In this study, we investigated how the tissue environment in which a solid tumor grows determines the effector cells contributing to therapeutic antibody–mediated control of tumor growth. Although it has become clear that myeloid cells play a key role for cytotoxic antibody–mediated depletion of normal and malignant B cells, for example, it is largely unknown how the environment of a solid tumor growing in different tissues affects the molecular and cellular pathways underlying antibody activity (30, 38, 39, 57).

By using a syngeneic melanoma model that allows us to induce tumor growth in the skin and the lung, we now show that different TAM subsets reside in melanoma tumors growing in different organ environments and that only select TAM subpopulations contribute to therapeutic antibody–dependent tumor cell killing.

In line with previous studies, the FcγR system was critical for therapeutic antibody activity irrespective of the organ in which the tumor grew (3639, 46, 47, 5760). However, the cell types responsible for antibody-dependent control of melanoma growth differed depending on the organ.

Thus, the recruitment of Ly6Chigh monocytes via the CCL2-CCR2 axis was critical for antibody-dependent depletion of melanoma and lymphoma cells in the skin, whereas the absence of CCR2 did not impair antibody-dependent depletion of melanoma cells in the lung.

Consistent with previous studies showing that recruitment of Ly6Chighmonocytes via CCR2 supports breast cancer metastasis in the lung (10, 11), we noted a reduced number of melanoma metastasis in the lung but not in the subcutaneous tumors, where a trend toward slightly larger tumor size became evident (Figs. 5 and 6). Thus, our results support the notion that, depending on the tissue environment, the recruitment of inflammatory monocytes via the CCL2-CCR2 axis can either promote tumor growth without contributing to antibody immunotherapy (in lung tumors) or may be a central component of antibody-mediated tumor rejection (in skin).

Consistent with the genetic data in CCR2-deficient animals, one distinguishing feature between the two tumor entities growing in a skin or lung environment was the much higher (or more sustained) level of CCL2 production in skin tumors and the predominant recruitment of TAM from the bone marrow (Figs. 3 and 5E). Although many tumor cells have been described to produce CCL2, B16F10 melanoma cells showed no autonomous production of this chemokine (3, 8, 61), which was rather produced by TAM. Functional evidence that bone marrow–derived cells are critical for controlling melanoma growth in the skin was provided by bone marrow chimeric mice in which the irradiation dose was titrated to predominantly exchange the hematopoietic system but to maintain tissue-resident macrophages in the skin. These studies demonstrated that activating FcγRs expressed on bone marrow–derived but not skin-resident macrophages were critical for therapeutic antibody activity.

Because the absence of neutrophils, mast cells, or basophils did not impair antibody activity and because the source of CCL2 was bone marrow–derived cells (Fig. 5F and fig. S2), a model in which inflammatory monocytes are recruited to the tumor where they differentiate into CCL2 producing TAM, which in turn leads to a positive feedback loop, seems likely. With respect to the lung, further studies will be necessary to investigate why CCL2 production by TAM is not maintained and why predominantly CD11chigh/CD68+/CD11bneg/MHCIIdim cells, most likely representing alveolar macrophages, can be found within the tumor. The titrated irradiation experiment to distinguish between bone marrow–derived and lung-resident macrophages did not yield clear results in the lung model, because alveolar macrophages (in contrast to skin-resident macrophages) became replaced by bone marrow–derived cells during the course of the experiment, consistent with previous results (44, 50, 56).

Arguing against an involvement of blood-derived monocytes, treatment with clodronate liposomes, which diminished blood monocyte numbers but retained alveolar macrophages, did not impair antibody activity (Fig. 6, D to F). To provide more direct evidence that alveolar macrophages contribute to antibody-dependent tumor immunotherapy, we further used Csfr2−/− mice deficient in granulocyte-monocyte CSF (GM-CSF) in which the development and function of alveolar macrophages are impaired (56). Supporting a model in which alveolar macrophages are responsible for controlling tumor growth in the lung, antibody-mediated control of lung tumors was impaired in GM-CSF–deficient mice, consistent with a reduced level of tumor-associated CD11chighCD68+ macrophages (Fig. 6, G to I).

However, caution is needed when interpreting results from inbred and knockout animal model systems, in which a general deletion of target genes occurs. Csfr2−/− mice, for example, not only show reduced lung-resident macrophage numbers but also develop a pronounced proteinosis during age, which may affect immigration of other immune cell subsets. Moreover, deletion of the GM-CSF receptor on Ly6Chigh monocytes has been shown to affect central nervous system inflammation in a model of experimental autoimmune encephalitis, suggesting that Ly6Chigh monocyte function may be altered (62).

In a similar manner, Gfi−/− mice show other alterations in immune system function apart from lacking mature neutrophils, such as altered B cell, T cell, and myeloid development (63). This may at least in part explain why an increase in tumor load was detectable in the lung model and why tumor eradication was not complete. Alternatively, we cannot completely rule out that neutrophils contribute at least in part in inhibiting tumor growth, as described previously (35). Despite the fact that we have noted a similar CCL2-dependent pathway underlying therapeutic antibody activity in two independent syngeneic skin tumor models, future studies in spontaneous tumor models growing in different locations will be necessary to identify which TAM subsets contribute to antibody-dependent tumor immunotherapy throughout the body. A previous study identified Kupffer cells as a possible effector cell population responsible for TA99-mediated depletion of melanoma metastasis in the liver (37). Thus, it seems plausible that, depending on the organ, either tissue-resident (lung and liver) or blood-derived (skin) TAM populations contribute to antitumor antibody activity. Last, future studies with samples of human tumors growing in the skin and the lung will need to show whether similar rules apply in the human system because immune cell subsets differ, especially those in the skin.

Together, our study shows that, despite a critical requirement of the same set of activating FcγRs for therapeutic antibody–dependent tumor cell killing, different cellular pathways were underlying tumor-specific antibody activity.

These results may suggest that the local tissue environment is a strong determinant of which immune effector cells contribute to tumor rejection by therapeutic antibodies and show that approaches that aim to deplete TAM subsets by targeting the CCL2-CCR2 pathway may also target an important effector cell population essential for therapeutic antibody activity at least if the tumor resides in the skin.

Our results may be helpful for optimizing clinical strategies that aim to limit the infiltration of tumor-supporting macrophages while maintaining TAM subsets that contribute to therapeutic antibody activity.