For isolation of in vivo–derived hybrids or assessment of CTCs, 5 × 105 B16F10 (H2B-RFP with or without Cre) cells were injected intradermally into R26R-YFP or actin-GFP mice, respectively. Once tumors reached 1 to 2 cm3 in diameter, they were surgically removed for immunohistochemical analyses or for FACS/flow analyses.

For demonstration that tumor cells can fuse with myeloid cells, 5 × 105 B16F10 (fl-dsRed-fl-eGFP) cells were injected intradermally into 6- to 8-week-old LysM-Cre transgenic mice. When tumors reached 1 cm3, primary tumors and lungs were removed for immunohistochemical analyses.

Immunohistochemical analysis of in vivo–derived tumors. B16F10 (H2B-RFP, Cre) primary tumors in Act-GFP or R26R-stop-YFP mice were fixed in 10% buffered formalin, frozen in optimum cutting temperature (OCT), and 5-μm sections were obtained. Tumors from R26R-stop-YFP mice were incubated with antibodies for GFP (1:500; Life Technologies) followed by detection with fluorescent secondary antibody (1:500, Alexa Fluor 488; Jackson ImmunoResearch). Nuclei were counterstained with Hoechst (1 μg/ml). Slides were digitally scanned with a Leica DM6000 B microscope and analyzed using Ariol software. Confocal images were acquired with a FluoView FV1000 confocal microscope (Olympus).

B16F10 (fl-dsRed-fl-eGFP) primary tumors and lungs from LysM-Cre mice were fixed in 4% paraformaldehyde for 2 hours at 20°C, washed, and cryopreserved in 30% sucrose for 16 hours at 4°C and then embedded in OCT. Primary tumors were stained as described in the paragraph above. Lung sections were cut to 8-μm thickness, baked for 30 min at 37°C, then subjected to antigen retrieval under standard conditions (R&D Systems, CTS016), blocked with DAKO Protein Block Serum-Free (Agilent, X090930-2), and incubated for 16 hours at 4°C with primary antibodies [anti-MITF (1:500; Abcam, ab12039), anti-dsRed (1:250; Clontech, 632496), and anti-GFP (1:1000; Abcam, ab13970)] in background-reducing antibody diluent (Agilent, S302281-2). Fluorescent-tagged secondary antibodies were applied, and then sections were mounted in a ProLong Gold antifade reagent (Molecular Probes, P36934). Antibody specificity was determined by immunostaining healthy lungs of nontumor-bearing mice and performing secondary antibody only controls.

FACS isolation and flow cytometric analyses of fusion hybrids. Tumors were diced and digested for 30 min at 37°C in DMEM + Collagenase A (2 mg/ml; Roche) + DNase (Roche) under stirring conditions. Digested tumor cells were filtered through a 40-μm filter and washed with PBS. For FACS isolation, hybrid and unfused cells were isolated by direct fluorescence on a Becton Dickinson InFlux sorter. For flow cytometric analysis, blood was collected retro-orbitally using heparinized microhematocrit capillary tubes (Fisher) into K2EDTA-coated tubes (BD Biosciences). RBC lysis was performed as described above. Cells were washed and resuspended in FACS buffer (PBS, 1.0 mM EDTA, and 5% FBS). Cells were incubated in PBS containing LIVE/DEAD Fixable Aqua (1:500; Invitrogen) with Fc Receptor Binding Inhibitor (1:200; eBioscience). Cells were then incubated in FACS buffer for 30 min with CD45-PeCy7 (1:8000; BioLegend), CSF1R-BV711 (1:200; BioLegend), F4/80-APC (1:400; BioLegend), and CD11b-AF700 (1:200; eBioscience). A BD LSRFortessa FACS machine was used for analyses. A statistical significance of P < 2.2 × 10−6 by unpaired t test was determined for CD45+ hybrid CTCs relative to CD45 hybrid, CD45+ unfused, and CD45 unfused CTCs. Technical duplicates of n = 5 or 6 mice were analyzed.

Tumorigenic analyses of FACS-isolated in vivo–derived hybrids. A total of 100 or 3000 FACS-isolated hybrids and unfused B16F10 cells were injected intradermally into C57BL/6J mice. For experiments with 100 cells, technical octuplicates with biologic duplicates, triplicates, or quadruplicates were performed, depending on the number of hybrids isolated form the primary tumor, for a total of n = 16 mice analyzed. For experiments with 3000 cells injected, technical triplicates were performed.

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