Although immune-checkpoint inhibitors have improved the survival of patients with various types of metastatic tumor, intra- and inter-patient heterogeneities in the response of multiple tumors to the therapy have significantly limited clinical benefits. Understanding the specific driving forces behind these heterogeneities will facilitate a better understanding of therapeutic resistance. Research has revealed that tumors in patients and established cancer cell lines are composed of multiple clones with different phenotypes. Our goal is to dissect these complexities and decipher mechanisms of tumor heterogeneities and resistance to immunotherapy. We have established single cell-derived clonal populations from parental, polyclonal 4T1, murine breast cancer cells. In vitro studies, while parental cells formed the heterogeneous types of colonies in a clonogenic assay, established clones created clone dependent uniform colonies. Each clone showed different growth rates, response to chemotherapy, and produced different amounts of cytokines. Then, 4T1 parental cells, clone 1, or clone 16 cells were injected into the spleen of mice to create multiple experimental liver metastases. Seven days after the inoculation, tumor-bearing mice were implanted with the window chamber above the liver, intravenously (iv) injected with fluorescently labeled anti-PDL1 IgG, and delivery of the antibody to individual liver metastasis was imaged using intravital microscopy (IVM). Then, mice were imaged using IVM for 7 days to determine the therapeutic effect on individual tumor growth. In another set of tumor-bearing mice, either isotype IgG or anti-PDL1 IgG were iv injected and the effect on the survival was evaluated. We found that the delivery of fluorescently labeled anti-PDL1 IgG and therapeutic effect was heterogeneous among liver metastases originated from 4T1 parental cells. We also found that there was an inverse correlation between the amount of the anti-PDL1 IgG delivered to the tumor and individual tumor growth. Liver metastases originated from clone 16 cells accumulated significantly more amount of the anti-PDL1 IgG and most of the tumors disappeared within 7 days. On the other hand, the anti-PDL1 IgG delivery to clone 1 cell-derived tumor was significantly reduced than parental and clone 16 cell-derived tumors and none of the tumors responded to the therapy. Thus, limited therapeutic efficacy can be attributed to the limited anti-PDL1 IgG delivery to the tumor. The survival of the tumor-bearing mice was correlated with the level of anti-PDL1 IgG delivery. While the anti-PDL1 therapy could extend the survival of mice-bearing clone 16 derived tumors, mice-bearing parental cells, and clone 1 cells didn’t respond to the therapy. To elucidate the mechanism for the difference in anti-PDL1 Ab delivery, the effect of the therapeutics on tumor growth, and survival, we evaluated the expression of PDL1 protein in tumors by immunohistochemical analysis. PDL1 expression was higher in clone 16 tumors compared to parental and clone 1 tumor. Next, we evaluated the amount of blood vessels in tumors, because the difference in the antibody delivery can be attributed to the amount of angiogenesis. Interestingly, there was no significant difference in the amount of blood vessels, indicating the expression level of the target protein is crucial to determine the amount of the antibody delivery. Regarding the immune microenvironment, there was no difference in the amount of CD8 cells inside tumors. Thus, heterogeneity in anti-PDL1 IgG delivery and its therapeutic efficacy was explained by heterogeneous PDL1 expression in polyclonal tumors. Evaluation of heterogeneity in PDL1 expression in the patient’s tumor shall be considered for personalizing the therapy.

Citation Format: Yan ting Liu, Shreya Goel, Megumi Kai, Thao Nguyen, Kenji Yokoi. Heterogeneous tumor microenvironment in metastatic breast cancer for insufficient anti-PDL1 IgG delivery and its efficacy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-42.