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

Abstract

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, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic 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 for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.

Figure 1. Analysis of mouse pancreatic CTCs identifies Wnt2 as a candidate CTC gene

a, Schematic representation of strategy for DGE of RNA isolated from primary mouse pancreatic tumour, metastatic ascites, and CTC-enriched blood. b, Immunofluorescence staining of mouse pancreatic CTC, leukocyte (WBC), and CTC cluster captured on ^HbCTC-Chip (DAPI, blue; Cytokeratin (CK), red; CD45, green). c, Quantitation of CK+ cells captured from control mice and pancreatic cancer bearing mice (dashed line - threshold of ≥ 6 CK+ cells/100 µL). d, DEGseq M-A plot of RNA sequence reads from mouse (MPANC-9) CTCs, comparing anti-EpCAM-captured versus IgG-coated ^HbCTC-Chips. (M - log2 fold change, A - averaged log2 reads, red dots – differentially expressed genes) e, RNA-ISH of CTCs and metastatic ascites cells co-expressing CK8+18 (green) and Wnt2 (red) transcripts. White arrowheads highlight Wnt2 signals. f, RNA-ISH of primary pancreatic tumours showing a small cluster of Wnt2 mRNA expressing cells near a pancreatic duct (D) (Wnt2, red dots; hematoxylin, light blue). Hematoxylin and eosin (H&E) stained serial section is shown (right). A second primary tumour imaged under fluorescence demonstrating Wnt2 expression in a subpopulation of tumour cells; high magnification shown (right, Wnt2, red; CK, green; DAPI, blue). (Scale bars=10 µm).

Figure 2. Wnt2 promotes anchorage-independent cell survival and pancreatic cancer cell metastasis

a, Number of GFP-positive metastatic nodules in the lungs of mice bearing subcutaneous tumours established with Vec- or Wnt2-expressing NB508 cells. b, Number of GFP-positive CTCs captured from the blood of mice described above. c, Number of GFP-positive lung metastatic nodules in mice, following tail vein injection with Vec- or Wnt2-NB508 cells tagged with both GFP and luciferase. d, Representative images of tumour spheres formed by Vec- or Wnt2-NB508 cells plated at 1,000 cells/well (scale bar = 250 µm). Quantitation of both tumour sphere number and diameter is shown plated at 100 cells/well (n=3). e, Immunoblotting analysis of Vec- and Wnt2-NB508 cells at time intervals following plating under non-adherent conditions. f, Suppression of tumour spheres formed by Wnt2-NB508 cells following infection with lentivirus encoding shRNA targeting Fn1 compared with non-target (NT) shRNA (n=3). Effectiveness of knockdown is shown by western blot above. (mean ± s.d.; * p < 0.01,** p < 0.001).

Figure 3. Association of Wnt2-prosurvival phenotype with non-canonical Wnt signaling and inhibition by suppression of Map3K7 (Tak1)

a, Suppression of Wnt2-induced, but not baseline, tumour sphere formation by NB508 cells, following treatment with 5Z-7-Oxozeaenol, a small molecule inhibitor of Tak1 (n=6). b, Immunoblot showing suppression of Fn1 expression following treatment of non-adherent cultures of Vec- and Wnt2-NB508 cells with increasing concentrations of 5Z-7-Oxozeaenol. c, Immunoblot analysis of Tak1, Fn1 and cleaved caspase 3 expression in Wnt2-NB508 cells following lentiviral infection with two different shRNAs targeting Tak1 or non-target (NT). d, Enumeration of tumour spheres formed by cells in c (n=6). e, Percentage of mice with detectable metastases by bioluminescence imaging for luciferase-producing tumor cells in mice 6 weeks following tail vein injection of Vec-NB508, Wnt2-NB508, and Wnt2-NB508 cells infected with three different shRNAs targeting Tak1 (n=12, 11, 14, 13, 12 respectively). Immunoblot of Tak1 knockdown by shRNAs B6, B7, and B4 is shown (right). (mean ± s.d., * p < 0.05)

Figure 4. Detection of Wnt2 mRNA expression and non-canonical Wnt signature in human pancreatic CTCs

a, Immunofluorescence staining of human pancreatic CTC, leukocyte (WBC), and CTC cluster captured on anti-human EpCAM ^HbCTC-Chip (DAPI nuclear stain, blue; CK and EpCAM cocktail, red; CD45, green). b, Enumeration of CK+EpCAM positive human CTCs (CTCs/mL) captured from patients with metastatic pancreatic cancer. Arrowheads indicate lower CTC numbers in patients responding to therapy. Blood samples from healthy donors were used to establish the threshold of ≥ 3 CK+EpCAM+/CK45− cells/mL (dashed line). c, RNA-ISH analysis of human pancreatic CTCs, showing co-expression of mRNAs for cytokeratins (CK) 8, 18, 19 and 23 (pooled probes in green) and Wnt2 (red). (DAPI, blue). (Scale bars = 10 µm). d, Induction of Wnt transcripts in pancreatic cancer cells grown as non-adherent tumour spheres compared to standard conditions (fold increase). e, Quantification of tumour spheres with or without Tak1 inhibitor (3µM 5-Z-7-Oxozeanol). (n=3; mean ± s.d.)