Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs.

2Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer 3 cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we 4 developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell 5 cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) 6 protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-7 amplification of WTA products, quantification of amplified cDNA yields and final qPCR 8 quantification, to identify the most reliable and accurate workflow for quantitation of gene 9 expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms 10 relative quantification. We then validated the performance of our method on single cells of 11 established breast cancer cell lines displaying distinct levels of HER2 protein. The different 12 protein levels were faithfully reflected by transcript expression across the tested cell lines 13 thereby proving the accuracy of our approach. Finally, we applied our method on patient-derived 14 breast cancer DCCs. Here, we were able to measure ERBB2 expression levels in all HER2-15 positive DCCs. In addition, we could detect ERBB2 transcript expression even in HER2-negative 16 DCCs, suggesting post-transcriptional mechanisms of HER2 loss in anti-HER2-treated DCCs. In 17 summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of 18 ERBB2 in DCCs. 19 66(27). Moreover, protocols for preparing qPCR samples are simpler and result in higher 67 sensitivity and reproducibility as compared to NGS-based approaches (7, 27). Importantly, 68 single-cell qPCR workflows exhibit high levels of reliability and wide and dynamic detection 69 ranges, making them exceptionally well-suited for targeted gene expression analyses in singles 70 cells, where sensitivity is essential and the amount of target genes is low (7, 27). Therefore, the 71 present study aimed to develop a single-cell qPCR assay to quantify gene expression changes in 72 single cells, specifically in patient-derived DCCs. We established a workflow comprised of 73 single-cell WTA, re-amplification of single-cell cDNA, post-WTA normalization of cDNA 74 quantities and qPCR-based data analysis. The new assay provides means for measuring 75 expression levels of individual pre-selected genes in WTA products generated from single cells 76 in an accurate and reliable fashion.77 6 78 Materials and methods 79 Cell lines 80 BT-474 (ACC 64) and MCF-7 (ACC 115) breast cancer cell lines were obtained from German 81 Collection of Microorganisms and Cell Cultures (DSMZ). MCF-10A (CRL-10317), a non-82 tumorigenic mammary epithelial cell line was obtained from American Type Culture Collection 83 (ATCC). ZR-75-1 (CRL1500, ATCC) and MDA-MB-453 (ACC 65, DSMZ) cells were 84 purchased from DSMZ. The identity of all cell lines was confirmed by DNA finger printing 85 analysis utilizing the GenePrint® 10 System (Promega). B...