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

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

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