Bulk and Single-Cell Profiling of Breast Tumors Identifies TREM-1 as a Dominant Immune Suppressive Marker Associated With Poor Outcomes

Ashok K Pullikuth; Eric D Routh; Kip D Zimmerman; Julia Chifman; Jeff W Chou; Michael H Soike; Guangxu Jin; Jing Su; Qianqian Song; Michael A Black; Cristin Print; Davide Bedognetti; Marissa Howard-McNatt; Stacey S O'Neill; Alexandra Thomas; Carl D Langefeld; Alexander B Sigalov; Yong Lu; Lance D Miller

doi:10.3389/fonc.2021.734959

Bulk and Single-Cell Profiling of Breast Tumors Identifies TREM-1 as a Dominant Immune Suppressive Marker Associated With Poor Outcomes

Front Oncol. 2021 Dec 8:11:734959. doi: 10.3389/fonc.2021.734959. eCollection 2021.

Authors

Ashok K Pullikuth¹, Eric D Routh^{1

2}, Kip D Zimmerman³, Julia Chifman^{1

4}, Jeff W Chou^{5

6}, Michael H Soike⁷, Guangxu Jin^{1

6}, Jing Su^{5

8}, Qianqian Song^{1

9}, Michael A Black¹⁰, Cristin Print¹¹, Davide Bedognetti¹², Marissa Howard-McNatt¹³, Stacey S O'Neill^{6

14}, Alexandra Thomas^{6

15}, Carl D Langefeld^{5

6}, Alexander B Sigalov¹⁶, Yong Lu^{6

17}, Lance D Miller^{1

6}

Affiliations

¹ Department of Cancer Biology, Wake Forest School of Medicine, Winston Salem, NC, United States.
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
³ Center for Precision Medicine, Wake Forest School of Medicine, Winston Salem, NC, United States.
⁴ Department of Mathematics and Statistics, American University, Washington, DC, United States.
⁵ Department of Biostatistics and Data Science, Wake Forest School of Medicine, Winston Salem, NC, United States.
⁶ The Comprehensive Cancer Center of Wake Forest University, Winston Salem, NC, United States.
⁷ Department of Radiation Oncology, University of Alabama-Birmingham, Birmingham, AL, United States.
⁸ Department of Biostatistics and Health Data Science, Indiana University School of Medicine, Indianapolis, IN, United States.
⁹ Center for Cancer Genomics and Precision Oncology, Wake Forest School of Medicine, Winston Salem, NC, United States.
¹⁰ Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
¹¹ Department of Molecular Medicine and Pathology and Maurice Wilkins Institute, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.
¹² Cancer Program, Sidra Medicine, Doha, Qatar & Department of Internal Medicine and Medical Specialties, University of Genoa, Genoa, Italy.
¹³ Surgical Oncology Service, Department of Surgery, Wake Forest School of Medicine, Winston Salem, NC, United States.
¹⁴ Department of Pathology, Wake Forest School of Medicine, Winston Salem, NC, United States.
¹⁵ Section of Hematology and Oncology, Department of Internal Medicine, Wake Forest Baptist Medical Center, Winston Salem, NC, United States.
¹⁶ SignaBlok, Inc. Shrewsbury, MA, United States.
¹⁷ Department of Microbiology & Immunology, Wake Forest School of Medicine, Winston Salem, NC, United States.

Abstract

Background: Triggering receptor expressed on myeloid cells (TREM)-1 is a key mediator of innate immunity previously associated with the severity of inflammatory disorders, and more recently, the inferior survival of lung and liver cancer patients. Here, we investigated the prognostic impact and immunological correlates of TREM1 expression in breast tumors.

Methods: Breast tumor microarray and RNAseq expression profiles (n=4,364 tumors) were analyzed for associations between gene expression, tumor immune subtypes, distant metastasis-free survival (DMFS) and clinical response to neoadjuvant chemotherapy (NAC). Single-cell (sc)RNAseq was performed using the 10X Genomics platform. Statistical associations were assessed by logistic regression, Cox regression, Kaplan-Meier analysis, Spearman correlation, Student's t-test and Chi-square test.

Results: In pre-treatment biopsies, TREM1 and known TREM-1 inducible cytokines (IL1B, IL8) were discovered by a statistical ranking procedure as top genes for which high expression was associated with reduced response to NAC, but only in the context of immunologically "hot" tumors otherwise associated with a high NAC response rate. In surgical specimens, TREM1 expression varied among tumor molecular subtypes, with highest expression in the more aggressive subtypes (Basal-like, HER2-E). High TREM1 significantly and reproducibly associated with inferior distant metastasis-free survival (DMFS), independent of conventional prognostic markers. Notably, the association between high TREM1 and inferior DMFS was most prominent in the subset of immunogenic tumors that exhibited the immunologically hot phenotype and otherwise associated with superior DMFS. Further observations from bulk and single-cell RNAseq analyses indicated that TREM1 expression was significantly enriched in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and M2-like macrophages, and correlated with downstream transcriptional targets of TREM-1 (IL8, IL-1B, IL6, MCP-1, SPP1, IL1RN, INHBA) which have been previously associated with pro-tumorigenic and immunosuppressive functions.

Conclusions: Together, these findings indicate that increased TREM1 expression is prognostic of inferior breast cancer outcomes and may contribute to myeloid-mediated breast cancer progression and immune suppression.

Keywords: TREM-1; breast cancer; cytokines; immune signature; immune suppression; transcriptomics; tumor infiltrating myeloid cells.