Abstract SY27-02: Heterogeneity of tumor-associated macrophages: Implications for antiangiogenic therapy
Authors: Palma Michele De , Naldini Luigi 
Published: May 2019

Abstract
Macrophages, mast cells, neutrophils and immature myeloid cells infiltrate developing tumors, where they seem to support angiogenesis by releasing factors that stimulate the growth and expansion of new blood vessels from the pre-existing vasculature (1). Given the heterogeneity of tumor-infiltrating myeloid cells, identifying the exact cell types involved in this process is proving increasingly difficult.
A classic example is the heterogeneity of tumor-associated macrophage (TAM) phenotypes seen in tumors (2). This may either reflect the existence of developmentally distinct subpopulations, or the influence of signals in the tumor microenvironment like hypoxia and/or various cytokines on a common monocyte precursor (or the combination of both). The type and concentration of cytokines and growth factors expressed in the tumor microenvironment may shape the phenotype and function of TAMs. This concept is exemplified by the “M1-M2” macrophage polarization paradigm (3), according to which TAMs may be driven by certain tumor- and T cell-derived cytokines (e.g. IL-4) to acquire a polarized “M2” phenotype, which favors tissue remodeling, angiogenesis, suppression of anti-tumor immunity and tumor cell dissemination to distant organs (4). Similarly, certain tumor-derived factors may drive neutrophils to acquire pro-tumoral and pro-angiogenic functions (5). This implies that neutralizing endogenous M2-polarizing molecules, as well as expressing M1-polarizing factors exogenously (6), may skew tumor-infiltrating myeloid cells toward an “M1” phenotype and blunt their pro-angiogenic and pro-tumoral activities. On the other hand, our recent studies suggest that, in mouse tumors, only a specific macrophage subset displays a profoundly “M2” skewed, pro-angiogenic phenotype, the Tie2-expressing macrophages (TEMs) (7, 8). These cells are characterized by the high expression of mannose receptor-1 (MRC1) and aptoglobin/hemoglobin scavenger receptor (CD163), and the low expression of several pro-inflammatory and anti-angiogenic molecules, such as IL-1beta, COX2, iNOS and IL-12 (as compared to other TAMs). In contrast, a substantial fraction of Tie2-negative TAMs express CD11c and produce higher amounts of pro-inflammatory molecules and lower amounts of pro-angiogenic factors (8). Our studies further indicate that different ratios of MRC1+ TEMs and CD11c+ TAMs are present in different tumor types. For instance, in the highly aggressive and metastatic Lewis lung carcinoma (LLC), a prominent proportion of TAMs express a MRC1+CD11c- (TEM) phenotype; conversely, in the slowing growing and non-metastatic N202 mammary carcinoma, the vast majority of TAMs express a MRC1-CD11c+ phenotype. In spontaneous MMTV-PyMT mammary carcinomas, the ratio between MRC1+ TEMs and CD11c+ TAMs changes along with tumor development. We are currently investigating if TAM heterogeneity in distinct tumor models correlates with the angiogenic properties of the tumors.
Several reports have shown that both mouse and human monocytes can be grouped into functional subsets. In murine blood, these include the Gr-1+ 'inflammatory' monocytes, which can give rise to macrophages (possibly including TAMs) and DCs under inflammatory conditions; as well as Gr-1- 'resident' monocytes, which appear to patrol blood vessels, promote tissue remodeling and vascular healing, possibly by differentiating toward tissue-resident macrophages (9). The developmental relationships among the distinct monocyte subsets and TAMs are poorly defined. We recently proposed that circulating inflammatory and resident monocytes might give rise to two distinct TAM subpopulations, the MRC1-CD11c+ classic TAMs and the MRC1+CD11c- TEMs, respectively (8). Indeed, resident monocytes isolated from the blood of tumor-free mice display a gene expression profile that is more similar to tumor-derived TEMs than classic TAMs, whereas inflammatory monocytes are more closely related to TAMs than TEMs. These data might suggest that inflammatory and resident monocytes are pre-committed to distinct fates in the tumor microenvironment. By performing lineage tracking and adoptive transfer studies, we are currently investigating whether the distinct monocyte subsets give rise to distinct macrophage subpopulations in developing tumors.
