Recruitment of myeloid cells is mediated by tumor cell clot formation. (A) In vivo assays of myeloid cell (green) recruitment. Cx₃cr1^gfp/+ mice were intravenously injected with 5 × 10⁵ CMAC-stained B16F10-wt cells (blue) and, on the opposite tail vein, with 9 × 10⁹ PKH26-stained platelets (red), isolated from C57BL/6 mice. At the indicated times, lungs were isolated and imaged as intact organ with a confocal microscope. Arrowheads indicate direct contact between tumor cells and GFP⁺ cells. Occasionally, clots were also observed associated with myeloid cells (star). Representative images from 3 independent experiments performed per time point. Ripley test was performed to analyze clustering of CX₃CR1-GFP cells during the time course. y-axis represents the sum of K(r)/area (r) (K = Ripley K, r = distance range 5-100 pixels). Clustering of CX₃CR1-GFP cells around tumor cells, peaking at 8 hours, was observed in 2 other independent experiments. (B) The fractalkine receptor is not involved in recruitment to tumor cells. Cx₃cr1^gfp/+ (top panels) or Cx₃cr1^gfp/gfp mice (middle panels) were intravenously injected with 5 × 10⁵ CMAC-stained B16F10-wt cells (blue) and, on the opposite tail vein, with 9 × 10⁹ PKH26-stained platelets (red), isolated from C57BL/6 mice. At 8 hours, lungs were isolated and imaged as intact organ with a confocal microscope. Representative images from 3 independent experiments performed are shown. Clot formation on tumor cells, recruitment of myeloid cells (green), and number of tumor cells observed per field were scored; n = 3 mice (Mann-Whitney). (C) Clot formation is required for subsequent myeloid cell recruitment. Myeloid cell (green) recruitment to tumor cells was assessed from images obtained from experiments described in Figure 1D and F. Representative images with B16F10-TFPI cells (blue; red, platelets; 8 hours after injection; top panels) and hirudin-treated mice (20 mg/kg; blue, tumor cell; red, platelets; middle panels) from 3 independent experiments are shown. Recruitment of myeloid cells to B16F10-wt and B16F10-TFPI tumor cells was scored at the indicated times (bottom left panel); n = 3 mice (Mann-Whitney). Recruitment of myeloid cells to tumor cells was scored in hirudin-treated mice (bottom right panel); n = 3 mice (Mann-Whitney). Scale bars represent 50 μm. White boxes define areas enlarged at right. (B-C) Data are mean + SD. *P < .05.