1D), we focused further study on these two subsets. Percentages of CD11b/Gr1mid and CD11b/Gr1low cells Sorafenib supplier in bone marrow, blood, and liver of tumor-bearing mice were analyzed at various time points during metastatic growth. Levels of CD11b/Gr1mid cells in bone marrow peaked at day 5, and decreased thereafter, which coincided with increasing levels in blood and liver.

Circulating and hepatic CD11b/Gr1mid cell numbers continued to rise by day 14 (Fig. 2B). In contrast, bone marrow and circulating CD11b/Gr1low cell numbers remained constant with time while increasing in the liver abruptly from day 12 (Fig. 2C). These results suggest that the CD11b/Gr1mid subset is recruited from bone marrow during development of liver metastasis, whereas the CD11b/Gr1low population

likely derived from expansion or differentiation of resident cells after metastases had established. To confirm the bone marrow origin of the CD11b/Gr1mid subset, GFP+ cells isolated from bone marrow of GFP transgenic mice were transferred intravenously into C57BL/6 mice 11 days after MC38 or PBS inoculation. Significantly more GFP+ bone marrow cells were found in MC38-inoculated tumor-bearing livers compared with PBS-inoculated controls (Fig. 2D). These GFP+ cells were in the peritumoral Fulvestrant supplier regions of liver metastases (Fig. 2E), and were CD11b+, CCR2+, and F4/80+ (Fig. 2F), markers expressed only by the CD11b/Gr1mid population. To investigate whether similar CD11b/Gr1mid and CD11b/Gr1low subsets are associated with liver metastasis of other cancer cell lines, we inoculated B16F1GFP+ and LLCGFP+ cells into C57BL/6 mice. Metastases were observed in the liver at day 14 when myeloid infiltrates were assessed. Formation of LLCGFP+ tumor colonies resulted

in a significant increase in the Tau-protein kinase CD11b/Gr1mid population, similar in extent to MC38GFP+ inoculation. In contrast, CD11b/Gr1mid cell numbers were not significantly altered after B16F1GFP+ colonization (Fig. 3A). LLCGFP+ inoculation also led to a substantial increase in CD11b/Gr1low cell numbers, whereas moderate increases were observed after B16F1GFP+ and MC38GFP+ inoculation (Supporting Fig. 3C). Thus, LLCGFP+ colonization was analogous to that of MC38GFP+ in recruiting CD11b/Gr1mid cells, whereas this recruitment was dispensable for B16F1GFP+ cells. To identify factors involved in recruitment of bone marrow-derived CD11b/Gr1mid cells to liver metastases, we compared the cytokine expression profile of MC38, B16F1, and LLC cells. MC38 cells expressed high levels of CCL2 and moderate levels of CXCL1, CXCL10, and tissue inhibitor of metalloproteinase 1 (TIMP-1). Moderate levels of CCL2, CXCL1, and TIMP-1 were also detected in culture medium of LLC cells. B16F1 cells produced moderate levels of CXCL10 and CCL5 but CCL2 was not detected (Fig. 3B). Additionally, we tested another B16 melanoma variant cell line, B16F10, and found it to have a similar cytokine expression profile as B16F1 (Supporting Fig.