Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.

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.

28Although thousands of breast cancer cells disseminate and home to bone marrow until 29 primary surgery, usually less than a handful will succeed in establishing manifest 30 metastases months to years later. To identify signals that support survival or outgrowth 31 in patients, we profiled rare bone marrow-derived disseminated cancer cells (DCCs) 32 long before manifestation of metastasis and identified IL6/PI3K-signaling as candidate 33 pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, 34DCCs lacked membranous IL6 receptor expression and mechanistic dissection 35 revealed IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells 36 via gp130. Responsiveness to IL6 trans-signals was found to be niche-dependent as 37 bone marrow stromal and endosteal cells down-regulated gp130 in premalignant 38 mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation 39 rendered cells independent from IL6 trans-signaling. Consistent with a bottleneck 40 function of microenvironmental DCC control, we found PIK3CA mutations highly 41 associated with late-stage metastatic cells while being extremely rare in early DCCs. 42Our data suggest that the initial steps of metastasis formation are often not cancer cell-43 autonomous, but also depend on microenvironmental signals. 44Word count: 175 45 46 47 48 low frequency of BM-DCCs (<10 -6 ), we either injected CD45 depleted or EpCAM-98 enriched BM cells or generated and transplanted spheres as these have a higher 99 engraftment-likelihood 9 . In total, we tested 42 patient samples and different routes of 100 application, including sub-cutaneous, orthotopic (site of origin), intra-femoral and intra-101 venous injection. We then assessed tumor formation at the cutaneous injection sites 102 and metastatic spread to lungs or BM. BM-derived DCCs from M1-stage patients 103 engrafted in two out of four cases. In contrast, early DCCs from 42 M0-stage patients 104 did not establish xenografts (Fig. 1a, b), neither at the injection sites nor in the lungs 105 (p = 0.006; Fisher's exact test). We also explored the presence of minimal systemic 106 cancer by testing for human cytokeratin (CK) or EpCAM-positive cells in murine BM. 107Interestingly, albeit DCCs of non-metastatic patients did not expand in mice, they 108 survived in murine BM in 4 out of 42 cases. We detected human EpCAM + or CK + DCCs 109 at a frequency of 1-5 DCCs/million BM cells 4-14 weeks after injection of CD45-110 depleted human BM cells (Fig. 1c). For one of these rare events we could not only 111 prove human and epithelial, but also malignant origin by single cell copy number 112 alteration (CNA) analysis (Fig. 1d). 113In summary and consistent with our findings in melanoma, early DCCs from patients 114 without manifest metastasis failed to generate xenografts. Besides lower absolute cell 115 numbers and fewer genetic alterations (see below), microenvironmental dependence 116 of early DCCs could account for these results. We therefore decided to retrieve ...

No abstract

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...