RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifyingWnt2 as a candidate gene enriched inCTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increasesmetastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment forWNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer. We established the CTC-Chip microfluidic capture platform to isolate CTCs from blood samples of a genetically engineered mouse pancreatic cancermodel, comparing their expression profile to that of simultaneously collected primary tumour and metastatic ascites cells (Fig. 1a and Supplementary Fig. 1). Captured cells were stained for epithelial cytokeratin (CK) and the leukocyte marker CD45, with CTCs scored asCK/CD45 (Fig. 1b). Eight control tumour-freemice had amedianof 1.3CK/CD45 cells per 100ml of blood (range, 0.2–5.6; mean6 s.d., 1.86 1.7) under the selected imaging parameters. Applying a threshold of$ 6 cells per 100ml, 7 out of 8 tumour-bearing mice were positive for CTCs (median, 31 cells per 100ml; range, 2–547; mean6 s.d., 1156 188) (Fig. 1b, c).MostCTCswere captured as single cells, although clusters of 5 to 10 cells were seen in ,50% of mouse blood samples. The on-chip purity of captured CTCs ranged from 0.1–6%, due to non-specific binding (NSB) by leukocytes (Supplementary Table 1). Given the minute amounts and variable purity of CTCs, we applied a sub-microgram RNA-based sequencing method using a singlemolecule ‘next generation’ platform, to derive a digital gene expression (DGE) profile. We processed each blood sample through paired EPCAMand mock IgG-functionalized CTC-Chips, allowing digital subtraction of matched NSB leukocyte reads from each CTCenriched DGE profile. Using the DEGseq statistical model applied to theMA-plotwith a false discovery rate (FDR) of 0.10, we identified 361 transcripts in mouse MPANC-9 cells that were increased more than twofold in anti-EPCAMversus IgG-chips, and absent in blood from non-tumour-bearing mice (Fig. 1d). This CTC candidate gene set was then compared with DGE profiles of 12mouse and 15 human primary pancreatic tumours, versus normal mouse and human pancreata, yielding a set of 9 tumour-associated genes whose expression was markedly increased in CTCs (Supplementary Table 2). One of these, Wnt2, was found to be consistently enriched in pancreatic cancers in the Oncomine database, leading us to pursue this gene as a prototype CTC-enriched transcript. RNA sequencing of metastatic cells in the mouse ascites demonstrated enrichment for Wnt2. A second mouse, MPANC-10, also demonstrated Wnt2 expression in both CTCs and metastatic ascites cells. The lower number of Wnt2 messenger RNA reads in MPANC-10 cells was correlated with the fewer cytokeratin transcript reads, indicating that both transcripts trackwith the number of CTCs in the blood specimen (Supplementary Table 3). Given the absence of antibodies for cellular imaging of WNT2, we developed fluorescent RNA in-situ hybridization (RNA-ISH) to verify Wnt2 expression in CTCs (Supplementary Figs 2 and 3). RNA-ISH analysis of CTCs using dual staining for Wnt2 and cytokeratin mRNAs identified Wnt2 transcripts in 64% of cytokeratin-expressing cells in 3 out of 4 mice. A comparableWnt2mRNA signal was observed in metastatic cells from ascites of mice bearing pancreatic tumours (Fig. 1e). In contrast, within primary tumour specimens, Wnt2 mRNA expression was only detectable in very small localized clusters of cells (1–5% of all cells) in 8 out of 14 primary tumour specimens (Fig. 1f). Histological analysis of rareWnt2-positive cells within pancreatic ductal adenocarcinomas (PDACs) did not reveal any obvious distinction from other tumour cells. The small number ofWnt2-positive cells in primary tumour specimenswas consistentwithDGEanalysis, which showed rare Wnt2RNA reads in bothmouse (8/12) andhuman (9/15) PDACs. Thus, Wnt2-positive cells are present within both CTC and metastatic ascites, and represent a rare subset of the primary tumour population. To test the functional consequences of WNT2 expression in vivo, lentiviral constructs were introduced into NB508 PDAC cells, which lack detectable endogenousWNT2 expression (Supplementary Fig. 4). Subcutaneous engraftment of green fluorescent protein (GFP)-tagged Wnt2-NB508 cells produced significantly more lung metastases in nude mice, compared with vector-transduced cells, despite the larger size of vector-expressing primary tumours (n5 8, P, 0.05) (Fig. 2a and Supplementary Fig. 4). EPCAM-captured CTCs showed a modest increase in numbers in mice bearing WNT2-expressing tumours, but this did not reach statistical significance (P5 0.25, Fig. 2b), even after normalization for the smaller size of WNT2-expressing tumours (P5 0.17, Supplementary Fig. 4). Thus,WNT2 expressionmay increase the metastatic potential of circulating cancer cells, without markedly increasing their generation from primary tumours. Consistent with this hypothesis, direct intravascular inoculation of cells into the tail vein of syngeneic mice, which bypasses the vascular invasion step, showed a marked increase in lung metastases for Wnt2-NB508 cells (5/6 Wnt2transduced versus 0/6 vector, P, 0.05) (Fig. 2c and Supplementary Fig. 4).