Abstract LB-271: Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s)
Authors: Suvilesh Kanve N. , Manjunath Yariswamy , Radhakrishnan Vijay , Avella Diego M. , Kimchi Eric T. , Staveley-O'Carroll Kevin F. , Li Guangfu , Kaifi Jussuf T. 
Published: September 2020

Introduction: Circulating tumor cells (CTCs) are liquid biomarkers with high potential for application in precision medicine cancer care. However, no robust methods have been developed to culture CTCs from early-stage cancer patients owing to their rarity and instability. Here, we describe a successful protocol to establish CTC culturing and CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer (NSCLC) patients. Further, comparative multi-omics characterization of these CDX tumors revealed novel diagnostic/therapeutic target(s). Methods: Surgically resected primary tumor fragments from patients with early stage NSCLCs were subcutaneously (s.c.) implanted to immunodeficient NOD SCID gamma (NSG) mice to generate patient-derived xenograft (PDX) models. Once palpable tumor reached a diameter of ~1.5 cm, animals were euthanized and blood was collected, CTCs were enriched with OncoQuick® and cultured in vitro. These patient-derived CTCs were further stained with anti-human antibodies against CK7, Napsin, TTF-1, CK5/6, PDL1 and CD45. Once human CTCs were confirmed, we then injected expanded CTCs s.c. to naïve NSG mice to establish CDX models. Single cell suspension from CDX tumors was cultured and fibroblasts were depleted using specific beads (Miltenyi biotec) to establish stable CTC lines. Further, advanced proteomics, single cell RNA sequencing (transcriptomics) and whole genome sequencing of patient-matched peritumoral and primary tumor tissues along with PDX and CDX tumors were carried out. Results: Human NSCLC primary tumor fragments implanted in NSG mice were tumorigenic at the s.c. implantation site, metastatic to distant organs, and passaged successfully to naïve NSG generations to maintain the PDX colonies. CTCs enriched from the PDX models were successfully cultured in vitro and positive for human NSCLC-specific markers but negative for human leukocyte marker CD45. Cultured PDX-derived CTCs that were xenografted into naïve NSG mice by s.c. demonstrated tumorigenicity and metastatic potential in CDX models. Stable CTC lines established using CDX tumors sustained &gt;30 passages. Global proteomic profiling of tumor tissues revealed 19 new proteins that were upregulated and 9 proteins that are down regulated. Single cell RNA sequence analysis depicts top rank genes that are overexpressed in tumor cluster. Whole genome sequencing of these samples identified somatic variants that needs further validation. Conclusion and future perspectives: This approach establishes CDX models from early-stage NSCLC patients at a high rate for personalized precision medicine. Models provide a unique opportunity to characterize rare patient-derived CTCs that helps in understanding CTC tumor biology and developing effective anti-cancer therapeutic strategies. Multi-omics characterization of these tissues identified a set of novel genes/proteins that needs to be validated in large patient population. Further, studying tumorigenic potential of established CTC lines after knockdown and overexpression of these genes will provide more information about translating these targets into therapeutics.

Citation Format: Kanve N. Suvilesh, Yariswamy Manjunath, Vijay Radhakrishnan, Diego M. Avella, Eric T. Kimchi, Kevin F. Staveley-O'Carroll, Guangfu Li, Jussuf T. Kaifi. Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-271.