Recent studies indicate that tumor refractoriness to both anti-angiogenic and cytotoxic therapies correlates with the marked accumulation of bone marrow-derived cells within the tumors (10, 11). The data suggest that some tumors may co-opt VEGF-independent, pro-angiogenic programs that are executed by tumor-infiltrating myeloid cells (10). Because TEMs express the angiopoietin receptor Tie2 and respond to Ang2 stimulation in vitro (12-14), we speculated that Ang2, which is secreted by angiogenic blood vessels, could regulate the attraction and/or activity of TEMs in tumors. We found that blocking Ang2 delays tumor progression in the MMTV-PyMT spontaneous breast cancer model and inhibits the growth of pulmonary metastases, both in early and late treatment trials. These results were confirmed in other tumor models, including human tumor xenografts. Interestingly, our data suggest that Ang2 regulates tumor progression by targeting both angiogenic blood vessels and tumor-infiltrating TEMs (Mazzieri, De Palma, Naldini, unpublished data). In collaboration with Claire Lewis and Gillian Tozer (University of Sheffield, UK), we studied the recruitment of TEMs and other TAM subpopulations to tumors following treatment with the vascular-disrupting agent, CA4-P. CA4-P induces a profound but reversible anti-tumor response in MMTV-PyMT mammary carcinomas. Interestingly, CA4-P-treated tumors contain increased numbers of Tie2+MMP9+ TEMs as compared to untreated tumors. We are currently investigating whether the selective elimination of TEMs by a conditional suicide gene strategy (7) delays tumor recovery and neo-angiogenesis following CA4-P treatment.
The promiscuous cell markers routinely used to identify myeloid cells in tumors, together with the limited availability of mouse models and experimental tools that allow dissecting the contribution of each of the distinct myeloid cells types involved, have limited our understanding of the role of myeloid cells in tumor angiogenesis. Both gene expression studies (8) and high-resolution, single-cell live imaging analysis (15, 16) of TAMs are unraveling an unexpected degree of functional heterogeneity, which has been anticipated only in part by standard immunophenotyping techniques. Assessing the specific contribution of distinct TAM subsets to tumor angiogenesis and progression may have important implications for the design of improved anticancer therapies.
References
1. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nature reviews 2008; 8: 618-31.
2. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer research 2006; 66: 605-12.
3. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature 2008; 454: 436-44.
4. DeNardo DG, Barreto JB, Andreu P, et al. CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer cell 2009; 16: 91-102.
5. Fridlender ZG, Sun J, Kim S, et al. Polarization of tumor-associated neutrophil phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer cell 2009; 16: 183-94.
6. De Palma M, Mazzieri R, Politi LS, et al. Tumor-targeted interferon-alpha delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis. Cancer cell 2008; 14: 299-311.
7. De Palma M, Venneri MA, Galli R, et al. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer cell 2005; 8: 211-26.
8. Pucci F, Venneri MA, Biziato D, et al. A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood "resident" monocytes, and embryonic macrophages suggests common functions and developmental relationships. Blood 2009; 114: 901-14.
9. Auffray C, Fogg D, Garfa M, et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science (New York, NY 2007; 317: 666-70.
10. Shojaei F, Ferrara N. Refractoriness to antivascular endothelial growth factor treatment: role of myeloid cells. Cancer research 2008; 68: 5501-4.
11. Shaked Y, Henke E, Roodhart JM, et al. Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer cell 2008; 14: 263-73.
12. Venneri MA, De Palma M, Ponzoni M, et al. Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer. Blood 2007; 109: 5276-85.
13. Murdoch C, Tazzyman S, Webster S, Lewis CE. Expression of Tie-2 by human monocytes and their responses to angiopoietin-2. J Immunol 2007; 178: 7405-11.
14. Lewis CE, De Palma M, Naldini L. Tie2-expressing monocytes and tumor angiogenesis: regulation by hypoxia and angiopoietin-2. Cancer research 2007; 67: 8429-32.
15. Egeblad M, Ewald AJ, Askautrud HA, et al. Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Disease models & mechanisms 2008; 1: 155-67.
16. Qian B, Deng Y, Im JH, et al. A distinct macrophage population mediates metastatic breast cancer cell extravasation, establishment and growth. PLoS One 2009; 4: e6562.
Citation Format: Michele De Palma, Luigi Naldini. Heterogeneity of tumor-associated macrophages: Implications for antiangiogenic therapy [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY27-02