Abstract 5592: Expansion and characterization on ALK positive NSCLC circulating tumor cells isolated using a size based inertial microfluidic Labyrinth device
Authors: Chen Yuru , Fouladdel Shamileh , Ball Harrison , Cheng Xu , Bao Liwei , Serhan Habib , Liu Albert , Vandenburg Bryce , Goo Laura , Merrill Nathan , Udager Aaron , Qin Angel , Merajver Sofia D. , Nagrath Sunitha 
Published: April 2023

Abstract
Introduction
Lung cancer (LC) is the leading cause of cancer deaths. 85% of LC are non-small cell lung cancer (NSCLC). Fusions involving anaplastic lymphoma kinase (ALK) are oncogenic drivers in ~ 7% of NSCLC. Previous studies have shown that drug resistance develops within ~2 years after ALK tyrosine kinase inhibitor targeted therapy. Due to resistance mechanisms and the biological heterogeneity that evolves over the treatment of ALK+ NSCLC, it is important to monitor and study those dynamic changes through the clinical treatment course to understand cancer progression.
Circulating tumor cells (CTCs) are rare cells, shed from cancer lesions into the bloodstream. They can be isolated from a patient's peripheral blood sample (~20ml) to enable study of tumor characteristics at multiple time points. They are promising biomarkers that not only offer phenotypic and genotypic information, but also present a potential to expand into patient derived ex vivo tumor models. In this study, we use a high throughput label-free microfluidic device, Labyrinth, developed in the Nagrath lab for cell size based CTC enrichment, followed by expansion of CTCs in organoid models, further molecular characterization, and growth in patient-derived xenograft (PDX) models.
Methods
Blood samples were collected from patients (n=22), three with known ALK status. CTC isolation was performed using Labyrinth at 2.0 mL/min. A part of the isolated CTCs were immunofluorescently stained for pan cytokeratin (pan CK), CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers for enumeration. Others were processed for expansion in ultralow attachment plates with three types of organoid culture media. The rest of the CTCs were used for molecular characterizations.
Growth curves were produced on expanded samples on Day 0, 7, 14, and 21 in different culture conditions. A live/dead assay and in situ hybridization (FISH) were also performed. On Day 7, selected samples were injected into NSG™ mice. Bulk RNA sequencing was conducted on the original tumor, Day 0 and 7 CTCs, and PDX samples.
Results
The median CTC concentration in this cohort (n=22) was 316.2 CTCs/ml of blood. Isolated CTCs from ALK+ NSCLC patients showed ALK fusions by FISH. We observed that the number of ALK CTCs was reduced over time consistent with decreased tumor size in CT scans. There were significant differences (p&lt;0.05) for vimentin positive CTCs compared to the other three types of CTCs, which indicated a higher prevalence of a mesenchymal state of NSCLC CTCs. One of the two samples successfully developed into PDX after two months. In summary, we demonstrated a successful workflow to enrich and expand CTCs from ALK+ NSCLC patients. The expanded CTCs can be further characterized and can generate tumors in PDX models.
Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Xu Cheng, Liwei Bao, Habib Serhan, Albert Liu, Bryce Vandenburg, Laura Goo, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Expansion and characterization on ALK positive NSCLC circulating tumor cells isolated using a size based inertial microfluidic Labyrinth device. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5592.