Published OnlineFirst March 31, 2014; DOI: 10.1158/2326-6066.CIR-13-0209 Cancer Research Article Immunology Research The Tumor Microenvironment Shapes Lineage, Transcriptional, and Functional Diversity of Infiltrating Myeloid Cells Kutlu G. Elpek1, Viviana Cremasco1, Hua Shen4,5, Christopher J. Harvey1, Kai W. Wucherpfennig1, Daniel R. Goldstein4,5, Paul A. Monach2, and Shannon J. Turley1,3 Abstract Myeloid cells play important regulatory roles within the tumor environment by directly promoting tumor progression and modulating the function of tumor-infiltrating lymphocytes, and as such, they represent a potential therapeutic target for the treatment of cancer. Although distinct subsets of tumor-associated myeloid cells have been identified, a broader analysis of the complete myeloid cell landscape within individual tumors and also across different tumor types has been lacking. By establishing the developmental and transcriptomic signatures of infiltrating myeloid cells from multiple primary tumors, we found that tumor-associated macro- phages (TAM) and tumor-associated neutrophils (TAN), while present within all tumors analyzed, exhibited strikingly different frequencies, gene expression profiles, and functions across cancer types. We also evaluated the impact of anatomic location and circulating factors on the myeloid cell composition of tumors. The makeup of the myeloid compartment was determined by the tumor microenvironment rather than the anatomic location of tumor development or tumor-derived circulating factors. Protumorigenic and hypoxia-associated genes were enriched in TAMs and TANs compared with splenic myeloid-derived suppressor cells. Although all TANs had an altered expression pattern of secretory effector molecules, in each tumor type they exhibited a unique cytokine, chemokine, and associated receptor expression profile. One such molecule, haptoglobin, was uniquely expressed by 4T1 TANs and identified as a possible diagnostic biomarker for tumors characterized by the accumulation of myeloid cells. Thus, we have identified considerable cancer-specific diversity in the lineage, gene expression, and function of tumor-infiltrating myeloid cells. Cancer Immunol Res; 2(7); 655–67. Ó2014 AACR. Introduction how they are recruited to and expand within growing tumors The tumor microenvironment contains a multiplicity of and metastases are of utmost importance to developing novel stromal cells of hematopoietic and nonhematopoietic develop- therapies and improving existing ones against cancer. mental origin, such as T cells, B cells, natural killer (NK) cells, Tumor growth is associated with the accumulation of a myeloid cells, fibroblasts, pericytes, adipocytes, and endothelial variety of myeloid cell types (2). Common myeloid cell pro- cells, which collectively shape the disease course (1–4). Although genitors in the bone marrow can give rise to myeloid cells with fi fi immunosuppressive potential, often referred to as myeloid- speci c roles have been identi ed for discrete stromal subsets, þ derived suppressor cells (MDSC). Monocyte-like CD11b factors controlling their recruitment, expansion, and function in low þ hi different tumors remain enigmatic. Therefore, a more complete Gr1 and granulocyte/neutrophil-like CD11b Gr1 subsets characterization of these subsets and a better understanding of of MDSCs have been reported to accumulate in the spleen, liver, blood, and bone marrow during tumor progression. Within tumors, myeloid cells with similar phenotypes are Authors' Affiliations: 1Department of Cancer Immunology and AIDS, referred to as tumor-associated macrophages (TAM) or neu- 2 Dana-Farber Cancer Institute; Boston University School of Medicine; trophils (TAN), possibly reflecting a more differentiated iden- 3Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts; Departments of 4Internal Medicine and tity. In vitro studies have shown that differentiation of bone 5Immunobiology, Yale School of Medicine, New Haven, Connecticut marrow progenitors into MDSCs requires a combination of Note: Supplementary data for this article are available at Cancer Immu- cytokines, particularly interleukin (IL)-6 and granulocyte col- nology Research Online (http://cancerimmunolres.aacrjournals.org/). ony-stimulating factor (G-CSF) or granulocyte macrophage Current address for K.G. Elpek: Jounce Therapeutics, Inc., Cambridge, MA colony-stimulating factor (GM-CSF), and the transcriptional 02138. regulator CCAAT/enhancer-binding protein (C/EBP; ref. 5). Corresponding Author: Shannon J. Turley, Dana-Farber Cancer Institute, Although splenic MDSCs are considered a reservoir for tumor- 44 Binney Street, D1440a, Boston, MA 02115. Phone: 617-632-4990; Fax: infiltrating myeloid cells (6), the exact