Dissecting the Autocrine and Paracrine Roles of the CCR2-CCL2 Axis in Tumor Survival and Angiogenesis

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX3CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX3CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX3CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx3cr1gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX3CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases

Correction: Dissecting the Autocrine and Paracrine Roles of the CCR2-CCL2 Axis in Tumor Survival and Angiogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0028305.]

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

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

β-mannosylceramide activates type I natural killer T cells to induce tumor immunity without inducing long-term functional anergy

PURPOSE: Most studies characterizing antitumor properties of iNKT cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, β-mannosylceramide (β-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether β-ManCer induces anergy of iNKT cells. EXPERIMENTAL DESIGN: Induction of anergy was determined by ex vivo analysis of splenocytes from mice pre-treated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model. RESULTS: β-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with β-ManCer, compared with α-GalCer. Moreover, while mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to two months, mice pretreated with β-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice. CONCLUSIONS: The lack of long-term functional anergy induced by β-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed. TRANSLATIONAL RELEVANCE: Activation of iNKT cells with α-galactosylceramide was very successful in preclinical mouse models of cancer; however, its success in clinical trials has been very limited. It has been very well-documented, that once iNKT cells are activated with α-galactosylceramide, they remain unresponsive to restimulation for months. This functional anergy could be a contributing factor to the failure of α-galactosylceramide clinically, as most therapeutics require multiple dosing to achieve maximum benefit. Here, we report that a different iNKT cell agonist, β-mannosylceramide, which is capable of inducing tumor immunity similarly to α-galactosylceramide but by a different mechanism, does not induce anergy. This suggests that β-mannosylceramide has the potential to work well clinically since it can be given in multiple doses without inducing anergy

Delicate Balance among Three Types of T Cells in Concurrent Regulation of Tumor Immunity

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here we addressed this question for Tregs and type II NKT cells in syngeneic models of colorectal and renal cancer. In mice with both type I and type II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Treg cells as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in three ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we demonstrated that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As cancer patients often have deficient type I NKT cell function, managing this delicate balance among three T cell subsets may be critical for the success of immunotherapy of human cancer