When crossed to CD11b-Cre mice, expressing Cre recombinase under the control of the myeloid specific CD11b promoter, Cre-mediated excision of the lacZ gene/stop cassette induced permanent GFP expression exclusively in cells having passed www.selleckchem.com/products/Belinostat.html through a CD11b-positive, myeloid stage [31]. Pancreatic sections of transplanted RT2;VC were stained for the lymphatic markers Podoplanin or LYVE-1 and for GFP, and double-positive cells were scored. In both transplantation settings, GFP+ cells were found integrated into the tumor lymphatic vasculature, demonstrating that cells of the myeloid lineage physically contributed to tumor lymphangiogenesis (Figure 4B, upper panels). We also tested whether CD11b+ cells integrated into tumor lymphatics without prior bone marrow transplantation by transplanting TRAMP-C1 cells into CD11b-Cre;Z/EG mice (Figure 1B).

Specific Cre-mediated recombination within the myeloid lineage of these mice was confirmed by FACS analysis of PB cells (Figure S3B). In the resulting tumors, GFP+ cells were found incorporated into LYVE-1+ and Podoplanin+ lymphatic vessel lining (data not shown). Triple-staining for LYVE-1, Prox-1 and GFP further showed that formerly myeloid cells expressed two lymphatic markers simultaneously (Figure 4C, arrowhead), indicating a significant differentiation towards a lymphatic endothelial phenotype. These results also revealed that integration occurred independently of prior irradiation, which had been previously reported to increase macrophage infiltration in human cancer [32]. In a second series of lineage-tracing experiments, FACS-sorted CD11b+/GFP+ cells were i.

v. injected into semi-lethally or non-irradiated RT2;VC mice (Figure 1A and C). 3 weeks after injection, adoptively transferred GFP+ cells were observed integrated into tumor lymphatics, identified by LYVE-1 or Prox-1 expression (Figure 4B, lower panels). The fact that the adoptive transfer of CD11b+/GFP+ cells into non-irradiated RT2;VC mice resulted into an integration of the injected cells into tumor lymphatics indicates that a full reconstitution of the hematopoietic system by stem cells is not a prerequisite for BMDC contribution to tumor lymphangiogenesis. To assess whether common myeloid progenitor cells (CMP) [33] provide the cells that incorporate into tumor lymphatics, FACS-sorted CMP cells (lin?/Sca-1?/IL7R��?/cKit+/GFP+; Figure 1E) were adoptively transferred into semi-lethally irradiated RT2;VC mice.

FACS analysis of PB cells 3 weeks post-injection revealed that transplanted CMP contributed to the generation of CD11b+/F4/80+ monocytes and CD11b+/F4/80? granulocytes but not to CD19+ B lymphocytes or CD3+ T lymphocytes (Figure S3C). Also here, GFP+ cells were found integrated into tumor-associated lymphatic endothelium, detected Anacetrapib by LYVE-1 or Podoplanin expression (Figure S4).