However, tumor cell numbers in MC38CCL2 KD-inoculated mice were comparable to controls by day 13 (Fig. 5A), mirroring the

lack of difference in CD11b/Gr1mid recruitment at this time point. Likewise, fewer MC38GFP+ cells were detected in CCR2 KO mice compared with controls (Fig. 5B), although the differences were not as striking. Overall, decreased accumulation of CD11b/Gr1mid and CD11b/Gr1low cells in liver metastases caused a substantial reduction in tumor burden. In an attempt to deplete the CD11b/Gr1mid and CD11b/Gr1low subsets, CD11b-DTR mice bearing a human diphtheria toxin receptor https://www.selleckchem.com/products/Bortezomib.html (DTR) transgene driven by a CD11b promoter were used. Here, conditional ablation of CD11b+ cells can be achieved by diphtheria toxin (DT) administration.18

DT was administered to CD11b-DTR mice on day 7 and 9 after MC38GFP+ inoculation, a time when metastatic colonies had formed, and mice were sacrificed on day 11. DT administration markedly depleted CD11b/Gr1mid and CD11b/Gr1low cells in the liver compared with treatment with PBS (Supporting Fig. 4A) and had little effects on levels of T (CD3+) or B (CD19+) cells (Supporting Fig. 4B). Neutrophils were shown to be unaffected by DT in CD11b-DTR mice19 and numbers of CD11b/Gr1high cells were similarly unaffected (Supporting Fig. 4A). Livers of control mice had large metastatic colonies, whereas metastases were much smaller in DT-treated mice (Supporting Fig. 4C) and correspondingly, markedly fewer MC38GFP+ tumor cells were detected in livers of DT-treated mice (Fig. learn more 5C). Administration of DT to wild-type C57BL/6 mice did not deplete CD11b/Gr1mid cells, affect the number of MC38GFP+ cells in the liver, or the formation of liver metastases compared with controls (Supporting Fig. 4D-F). Tumor cell proliferation was assessed by staining liver tissue sections of CD11b-DTR mice after DT or PBS treatment. A pronounced two-fold reduction in both bromodeoxyuridine out incorporation (BrDu) and Ki67-positive cells (Fig. 5D,E) were observed

after DT treatment compared with controls. Overall, depletion of the CD11b/Gr1mid and CD11b/Gr1low subsets minimized metastatic growth, causing an appreciable reduction in tumor burden. We considered the possibility that CD11b+ cell depletion could instigate an adaptive immune response leading to decreased tumor burden. However, T cell and B cell numbers were comparable between DT-treated CD11b-DTR mice and controls (Supporting Fig. 4B). We also assessed myeloid infiltrates 14 days after MC38GFP+ inoculation in SCID mice (Supporting Fig. 5) and found myeloid subsets similar to those observed in wild-type C57BL/6 mice (Fig. 5F). Taken together, these findings suggest that accumulation of the CD11b/Gr1mid and CD11b/Gr1low subsets and decreased tumor growth after their depletion did not involve an adaptive immune response.