Bone marrow derived CX₃CR1+ cells are drivers of tumor angiogenesis.

<p>(<b>A</b>) FACS analysis of <i>cx₃cr1</i>^gfp cells purified by FACSAria Cell-Sorting System from BM of CCR2+ CD45.1 donor mice before their transfer to CCR2−/− mice (<b>B</b>) Imaging (IVIS) of the primary tumor on day 60, as recorded by the IVIS camera using – luciferin filter (recording luciferase activity of the cancer cells) as follows: CCR2+/+ C57BL/6 mice (WT) (a), CCR2−/− mice (b), CCR2−/− transplanted with GFP+ cells from BM of CCR2+ donor mice (c). All photos show a representative mouse per group (1 of 6 mice). (<b>C</b>) Computerized CCCD analysis of six mice per group. Results of six mice per group are shown as total flux (p/s ×10⁴) ±SE. * Indicates p<0.001. (<b>D</b>) Representative primary tumor sections were then analyzed by to immunostaining using different colors for CD45.1 (red color, only transferred <i>cx₃cr1</i>^gfp cells) and CD11b+ (green). (<b>E</b>) Analysis of 60 sections from six mice per group for the relative number of CD11b+ cells at tumor sections from each group, and of CD45.1 cells following cell transfer * Indicates p<0.001.</p

<i>Bone marrow derived CD11b+CCR2+ cells are essential to support tumor development and angiogenesis.</i>

<p>(<b>A</b>) CD11b+ BMD cells from <i>cx₃cr1</i>^gfp CCR2+ CD45.1 mice were purified (left panel), analyzed fro the relative mummer of GFP+ cells (right panel) and transferred to CCR2−/− mice bearing CCR2+ tumor (<b>B</b>) shows imaging (IVIS) of a representative mouse as recorded using a GFP filter. (<b>C</b>) Imaging (IVIS) of the primary tumor on day 60, as recorded by the IVIS camera using – luciferin filter (recording luciferase activity of the cancer cells) as follows: CCR2+/+ C57BL/6 mice (WT) (a), CCR2−/− mice (b), CCR2−/− transplanted with BM of WT mice(c) and CCR2−/− transplanted with BM of CCR2−/−mice. All photos show a representative mouse per group (1 of 6 mice). (<b>D</b>) The computerized CCCD analysis of six mice per group. Results are shown as total flux (p/s ×10⁴) ±SE. * Indicates p<0.001 (<b>E</b>) Histological, Immunohistochemical and immunofluorescence analyses of primary tumors from CCR2^+/+ C57BL/6 mice (WT), CCR2^−/− C57BL/6 mice and BM transplanted CCR2^−/− mice. Panel a–c show H&E staining, d–f show anti -PCNA staining, g–i show anti F4/80, j–l show anti VEGF and m–o show anti CD31.</p

mE3-mIg inhibits the development of primary tumor in CCR2^−/− mice.

<p>(A) Three groups of CCR2^−/− and one of C57BL/6 mice were administered 7×10⁶TRAMP C1-luc cells. 25 days later, mice were repeatedly administered (every 3 days) with 200 µg mE3-Ig, isotype-matched control mIgG or PBS and monitored for the development of the primary tumor. Results are shown as tumor volume ± SE. * Indicates p<0.001. (B) Imaging of the primary tumor was done on day 65, as recorded by the CCD camera(IVIS).Panels a, b & c show representative photos of a CCR2^+/+ C57BL/6 mouse (a), CCR2^−/− C57BL/6 mouse (b) and CCR2−/− mouse treated with mE3-mIg (c) which were i.p injected with 200 µl luciferin 5 min before the exposure . (C) Summery of the computerized CCCD analysis of six mice per group of control mice (WT), CCR2^−/− mice and those treated with mE3-mIg. Results are shown as total flux (p/s ×10⁴) ±SE. * Indicates p<0.001 when comparing b and c to a, ** Indicates p<0.001 p<0.005 c to b.</p

CCR2−/− mice display impaired development of CCR2+ primary tumors that become non-metastatic.

