ex Vivo Patient-Derived Tumor Organotypic Spheroids to Identify Combination Therapies for HER2 Mutant Non–Small Cell Lung Cancer Elena Ivanova1,2, Mari Kuraguchi1,2, Man Xu1,2, Andrew J. Portell1,2, Luke Taus2, Irmina Diala3, Alshad S. Lalani3, Jihyun Choi1, Emily S. Chambers1, Shuai Li1, Shengwu Liu1, Ting Chen1, Thanh U. Barbie4, Geoffrey R. Oxnard1,5, Jacob J. Haworth1, Kwok-Kin Wong6, Suzanne E. Dahlberg7, Amir A. Aref1, David A. Barbie1,2,5, Magda Bahcall1,2, Cloud P. Paweletz1,2, and Pasi A. Janne€ 1,2,5 ABSTRACT ◥ Purpose: Evaluating drug responses using primary patient- validated in vivo using the DFCI359 and DFCI315 PDXs and a derived cells ex vivo represents a potentially rapid and efficient HER2YVMA genetically engineered mouse model. approach to screening for new treatment approaches. Here, Results: Both neratinib and afatinib, but not gefitinib, induced we sought to identify neratinib combinations in HER2 mutant cell death in DFCI359 XDOTS. The combinations of neratinib/ non–small cell lung cancer (NSCLC) patient xenograft-derived trastuzumab and neratinib/temsirolimus enhanced the therapeu- organotypic spheroids (XDOTS) using a short-term ex vivo tic benefit of neratinib alone in DFCI315 and DFCI359. The system. combination of neratinib and trastuzumab in vivo was more Experimental Design: We generated two HER2-mutant effective compared with single-agent neratinib or trastuzumab NSCLC PDX models [DFCI359 (HER2 exon19 755_757LREde- and was associated with more robust inhibition of HER2 and linsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)] and downstream signaling. used the PDX tumors to generate XDOTS. Tumor spheroids were Conclusions: TheXDOTSplatformcanbeusedtoevaluate growninamicrofluidicdeviceandtreatedex vivo with neratinib- therapies and therapeutic combinations ex vivo using PDX based drug combinations. Live/dead quantification was per- tumors. This approach may accelerate the identification and formed by dual-labeling deconvolution fluorescence microscopy. clinical development of therapies for targets with no or few The most efficacious ex vivo combination was subsequently existing models and/or therapies. Introduction correlation with responses to tyrosine kinase inhibitors (TKI; refs. 2–6). Nevertheless, similar success is yet to be seen in a number of other Tumor genotyping has expanded the repertoire of biomarkers in NSCLC genotypes, including in-frame exon 20 insertions in HER2. non-small cell lung cancers (NSCLC) and as such has become the The irreversible dual EGFR/HER2 inhibitors afatinib (NCT00796549), primary diagnostic tool in predicting responses to targeted thera- dacomitinib (NCT00548093), and neratinib (NCT01827267) showed pies (1). Notably, expression of mutant EGFR, or oncogenic fusions little clinical benefit as monotherapies in HER2-mutant lung cancer, all such as those of anaplastic lymphoma kinase (ALK), ROS1, RET,or with response rates of <10% (7–9). Although the combination of NTRK1 receptor tyrosine kinases (RTK) has demonstrated good afatinib and rapamycin demonstrated promising combination efficacy in a preclinical in vivo study using a genetically engineered mouse model (GEMM) of HER2-mutant NSCLC, the subsequent clinical trial 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, (NCT01822767) evaluating the combination of neratinib and temsir- Massachusetts. 2Belfer Center for Applied Cancer Science, Dana Farber Cancer Institute, Boston, Massachusetts. 3Puma Biotechnology Inc., Los Angeles, olimus resulted in only a minimal improvement in response rate (14% California. 4Department of Surgery, Brigham and Women's Hospital, Boston, vs. 0%) compared with single-agent neratinib (10, 11). More recently, Massachusetts. 5Lowe Center for Thoracic Oncology, Dana Farber Cancer the trastuzumab-based antibody drug conjugate (ADC), ado- Institute, Boston, Massachusetts. 6Department of Medicine, Division of Hema- trastuzumab (T-DM1), has demonstrated some encouraging activity tology and Medical Oncology, New York University Langone Medical Center, in HER2 mutant NSCLC and next-generation ADCs (DS-8201a; New York, New York. 7Department of Biostatistics and Computational Biology, NCT03505710) are undergoing clinical evaluation (12, 13). On the Dana-Farber Cancer Institute, Boston, Massachusetts. basis of the current landscape of available HER2-directed therapies, Note: Supplementary data for this article are available at Clinical Cancer and the lack of approved therapies for HER2 mutant NSCLC, addi- Research Online (http://clincancerres.aacrjournals.org/). tional studies are needed to identify therapeutic strategies for this Prior