CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma Jennifer Eom 1,2 , Saem Mul Park 1,2 , Vaughan Feisst 1,2 , Chun-Jen J. Chen 1,2 , Joanna E. Mathy 1,2 , Julie D. McIntosh 1,2 , Catherine E. Angel 1,2 , Adam Bartlett 2,3 , Richard Martin 4 , Jon A. Mathy 3,5 , Jonathan S. Cebon 6 , Michael A. Black 2,7 , Anna E.S. Brooks 1,2 , and P. Rod Dunbar 1,2 ABSTRACT ◥ Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases mod- ulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90 þ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90 þ podoplanin þ CD105 þ CD146 þ CD271 þ VCAM-1 þ ICAM-1 þ a-SMA þ ) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90 þ CD34 þ CD105 þ CD271 þ ) represents a novel population of CD34 þ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demon- strated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90 þ CD146 þ CD36 þ NG2  pericytes in the walls of high endothelial venules and other small vessels, and CD90 þ CD146 þ NG2 þ CD36  pericytes in the walls of larger vessels. Distinguishing between these CD90 þ SC sub- populations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes. Introduction Cancer cells can enter lymphatic vessels and travel to lymph nodes (LN) where they can form LN metastasis. In normal LNs, different populations of stromal cells (SC) of mesenchymal origin, including fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), and marginal reticular cells (MRC), construct an elaborate LN architecture, supporting various LN functions (1–6). For example, FRCs produce factors that support T-cell survival and form a conduit system deliv- ering small antigens and signaling molecules to immune cells within the LN (7, 8). However, following tumor infiltration into LNs, tumor- subverted SCs contribute to tumor progression by mechanisms like the production of tumor growth factors, stimulation of angiogenesis, and suppression antitumor immune responses (9–11). Although nine or more distinct nonendothelial SC subsets have been identified in murine LNs (12, 13), it remains unclear how many SC subpopulations are present in human LNs and how they respond to tumor infiltration, in part, due to the technical challenges of isolating SCs ex vivo and the scarcity of human LNs for such studies (14, 15). Earlier work in human LNs revealed the complex heterogeneity within lymphatic endothelial cells and identified the unique characteristics of MRCs, highlighting key differences between murine and human LN SCs (16, 17). Here, we further dissected the diversity within human LN SCs, and identified the presence of additional SC subpopulations (CD34 þ SCs and subsets of pericytes) that were previously unchar- acterized. We demonstrated the presence of distinct subsets of tumor- associated CD90 þ SCs including FRCs and CD34 þ SCs in melanoma- infiltrated LNs (MILN), which differ from their counterparts in normal LNs. Finally, we showed that FRCs and CD34 þ SCs in MILNs displayed transcriptionally distinctive profiles, although both of them can express fibroblast activation protein (FAP), a widely used marker to identify FRCs in murine LNs and cancer-associated fibroblasts (CAF) in human cancers. Collectively, our findings demonstrated that the composition of human LN stroma is more complex than previously appreciated and that melanoma infiltration induced changes in LN SCs. Our data also revealed heterogeneity within the SCs in melanoma metastases and provide a molecular map to precisely identify distinctive SC subpopulations. Materials and Methods LN tissue and cell samples Normal LNs with mild reactive changes (18, 19) were excised from the axillary, inguinal, cervical, mesenteric, and mediastinum regions from 9 donors undergoing surgery. MILNs were obtained from 11 patients with metastatic melanoma undergoing elective surgery, and clinical details of the MILNs studied are available in Supplementary Table S1. Written informed consent from patients or next of kin was obtained in accordance with the Declaration of Helsinki, under 1 School of Biological Sciences, University of Auckland, Auckland, New Zealand. 2 Maurice Wilkins Centre, University of Auckland, Auckland, New Zealand. 3 Department of Surgery, Faculty of Medical Health Sciences, University of Auckland, Auckland, New Zealand. 4 Department of Surgery, Waitemata District Health Board, Auckland, New Zealand. 5 Auckland Regional Plastic, Reconstruc- tive & Hand Surgery Unit, Auckland, New Zealand. 6 Olivia Newton-John Cancer Research Institute, La Trobe University School of Cancer Medicine, Heidelberg, Victoria, Australia. 7 Department of Biochemistry, University of Otago, Dunedin, New Zealand. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). J. Eom and S.M. Park contributed equally to this article. A.E.S. Brooks and P.R. Dunbar contributed equally as co-senior authors of this article. Corresponding Author: P. Rod Dunbar, University of Auckland, 3a Symonds Street, Auckland 1003, New Zealand. Phone: 649-373-7599; Fax: 649-308-2317; E-mail: r.dunbar@auckland.ac.nz Cancer Immunol Res 2020;8:990–1003 doi: 10.1158/2326-6066.CIR-19-0796 Ó2020 American Association for Cancer Research. AACRJournals.org | 990 Downloaded from http://aacrjournals.org/cancerimmunolres/article-pdf/8/8/990/2356819/990.pdf by guest on 16 June 2022