relationship between 617-582-7999; E-mail: [email protected] these cells remains elusive. Accumulating evidence indicates doi: 10.1158/2326-6066.CIR-13-0209 that MDSCs, whether in the spleen or in the tumor, have direct Ó2014 American Association for Cancer Research. suppressive effects on cytotoxic leukocytes. In addition to www.aacrjournals.org 655 Downloaded from cancerimmunolres.aacrjournals.org on September 23, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst March 31, 2014; DOI: 10.1158/2326-6066.CIR-13-0209 Elpek et al. þ MDSCs, conventional and plasmacytoid dendritic cells (DC) PtenL/ ) were kindly provided by Dr. DePinho [while at the may exert immunoregulatory effects in tumors (2) using a Dana-Farber Cancer Institute (Boston, MA); currently at the variety of mediators such as indoleamine 2,3-dioxygenase MD Anderson Cancer Center (Houston, TX); ref. 11]. (IDO), inducible nitric oxide synthase (iNOS), and arginase I to suppress T-cell proliferation, cytotoxicity, and effector Cell lines and tumor models cytokine production. B16, EL4, 4T1, A20, and CT26 cells were obtained from Given that myeloid cells with protumorigenic and immu- American Type Culture Collection. Pan02 cells were obtained nomodulatory functions have been observed in multiple ani- from the NCI Developmental Therapeutics Program reposito- mal tumor models and in patients with cancer, they represent ry. The 4T07 cells were obtained from Dr. J. Lieberman (Har- important targets for immunotherapy. Efforts are under way to vard University, Cambridge, MA); they were expanded, ali- identify myeloid-focused strategies. Approved chemotherapy quoted, and banked in liquid nitrogen. No additional authen- agents, such as gemcitabine (7), 5-fluorouracil (8), and suni- tication was performed on these cells. The Her2 cell line was tinib (9), can eliminate or prevent the accumulation of MDSCs, derived from a spontaneous breast tumor in a Balb/c Her-2/ especially in lymphoid organs, and retard tumor progression. neu–transgenic mouse that was found to lose Her-2/neu Likewise, agents that block myeloid recruitment to tumors, expression in vitro. Transgenic rat Her-2/neu sequence was such as CSF1R inhibitors (10), hold clinical promise. However, inserted into a pMFG retroviral vector to obtain a stably to improve current strategies and identify new universal overexpressing cell line. Cell lines were maintained in Dulbec- targets for therapeutic intervention, it is essential to under- co's Modified Eagle Medium (B16 and EL4) or RPMI (4T1, Her2, stand how each myeloid cell subset from one tumor relates to A20, Pan02, CT26, and 4T07) supplemented with 10% FBS and the same population in other tumor types. 1Â penicillin/streptomycin. In this study, we analyzed myeloid subsets in multiple For subcutaneous tumors, 1 to 2.5 Â 105 cells were injected murine tumors to study how phenotype, frequency, and tran- into the flanks of na€ve mice (4T1, Her2, CT26, and 4T07 into scriptional profiles relate within different tumors, using triple- Balb/c; B16 and Pan02 into C57BL/6; A20 into Balb/cxCD45.1; þ negative 4T1 breast cancer, Her2 breast cancer, and B16 and EL4 into C57BL/6xCD45.1), and tumor growth was moni- melanoma as models. Strikingly, each tumor type contained tored using calipers until tumor size reached 0.5 to 1 cm diameter. a distinct myeloid cell landscape, with TAMs, TANs, and DCs For transgenic models, tumors were monitored and analyzed at represented in all tumors but at markedly different ratios, different time points based on tumor growth (RIP-Tag2 at 11–12 while systemic MDSC accumulation was exquisitely tumor- weeks, and PDA and Her2 transgenic at 11–24 weeks). specific. Our data suggest that tumor type, rather than ana- tomic location, dictates myeloid composition in the tumor Cell preparation and flow cytometry lesion. Furthermore, although each subset exhibits similar Spleens and tumors were processed by gentle mechanical transcriptional signatures associated with its identity in dif- disruption with forceps followed by enzymatic digestion using ferent tumors, our study demonstrates that functional differ- 0.2 mg/mL collagenase P (Roche), 0.8 mg/mL dispase (Invitro- ences exist across myeloid subsets from different tumors. In gen), and 0.1 mg/ML DNase I (Sigma). Tissues were incubated in particular, our data suggest that haptoglobin may represent a digestion medium at 37C for 30 minutes; released cells were biomarker for tumors characterized by systemic accumulation collected and filtered, and the remaining tissue was further pro- of myeloid cells. Thus, our study provides important insights cessed. Blood was collected into tubes containing 2 mmol/L into the identity and functional characteristics of tumor-asso- EDTA, and