Analyses of human solid tumors. The presence of cell fusion between Y chromosome–containing blood cells and host tumor epithelium was evaluated by dual FISH and immunohistochemical analyses. X and Y chromosome FISH probes were hybridized to 5-μm formalin-fixed paraffin-embedded primary human tumor sections using CEP X (DXZ1 locus) and CEP Y (DYZ1 locus) probes (Abbott Molecular) following the manufacturer’s protocols. Briefly, the tissue was treated with Retrievagen A solutions (BD Biosciences) and Tissue Digestion Kit II reagents (Kreatech) and then hybridized with a probe at 80°C for 5 min and 37°C for 12 hours. Tissue sections were permeabilized with graded detergent washes at 24°C and then processed for immunohistochemical staining. Tissue was incubated with antibodies to pan-cytokeratin (Fitzgerald) and counterstained with Hoechst dye (1 μg/ml). Two slides were analyzed for each tumor section. Slides were digitally scanned and quantified by two independent investigators. Areas with Y chromosome positivity were analyzed by confocal microscopy. H&E staining was conducted on adjacent sections.

In situ analyses of human peripheral blood analyses. Patient peripheral blood was collected in heparinized Vacutainer tubes (BD Biosciences), and then, lymphocytes and peripheral mononuclear cells were isolated using density centrifugation and LeucoSep Centrifuge Tubes (Greiner Bio-One) according to the manufacturer’s protocol. Cells were then prepared for antibody staining. Briefly, cells were adhered to poly-d-lysine–coated slides, fixed, and permeabilized before staining for CD45 and cytokeratin expression using antibodies to CD45 (eBioscience) and human pan-cytokeratin (Fitzgerald). Phenotypes of CHCs were evaluated with additional antibody staining, including to CD66b (BD Pharmingen), CD68 (Abcam), CD163 (Neomarkers), CSF1R (Abcam), and EPCAM (1:200; US Biological). Tissue was developed with appropriate fluorescent-conjugated secondary antibodies [anti-mouse Cy3 (Jackson ImmunoResearch), goat anti-guinea pig 488 (Invitrogen), goat anti-guinea pig 555 (Invitrogen), anti-rabbit A647 (1:500; Thermo Fisher Scientific), and anti-mouse Cy5 (1:500; Jackson ImmunoResearch)] and then was stained with Hoechst (1 μg/ml). Slides were digitally scanned with a Leica DM6000 B microscope or a Zeiss AxioObserverZ1 microscope and analyzed using Ariol or Zeiss Zenblue software.

To determine whether circulating CD45+/CK+ or CD45+/EPCAM+ cells were cell fusion products, patient peripheral blood was subjected to FISH/immunohistochemical analyses as described for solid tissues (see above). Processed peripheral blood was interrogated with Y chromosome FISH (DYZ1 locus) probes (Abbott Molecular) following the manufacturer’s protocols. Briefly, cells were hybridized with a probe at 42°C for 16 to 20 hours and then subjected to graded detergent washes. Cells were then subjected to antibody staining with anti-CD45 conjugated to fluorescein isothiocyanate (FITC) (1:100; BioLegend) and EPCAM (1:100, US Biological) and processed with anti-rabbit AF647 secondary antibodies (1:250; JacksonImmuno Research). Cells were imaged on a Zeiss AxioOberverZ1 microscope. Images were postprocessed to rule out nonspecific staining. Briefly, CZI files were opened using ZEN 2.3 Lite (Blue Edition) and saved as single-channel TIF files [four channels per CZI: EPCAM (white), Y chromosome (red), CD45 (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue)]. Single-channel TIF files were loaded into MATLAB as UINT8 matrices containing RGB information at each pixel. To create binary images, pixel intensity thresholds were set for each channel image separately: Any pixel with a value above the threshold was turned ON (that is, maximum intensity), and the remaining pixels were turned OFF (that is, zero intensity). Two binary channel images were reassigned colors (EPCAM: white → yellow, Y chromosome: red → white); all binary channel images were then overlaid.

Quantification of CHCs in patient blood. Manual quantification by three independent investigators of randomly selected regions containing 2000 cells evaluated CD45 and cytokeratin status of Hoescht+ cells. Percentages of CHCs in the buffy coat correlate with disease stage with significance determined by overall ANOVA post-test [P < 6.3 × 10−8; P values for no nodal-met (0.00035), nodal-met (0.05), and no nodal-nodal (0.15)], while none of the conventional CTCs (that is, CD45−ve) comparisons across stage were statically significant [P values for no nodal-met (0.31) and nodal-met (0.9)]. Survival analysis was conducted on 18 of 20 pancreatic patients (2 patients were lost to follow-up, 9 patients have high levels, and 9 patients have low levels) to correlate CHCs or CTCs with time to death using Kaplan-Meier curve and log-rank test using dichotomized biomarkers based on a median value. High CK+/CD45+ (median, >0.808) was associated with a statistically significant increased risk of death (log-rank test, P = 0.0029) with a hazard ratio of 8.31, but high CK+/CD45−ve (median, >0.101) did not have a statistically significant effect on time to death (log-rank test, P = 0.95).

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