presentation: Part of the work was previously presented at the 2017 World subset of patients with NSCLC. Congress of Lung Cancer and the 2018 American Association for Cancer Models generated directly from cancer patients’ tumors including Research Annual Meeting. cell lines, patient-derived xenografts (PDX), or organoids are increas- € Corresponding Authors: Pasi A. Janne, Dana-Farber Cancer Institute, 450 ingly being used to screen for novel treatment approaches (14–16). Brookline Avenue, LC4114, Boston, MA 02215. Phone: 617-632-6036; Fax: 617- However, the process of cell line development and the cells’ adaptation 582-7683; E-mail: [email protected]; and Cloud P. Paweletz, [email protected] to growth in a two-dimensional environment can render some tumors that were drug-sensitive in vivo resistant in vitro. PDX experiments can Clin Cancer Res 2020;26:2393–403 often take months to complete due to slow tumor growth kinetics, and doi: 10.1158/1078-0432.CCR-19-1844 are impractical to perform in scale requisite for more comprehensive Ó2020 American Association for Cancer Research. drug combination screens. In addition to these intrinsic limitations of AACRJournals.org | 2393 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 7, 2020; DOI: 10.1158/1078-0432.CCR-19-1844 Ivanova et al. Generation of HER2-mutant Ba/F3 and NIH-3T3 cells Translational Relevance The HER2 755_757LREdelinsRP mutation was introduced via site The development of anticancer therapies requires appropriate directed mutagenesis into the pDNR-Dual Donor Vector (Clontech) preclinical models in which to test treatments. HER2-mutant non– containing wild type HER2 using the QuikChange II XL Site-Directed small cell lung cancer (NSCLC) occurs in 3% of patients with lung Mutagenesis Kit (Agilent Tech) according to the manufacturer's adenocarcinoma, and very few models of HER2-mutant NSCLC instructions. Two rounds of mutagenesis were necessary to create the are available for preclinical therapeutic development. We gener- HER2 755_757LRE delinsPR mutation. The first round introduced the ated 2 patient-derived HER2-mutant xenograft (PDX) models and HER2_L755P substitution mutation using primers F-50-AAATTC- developed a platform to rapidly test HER2-targeted therapies CAGTGGCCATCAAAGTCCCGAGGGAAAACACATCCCCC-30 ex vivo using the PDX tumors. Our methods may accelerate the and R-50-GGGGGATGTGTTTTCCCTCGGGACTTTGATGGC- identification and prioritization of promising anticancer therapies CACTGGAATTT-30. The second round introduced the E757 dele- to pursue further in clinical trials. tion using primers F-50-GCCATCAAAGTCCCGAGdelAAACA- CATC CCCCAAAG-30 and R-50-CTTTGGGGGATGTGTTT- delCTCGGGACTTTGATGGC-30. All constructs were confirmed by DNA sequencing. The constructs were shuttled into the retro- the preclinical models, the identification of effective HER2-directed viral vector JP1540 or lentiviral vector JP1698 using the BD Creator treatments is further complicated by the commercial availability of System (BD Biosciences) and Ba/F3 and NIH-3T3 cells were only one HER2-mutant NSCLC cell line, H1781 (17). infected with lentivirus according to standard protocols and as We recently described a novel ex vivo system of organotypic described previously (23). Stable clones were obtained by selection tumor spheroids growth in a 3-dimensional microfluidic device in puromycin (1 mg/mL). (DOTS; refs. 18, 19). The device is designed to support short-term (≤7 days) culture of freshly harvested primary tumor cells and Focus formation assay associated immune cells resuspended in collagen for the duration of NIH/3T3 cells expressing WT HER2 or the HER2 755_757LRE- a screen, and allows for conventional immunofluorescence and delinsRP mutation were cultured in DMEM with 10% bovine calf microscopy based analysis. Thanks to its scalability, the DOTS serum. Once cells reached confluency, serum was reduced to 5%. Plates system allows for rapid evaluation of multiple different therapies were stained with 0.5% crystal violet solution two weeks later. using biopsies derived from mouse models; primary murine models (MDOTS) or patient derived xenografts (XDOTS) or Patient-derived lung tumor xenografts and genetic engineered directly from patients (PDOTS) for which preclinical models mouse models cannot be established or do not exist. In the current study, we PDX models were generated from core needle biopsies, surgical have adopted this system to study new therapeutic approaches for biopsies, or pleural effusions from lung cancer patients at the Dana- HER2 mutant NSCLC using tumors derived from HER2-mutant Farber Cancer Institute (DFCI, Boston, MA) at the Belfer Center