<p>(<b>A</b>) Six CCR2^+/+ C57BL/6 mice (WT) and six CCR2^−/− C57BL/6 mice were administered with 7×10⁶ TRAMP C1-luc cells. Imaging of primary tumor was done on day 60, as recorded by the CCD camera (IVIS). Panels a & b show representative photos of CCR2^+/+ C57BL/6 mice (WT) (a) and CCR2^−/− C57BL/6 mice (b) which were i.p injected with 200 µl luciferin 5 min before the exposure. (<b>B</b>) Computerized CCCD analysis of six mice per group. Results of six mice per group are shown as total flux (p/s ×10⁴) ±SE. * Indicates p<0.001. (<b>C</b>) Starting day 25, the two groups of mice were monitored for the development of the primary tumor. Results are shown as tumor volume ± SE. (<b>D</b>) Micro-metastases luminometer analysis of luc+ counts in organ sections obtained on day 50 from brain, heart, lungs, bones and primary tumor of CCR2+/+ C57BL/6 mice (WT) and CCR2−/− C57BL/6 mice administrated with 7×10⁶ C1-luc cells i.v, and the same number of cells s.c. to form primary tumor. Results are shown as mean relative light units per µg total protein, 9RLU/µg) ±SE. * Indicates p<0.001 (<b>E</b>) Histological and Immunohistochemical analyses of primary tumors from CCR2^+/+ C57BL/6 mice (WT) and CCR2^−/− C57BL/6 mice. Panels a, b show H&E staining (×10) taken by fluorescence microscope, c–f show anti -PCNA staining; c, d (×10), e, f (×40). (<b>F</b>) Immunohistochemical and immunofluorescence analysis of primary tumors from CCR2^+/+ C57BL/6 mice (WT) and CCR2^−/− C57BL/6 mice. Panels a–d show anti F4/80 staining; a, b (×10), c, d (×40) , e–h show anti VEGF staining; e, f (×10), g, h (×40) and i–j show anti CD31 staining (×40).</p

Exposing tumor cells to anti-VEGF-A antibodies reduces the level of VEGF-A in TMPs without affecting the number of TMPs.

<p>(A) A representative flow cytometry dotplot for TMP quantification. TMPs are approximately 1 µm, and counting beads are 7.35 µm. The number of TMPs per sample was calculated as the ratio between the number of events collected in the counting beads gate and the number of events collected in the TMPs gate over the total number of counting beads loaded in the sample. (B) EMT/6, 4T1 and MDA-MB231 breast carcinoma cells were either left untreated or exposed to 2 µg/ml B20 antibody for 48 h. TMPs were purified from conditioned medium and quantified by flow cytometry. Shown are the means ± S.D. of triplicates. (C) An equal number of TMPs (100,000) from untreated or B-20-exposed EMT/6, 4T1 and MDA-MB231 breast carcinoma cells were used to quantify the level of VEGF-A by ELISA. In some experiments control for B20 antibodies was used in a form of IgG in culture. Shown are the means ± S.D. of triplicates. **, 0.01>p>0.001.</p

Representative flow cytometry plots of viable CEPs, hemangiocytes, and myeloid derived suppressor cells.

<p>An example of the analysis of flow cytometry data obtained from peripheral blood of BALB/c mice is presented. Viable CEPs are determined as (a) positive for VEGFR2 and negative for CD45 as well as (b) positive for CD117 and negative for 7-AAD. Hemangiocytes are determined as (c) positive for CD45 and CXCR4 as well as (d) positive for VEGFR1. Myeloid derived suppressor cells (MDSCs) are determined as positive for (e) both Gr-1 and CD11b.</p

TMPs from cells exposed anti-VEGF-A antibody do not promote angiogenesis in tumors.

<p>(A) Matrigel plugs containing an equal number of TMPs (0.5×10⁶) from untreated or B20-exposed EMT/6 cells were implanted into the flanks of 8–10 week old BALB/c mice. Matrigel plugs containing PBS were used as a negative control. Ten days later, plugs were removed and prepared as single cell suspensions. The extracted cells were immunostained for endothelial cells, hemangiocytes and MDSCs and analyzed by flow cytometry. Results are presented as the number of cells per 1 mg Matrigel. (B–E) Eight to ten week old BALB/c mice (n = 4 mice/group) were implanted with 0.5×10⁶ EMT/6 cells into the flanks. When tumors reached a size of approximately 50 mm³, injections with 0.5×10⁶ TMPs from untreated or B20-exposed EMT/6 cells were performed twice weekly. Control mice were injected with PBS. (B) Tumor growth was assessed by a Vernier caliper using the formula, width²×length×0.5. Tumors were removed at endpoint, and subsequently were either (C) stained for CD31 (in red), CD45 (in green), and Hoechst (in blue) for the evaluation of (D) microvessel density and perfusion (scale bar = 100 µm), or (E) prepared as single cell suspensions for the evaluation of MDSCs and hemangiocytes colonization of tumors using flow cytometry. **, 0.01>p>0.001; ***, p<0.001.</p

TMPs from cells exposed to anti-VEGF-A antibody do not induce viable CEP and hemangiocyte mobilization.

