Abstract LB-132: A new preclinical model for immuno-oncology: Combination of tumor, bone microenvironment and immune system
Authors: Kähkönen Tiina E. , Suominen Mari I. , Mäki-Jouppila Jenni , Halleen Jussi M. , Tanaka Azusa , Seiler Michael , Bernoulli Jenni 
Published: July 2017

Abstract
Breast and other solid tumors commonly metastasize to bone. Bone microenvironment regulates hematopoietic stem cells and thereby immune cell differentiation. Therefore, a system with functional interaction between bone, immune system and cancer is a prerequisite in developing and in preclinical testing of novel immunotherapies against osseous tumors. Our aim was to establish a model where tumor, bone microenvironment and immune system would be combined to functional entity.
An intratibial injection of 1x106 of BT-474 (ER+, PR+, HER2+) human breast cancer cells were given to huNOG mice (HSCFTL-NOG-F, provided by Taconic Biosciences). These humanized mice were produced through engrafting hCD34+ hematopoietic stem cells (HSC) into CIEA NOG mouse® (NOD.Cg-Prkdcscid Il2rgtm1Sug/ JicTac, Taconic Biosciences). The tumor induced bone changes were followed by radiography at 4, 6 and 8 weeks. Bone mineral density (BMD) and bone mineral content (BMC) were quantified by dual x-ray absorptiometry (DXA) at end of study. Bone turnover markers CTX-I, TRACP5b and PINP were measured from serum in the beginning and at sacrifice. Spleen, thymus, lymph nodes and hind limbs were collected, tumor and bone areas, as well as the expression of human CD3, CD4, CD8, CD16/56, CD20, CD45, CTLA-4 and PD-L1 in immune cells was analyzed.
Tumor-induced osteoblastic new bone growth was observed in all tumor-bearing tibias. Osteoblastic and tumor areas were larger in huNOGs compared to NOG mice. Correspondingly, BMC was higher in huNOGs than in NOG mice, while BMD was comparable. Increased BMC was mainly due to a rise in trabecular bone volume quantified from histological sections. Measurements of bone turnover markers revealed an increased bone resorption rate in NOG mice compared to huNOGs. Spleen weight was markedly increased in huNOGs compared to NOG mice. Strong expression of CD3, CD4, CD8 and CD45 was observed in spleen and lymph nodes of huNOG mice indicating high prevalence active human immune cells. CD45-positive tumor-infiltrating lymphocytes (TILs) and CD4-positive T-helper cells were observed in 80% of the mice. Furthermore, PD-L1 was expressed in 50% of tibias and no CTLA-4 expression was observed in this model.
To our knowledge, the first humanized mouse model of tumor growth in bone was established in this study. The model is characterized by tumor growth, extensive tumor-induced osteoblastic changes and tumor-infiltrating human immune cells in bone, and mimics the late stage of breast cancer metastasized to bone. This humanized mouse model provides a completely new platform for preclinical testing of cancer immunotherapies, particularly the therapies targeting cancers metastasizing to or growing in bone.
Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni Mäki-Jouppila, Jussi M. Halleen, Azusa Tanaka, Michael Seiler, Jenni Bernoulli. A new preclinical model for immuno-oncology: Combination of tumor, bone microenvironment and immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-132. doi:10.1158/1538-7445.AM2017-LB-132