Monocyte chemoattractant protein-1 blockade inhibits lung cancer tumor growth by altering macrophage phenotype and activating CD8+ cells

Abstract

The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non-small-cell lung cancer (NSCLC). Anti-murine-CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8(+) T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.

Figure 1.

Monocyte chemoattractant protein–1 (CCL2) blockade inhibits the growth of thoracic malignancies. (A and B) Mice (n = 5–8 for each group) bearing 200-mm³ flank tumors were treated with either saline (Control) or intraperitoneal α-CCL2/CCL12 monoclonal antibodies (mAbs) twice per week (a-CCL2). (A) Growth curves of tumors with these treatments in the non–small-cell lung cancer (NSCLC) cell line tissue culture 1 (TC1) (*P < 0.01). (B) Differences in tumor volume between control and α-CCL2–treated mice, at the time that the size of tumors in control mice was above 10% of their weight, in three NSCLC cell lines (TC1 and Lewis lung carcinoma [LLC] at 25 days, and lung cancer K-ras (LKR) at 40 days), and two mesothelioma cell lines (AB12 and AE17 at 35 days) (*P < 0.05 versus control for each). (C and D) Effects of α-CCL2 blockade were evaluated in two orthotopic NSCLC tumors: intravenous (IV) LLC (C) or a k-ras mutation model (D). Mice (n = 7–9 for each group) were treated with either saline (control) or intraperitoneal α-CCL2 mAb twice per week (a-CCL2). A trend toward increased survival was evident with CCL2 blockade.

Figure 2.

CCL2 blockade inhibited spontaneous metastases in a model of NSCLC. LKR-M flank tumors were allowed to reach an average size of 200–250 mm³. Mice were then treated with either saline (Control) or intraperitoneal α-CCL2/CCL12 mAbs twice weekly (a-CCL2). After 5 or 6 weeks of treatment, mice were evaluated for lung metastases. (A) Macroscopic (top) and hematoxylin-and-eosin–stained section (bottom) of lungs from control or a-CCL2–treated mice. (B and C) Changes in lung weight compared with lungs from naive mice (B), and the percentages of lung occupied by metastases (C) in each group, showed a significant reduction in tumor burden in mice treated with a-CCL2. (D) Number of nodules in each of the two groups at 6 weeks. No significant difference was evident in the number of nodules. (B–D) Each dot represents one mouse. *P < 0.05.

Figure 3.

CCL2 blockade skews the ratio of M1/M2 tumor-associated macrophage (TAM) phenotype. Mice bearing large TC1 tumors were treated with either saline (Control) or intraperitoneal α-CCL2 mAb (A-CCL2). Tumors were analyzed when tumor volume curves started to diverge. (A) Percentages for each of three phenotypes: M0 (CD11b⁺/Ly6G⁻/F4/80⁻), M1 TAMs (CD11b⁺/Ly6G⁻/F4/80⁺/CD206⁻), and M2 TAMs (CD11b⁺/Ly6G⁻/F4/80⁺/CD206⁺) out of all tumor cells. The percentage of M2 TAMs was significantly reduced with CCL2 blockade, with a trend toward increased M0 TAMs. (B) Change in ratio of non-M2 to M2 macrophages showed an increased ratio in mice treated with a-CCL2. (C) Reduction was evident in mean fluorescence intensity (MFI) of the M2 TAM marker (the mannose receptor CD206) in mice treated with α-CCL2. (D) Fold changes were evident in the expression of mRNA of several known markers of M1/M2 TAMs in mice treated with α-CCL2, compared with control mice. Three markers of M2 TAMs (solid bars) were reduced to 55–66% of control levels in mice treated with α-CCL2, whereas the M1 marker inducible nitric oxide synthase (iNOS) (open bar) was increased by 15%.

Figure 4.

Effect of CCL2 blockade on thoracic malignancies is dependent on intratumoral CD8⁺ T cells. (A) CB-17 severe combined immunodeficient (SCID) mice (n = 5–6 for each group) bearing 200-mm³ TC1 tumors were treated with saline (Control) or intraperitoneal α-CCL2 mAb (a-CCL2). No difference in the pattern of tumor growth was evident between the two groups. (B) Immunocompetent C57bL/6 mice (n = 5–6 for each group) bearing large TC1 tumors were treated with: (1) saline (diamonds, Control); (2) intraperitoneal α-CCL2 mAb (a-CCL2) (squares, a-CCL2); (3) saline, and injected with 300 μg of an anti-CD8 mAb intraperitoneal twice weekly (crosses, a-CD8); and (4) α-CCL2 mAB and depletion of CD8⁺ cells (circles, a-CD8 a-CCL2). Control and a-CCL2 groups were treated with an intraperitoneal control IgG antibody. The effect of CCL2 blockade on tumor growth disappeared completely in mice depleted of CD8⁺ T cells. (C) SCID mice (n = 7–8 for each group) were injected on the right flank with the NSCLC cell line LKR-M. After flank tumors reached an average size of 200–250 mm³, mice were treated with either saline (Control) or intraperitoneal α-CCL2/CCL12 mAb twice weekly (a-CCL2). After 5 weeks of treatment, lungs were evaluated for metastases. The percentage of lung occupied by metastases did not differ between the two groups. Each dot represents one mouse. (D) Percentages of intratumoral CD8⁺ T cells expressing the two activation markers, CD25 (left) and 4-1BB (right). The percentage of activated cells out of all CD8⁺ T cells was doubled in mice treated with a-CCL2 mAb (*P < 0.05).