CRL-2505 prostate cancer cells promote CEP migration in vitro. (A): CRL-2505 CM and confluent CLs significantly promote the migration of DilC18-labeled CEPs (CEP-DilC18) in vitro compared with SW620 colon cancer cells and serum-free TCM controls (∗, p < .001). (B): In CL cultures, in addition to CEPs scored in suspension (arrow), a significant number of migrated CEP-DilC18s also extend cytoplasmic processes on the underlying tumor cell layer after 48 hours (magnification, ×20). (C): In SW620 CL cultures, only CEP-DilC18s in suspension are observed (magnification, ×20). Abbreviations: CEP, circulating endothelial precursor cell; CL, cell layer; CM, conditioned medium; CL, cell layer; TCM, tissue culture medium.

Immunophenotype of human CEPs expanded in vitro. (A): Schematic representation of surface antigenic changes in different characterized endothelial cell progenitor populations. Highly proliferative cell lineages are colored red. (B): Representative flow cytometric staining of highly proliferative, low and high passage CEPs show stable expression of CD146, CD141, CD31, and VEGFR2, low levels of CD34, and a lack of CD14, CD45, and CD133 antigen expression. Abbreviations: BOEC, blood outgrowth endothelial cell; CEC, circulating endothelial cell; CEP, circulating endothelial precursor cell; CFU-EC, colony forming unit-endothelial cell; ECFC, endothelial colony-forming cell; EPC, endothelial progenitor cell; HPC, hematopoietic progenitor cell.

Adenovirus construction and circulating endothelial precursor cell (CEP) infection. (A): Marker adenovirus infection of endothelial progenitor cells at a multiplicity of infection of 100 results in expression of red fluorescent protein in more than 74 ± 13% of cells assessed by flow cytometry and fluorescence microscopy. (B): An (s)CD115 receptor was constructed by cloning the ligand binding extracellular domains of CD115 proximal to the transmembrane domain (dotted line) into the pDNR donor vector as p(s)CD115 and inserted into an adeno-X adenoviral backbone as Ad(s)CD115. Both p(s)CD115 transiently transfected HEK 293 cells and Ad(s)CD115-infected CEPs secrete the 72-kDa (s)CD115 protein in vitro shown in representative Western blots. (C): Serum-free supernatants from p(s)CD115-transfected and Ad(s)CD115-infected cultures significantly inhibit CSF-1-driven proliferation of CRL-2470 mouse macrophages in vitro (∗, p < .001). (D): Representative images of the effect of p(s)CD115 supernatants on CRL-2470 are shown (magnification, ×20). Serum-free supernatants from Ad(s)CD115-infected CEPs significantly inhibit the migration of CRL-2470 in vitro (∗, p < .001). Abbreviations: CSF-1, colony-stimulating factor-1; (s), soluble.

CEPs preferentially home to CRL-2505 prostate cancer xenografts in vivo. CEP-DilC18s were introduced into mice bearing established CRL-2505 xenografts. (A): Representative flow cytometric side scatter plot analysis showing that 2.4 ± 1.3% of tumor material is comprised of CEP-DilC18s 3 days after administration compared with control xenografts. (B): Representative fluorescence microscopy images of exogenous CEP-DilC18s migrated to xenotransplanted CRL-2505 tumors shows CEP-DilC18s are found throughout the tumor (left panel; magnification, ×40; bar, 20 μm). (C): Representative images show that CEP-DilC18s colocalize with tumor cells staining for Ki-67⁺ (Alexa-fluor 488, green; nuclear counterstain, diamidino-2-phenylindole, blue) in the peritumoral space within 20 cell layers of the TB 13 days after injection. (D): In major organs, exogenous CEP-DilC18s are primarily found in the tumor (∗, p < .001) compared with all other organs after 13 days. (E): Detection of CEP-DilC18s in peripheral blood and tumors 36 and 72 hours after injection (∗, p = .03). (F): A representative fluorescent activated cell sorter histogram showing levels of CEP-DilC18s in peripheral blood after 72 hours compared with control mice. Abbreviations: CEP, circulating endothelial precursor cell; Cntl, control; TB, tumor boundary.

Cellular therapy targeting tumor-associated macrophages. (A): Intracardial injection of ringer solution (Cntl) and exogenous CEP-DilC18s infected with AdCntl or Ad(s)CD115 virus significantly influenced the growth of established CRL-2505 xenografts at the indicated time points (^‡, p < .05, ^†, p < .02; data ± SEM) after CEP-DilC18 injection. At termination on day 13 after adenovirus-infected CEP-DilC18 injection, a significant reduction in the tumor masses of CRL-2505 was observed in the Ad(s)CD115 group (∗, p < .03 compared with Cntl and AdCntl groups, data ± SEM; bar, 10 mm). (B): In CRL-2505 prostate tumors, representative images show TAMs staining positive for F4/80 antigen (Alexa-488, arrowheads) primarily localized in perivascular regions within the tumor in the vicinity of AC areas of extracellular matrix remodeling (left panel; magnification, ×100; bar, 20 μm) or in the peritumoral compartment (right panel; magnification, ×100; bar, 20 μm) in the vicinity of the TB colocalizing with CEP-DilC18. The autofluorescence of erythrocytes denotes the location of a tumoral capillary (nuclear counterstain diamidino-2-phenylindole). Quantification of F4/80⁺ TAM levels shows significant (p < .05) decreases in the Ad(s)CD115 group compared with the AdCntl group. (C): Representative images (magnification, ×40; bar, 20 μm) and quantification show an associated significant decrease (p < .01) in the numbers of proliferating Ki-67-positive cells (arrowheads) in the Ad(s)CD115 group compared with the AdCntl group. (D): Representative images (magnification, ×60; bar, 20 μm) of von Willebrand factor staining and quantification show a significant (p < .03) reduction in capillary densities in the Ad(s)CD115 group compared with the AdCntl group. (E): Kupffer cells isolated from AdCntl and Ad(s)CD115 groups show a similar degree of fluorescent latex bead phagocytosis (magnification, ×4; bar, 10 μm; in vitro) 3 days after CEP administration. Abbreviations: AC, acellular; CEP, endothelial precursor cell; Cntl, control; TB, tumor boundary.