Immuno-oncology (IO) has provided groundbreaking results in cancer treatment. Triple-negative breast cancer (TNBC) tumors attract immune cells, and the presence of tumor-infiltrating lymphocytes (TILs) is linked to improved survival. Programmed cell death 1 (PD-1) is expressed by TILs and its ligand (PD-L1) by TNBC cells, and targeting of PD-1 has shown promising results in treatment of patients with primary TNBC. High frequency of bone metastases is typical in TNBC patients. As immune regulation is different in bone than in other organs it is essential to study the efficacy of IO therapies in bone microenvironment. The aim of this study was to assess the efficacy of anti-PD-1 therapy (pembrolizumab, Keytruda) in the growth of primary and bone metastatic TNBC in preclinical models.
MDA-MB-231(SA)-luc human TNBC cells were inoculated into the mammary fat pad (orthotopic model) or tibia bone marrow (bone model) of female huNOG mice engrafted with CD34+ cells from two different donors. Treatments with pembrolizumab or human IgG4 isotype control (5 mg/kg, i.p., Q5D, n=8) were started 3 days after the inoculations. Tumor growth was monitored by bioluminescence imaging (BLI), orthotopic tumor volume by caliper, and tumor induced changes in bone by X-ray imaging for 21-24 days. Tumor samples were processed to immunohistochemical (IHC) analysis of TILs, PD-1 and PD-L1. Bone volume was analyzed ex vivo by micro-computed tomography, and serum TRACP5b values were determined as a marker of osteoclast number.
Tumor growth at treatment start was confirmed by BLI. Pembrolizumab decreased tumor growth in the orthotopic model. 37.5% of the mice had partial response and 12.5% had tumor rejection. The mice exhibited donor-related differences in efficacy but the overall response was similar. IHC showed low to moderate PD-L1 expression in the TNBC tumors (tumor proportion score 1-49%) and moderate number of CD4+ and CD8+ TILs. No PD-1 expression was observed in pembrolizumab treated mice due to antibody blocking of the epitope. In the bone model, pembrolizumab had no effect in tumor growth or any bone parameters studied. PD-L1 expression was comparable in orthotopic and bone tumors. In bone tumors, CD4+ cells were dominant and CD8+ and PD-1+ cells were rarely observed.
In orthotopic model the response rate was similar to what has been observed in TNBC patients, but the bone metastatic growth could not be inhibited by the PD-1 blockage. Bone marrow has a unique microenvironment and immune cell compartment compared to any other organ, and it is known to be involved in immunosurveillance and to favour tumor cells to become immune evasive. The lack of efficacy of immunotherapy in bone compared to orthotopic tumor may be due to these differences. These results highlight the importance of using both orthotopic and metastasis models in preclinical oncology studies.
Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Teppo Haapaniemi, Azusa Tanaka, Michael Seiler, Jenni Bernoulli. Differential efficacy of PD-1 targeted immunomodulation in preclinical models of primary and bone metastatic triple-negative breast cancer [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 1677.