Abstract 3374: Large-scale single-cell whole transcriptomic analyses reveal distinct malignant phenotypes of CTCs from NSCLC patients
Authors: Rieckmann Lisa-Marie , Spohn Michael , Selbuz Ekaterina , Schubert Claudia , Agorku David , Becker Lisa , Borchers Alina , Krause Jenny , Ruff Lisa , Heinemann Sarina , Kobus Franca , Hille Jurek , Tehrany Andia Louisa , Velthaus-Rusik Janna-Lisa , Franzenburg Sören , Christopoulos Petros , Winter Hauke , Thomas Michael , Riethdorf Sabine , Gagliani Nicola , Bokemeyer Carsten , Krebs Christian F. , Sprick Martin , Trumpp Andreas , Peine Sven , Hardt Olaf , Stoecklein Nikolas H. , Pantel Klaus , Rosenstiel Philipp , Loges Sonja , Janning Melanie 
Published: June 2022

Circulating tumor cells (CTC) hold great promise for representation of intratumoral heterogeneity and increasing our understanding of resistance mechanisms. However, especially in non-small cell lung cancer (NSCLC), numbers of CTCs detected and isolatable from peripheral blood samples using standard EPCAM-based techniques are too low to perform robust multi-omics analyses. Recent data suggested that diagnostic leukapheresis (DLA) can be used to increase CTC numbers by enriching them from larger blood volumes. Here, we present single-cell RNA sequencing data (scRNAseq) from 3172 NSCLC CTCs isolated by DLA.

Between 3.1 and 8.0 liters of blood volume was processed with DLA and mononuclated cells were collected from six stage IV NSCLC patients. A total of 80x108 cells (≈33% of the DLA) were used for magnetic depletion of CD31+, CD3+, CD16+, CD235a+ and CD45+ cells followed by FACS sorting for CD45 negativity. Sorted cells were subjected to scRNAseq analysis using 10X Genomics. CTC transcriptomes were identified by marker gene expression (Satija Lab, US and R package SingleR). Gene set enrichment analysis (GSEA) on hallmark gene sets were performed to compare CTC transcriptomes and sc transcriptomes from primary NSCLC tumors.

Unsupervised dimensional reduction and clustering revealed 7 distinct CTC cluster. Inferred copy number variation (CNV) analyses confirmed greater CNV variability compared to hematopoietic cells and a high degree of heterogeneity consistent with tumor cells. Also, CTC transcriptomes showed significantly higher expression of cancer-associated genes like Cyclin D1 and metastasis-associated protein 2 compared to normal hematopoietic cells. CTC clustering was independent of patient or histology, thus indicating a potential function-based clustering. Pseudotime analyses of all scCTC transcriptomes revealed three principal CTC phenotypes: (i) epithelial-like (expression of E-Cadherin), highly proliferative (expression of KI67 and E2F targets pathway) and immune responsive (enriched for IL1B, Interferon-α/γ-response pathways), (ii) mesenchymal/invasive (expression of Vimentin, mTORC1, hypoxia and glycolysis pathways) and (iii) mesenchymal/cancer stem cell-like (enrichment of genes including ALDH1A3 and oxidative phosphorylation and adipogenesis pathways). Compared to a set of scRNAseq data from primary NSCLC tumors (n=46), GSEA revealed an enrichment of pathways involved in cell cycle, anti-apoptosis and invasion in CTCs suggesting higher malignant potential compared to tissue-resident NSCLC tumor cells.

Performing CTC enrichment using DLAs resulted in generation of an unprecedented number of transcriptomes from individual CTCs derived from NSCLC patients. Our data should lead to a better understanding of the heterogeneity of blood circulating CTCs and their associated biology and may allow rational design of CTC-targeting drugs.

Citation Format: Lisa-Marie Rieckmann, Michael Spohn, Ekaterina Selbuz, Claudia Schubert, David Agorku, Lisa Becker, Alina Borchers, Jenny Krause, Lisa Ruff, Sarina Heinemann, Franca Kobus, Jurek Hille, Andia Louisa Tehrany, Janna-Lisa Velthaus-Rusik, Sören Franzenburg, Petros Christopoulos, Hauke Winter, Michael Thomas, Sabine Riethdorf, Nicola Gagliani, Carsten Bokemeyer, Christian F. Krebs, Martin Sprick, Andreas Trumpp, Sven Peine, Olaf Hardt, Nikolas H. Stoecklein, Klaus Pantel, Philipp Rosenstiel, Sonja Loges, Melanie Janning. Large-scale single-cell whole transcriptomic analyses reveal distinct malignant phenotypes of CTCs from NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3374.