Abstract 1738: PD-1/PD-L1 expression and tumor-infiltrating immune cells in triple-negative breast cancer: Characterization of preclinical primary tumor and bone metastasis models in humanized mice
Authors: Kähkönen Tiina E. , Suominen Mari I. , Halleen Jussi M. , Tanaka Azusa , Seiler Michael , Haapaniemi Teppo , Bernoulli Jenni 
Published: August 2018

Abstract
Expression of programmed cell death-1 receptor (PD-1), programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been shown in many studies supporting development of immunotherapies against TNBC. In order to develop novel immunotherapies especially against bone metastatic TNBC, better understanding of PD-1/PD-L1 axis, TILs, and tumor associated macrophages (TAMs) in the bone microenvironment is warranted. Aim of this study was to characterize immune cell reaction between TNBC primary tumor and bone metastasis in humanized mice to support immunotherapy drug discovery against bone metastasis.
MDA-MB-231(SA) human breast carcinoma cells were inoculated into mammary fat pad or tibia of female CIEA NOG mice engrafted with human pluripotent CD34+ cells and tumor growth was followed for 3 weeks. Immunohistochemical stainings of primary tumors and intratibial tumors were performed against human specific antigens to characterize expression of PD-1, PD-L1, Granzyme B, CD4, CD8 and CD68/CD163. TILs and TAMs were assessed by 4-scale immunoscoring system and PD-L1 expression was determined by Tumor Proportion Scoring (TPS). PD-L1 positivity of the TAM's was omitted in TPS interpretation.
Carcinoma cells of orthotopical tumors exhibited mainly low or moderate heterogeneous expression of PD-L1 (TPS score 1-49%). Moderate or high numbers of CD4 and CD8 positive TILs (scoring 2-3) and low PD-1 expression were observed. Granzyme B expression correlated with CD8 positivity. In comparison, when MDA-MB-231(SA) carcinoma cells were inoculated into bone marrow, tumors had corresponding PD-L1 expression and tumor-infiltrating CD4+ and granzyme B+ human immune cells were observed. However, in the bone microenvironment less CD8+ immune cells were observed and PD-1 expression was negative. When analyzing all tumor area, intratumoral and peritumoral variation of the marker expression and cell location of TILs and TAMs was observed especially in the bone tumors but also in the primary tumors. TILs and TAMs occurred in scattered as well as aggregate form in tumor area.
Taken together, TNBC primary tumors and bone metastases had mainly low or moderate, but clear membranous expression of PD-L1. TILs consisted of CD4+, CD8+, and granzyme B+ cells in the primary tumors, but less CD8+ cells were observed in the bone metastases. PD-1 expression was negative in bone metastases. The obtained results comparing primary tumor and bone metastasis highlights the importance of understanding the influence of tumor microenvironment. Immune system is not only strong regulator of cancer progression, but it also regulates bone turnover. Therefore, it is crucial to use predictive preclinical models when assessing novel immunotherapies.
Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Azusa Tanaka, Michael Seiler, Teppo Haapaniemi, Jenni Bernoulli. PD-1/PD-L1 expression and tumor-infiltrating immune cells in triple-negative breast cancer: Characterization of preclinical primary tumor and bone metastasis models in humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1738.