<p>(A) An equal number of TMPs (0.5×10⁶) from untreated (CONT) or B20-exposed EMT/6 cells was injected into the tail vein of 8–10 week old non-tumor bearing BALB/c mice (n = 4 mice/group). Control mice were injected with PBS (PBS). One hour later, blood was drawn from the retro-orbital sinus for the evaluation of viable CEPs (CD45−/VEGFR2+/CD117+/7AAD−), MDSCs (Gr1+/CD11b+), and hemangiocytes (CD11b+/CXCR4+/VEGFR1+) using flow cytometry. (B) Half a million TMPs from untreated (CONT) or B20-exposed cells were tagged with PKH26, and subsequently injected into the tail vein of BALB/c mice (n = 4 mice/group). Control mice were injected with PBS. One hour later, blood was drawn by cardiac puncture and total BMDCs (CD45+), viable CEPs, hemagiocytes, and MDSCs were analyzed by flow cytometry. The percentage of the different cell types positive for tagged TMPs was plotted. **, 0.01>p>0.001; ***, p<0.001.</p

TMPs from cells exposed to anti-VEGF-A antibody exhibit reduced ability to promote endothelial cell activity.

<p>Matrigel plugs containing an equal number of TMPs (0.5×10⁶) from untreated or B20-exposed EMT/6 cells were implanted into the flanks of 8–10 week old BALB/c mice. Matrigel plugs containing PBS were used as a negative control. Ten days later, plugs were removed and then sectioned. (A) Slides were stained with H&E or immunostained with the endothelial cell marker CD31 (designated in red) (scale bar = 100 µm). (B) Microvessel density in the plugs was calculated by counting vessel structures. (C–D) An equal number of TMPs (5×10⁶) from untreated or B20-exposed EMT/6 cells were tested for HUVEC migration (C) and invasion (D) using the modified Boyden chamber assay. PBS was used as a negative control. Cells invading the membrane of the Boyden chamber were stained with Crystal Violet and images were captured using a Leica CTR 6000 microscope. The number of cells invading the membrane were counted and plotted (n>8/group). (E) Aortic rings from BALB/c mice (n = 4/group) were cultured in medium containing 0.1×106 TMPs from untreated or B20-exposed EMT/6 cells. Endothelial cell medium (ECGS) was used as a positive control. Images were captured using an inverted light microscope system (Leica CTR 6000 system) (Scale bar = 500 µm). *, 0.05p>0.001; ***, p<0.001.</p

Identification of Dormancy-Associated MicroRNAs for the Design of Osteosarcoma-Targeted Dendritic Polyglycerol Nanopolyplexes

The presence of dormant, microscopic cancerous lesions poses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized pairs of dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93, and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients’ tumor samples compared to normal bone, making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge; hence, we synthesized an aminated polyglycerol dendritic nanocarrier, dPG-NH₂, and designed dPG-NH₂-microRNA polyplexes to target cancer. Reconstitution of these microRNAs using dPG-NH₂ polyplexes into Saos-2 and MG-63 cells, which generate fast-growing osteosarcomas, reduced the levels of their target genes, MET proto-oncogene, hypoxia-inducible factor 1α, and moesin, critical to cancer angiogenesis and cancer cells’ migration. We further demonstrate that these microRNAs attenuate the angiogenic capabilities of fast-growing osteosarcomas <i>in vitro</i> and <i>in vivo</i>. Treatment with each of these microRNAs using dPG-NH₂ significantly prolonged the dormancy period of fast-growing osteosarcomas <i>in vivo</i>. Taken together, these findings suggest that nanocarrier-mediated delivery of microRNAs involved in osteosarcoma tumor–host interactions can induce a dormant-like state