Background: Tumor-associated macrophages (TAMs) play key roles in the progression of cancers and their response to various treatments, for example through secretion of pro-survival growth factors; contribution to angiogenesis; and modulation of the tumor immune response via antigen presentation and expression of factors such as PD-L1. TAM effects can be highly localized within the tumor microenvironment, for example through cell-cell contact, yet it has been difficult to directly study interactions between TAMs and tumor cells at high resolution, in orthotopic sites of cancer development, and in real-time as cells respond to therapeutic treatment. To understand the biology underlying heterogeneous interactions between TAMs and tumor cells, we used in vivo fluorescence imaging at single-cell resolution to study pharmacodynamic (PD) response of tumor cells and TAMs to chemotherapeutic treatment.
Methods: To directly image host leukocytes in a variety of tumor models, we used a combination of fluorescent genetic reporter host animals (CX3CR1GFP/+ reporter mice) and macrophage labeling via well-validated fluorescent and magnetic dextran-coated nanoparticles. For direct visualization of therapeutic response in cancer cells themselves, we used 53BP1-mApple as a transgenic fluorescent reporter to quantify DNA damage responses at the single-cell level. Automated computational segmentation of 3D images enabled precise quantification of statistical correlation between spatial TAM localization and cellular DNA damage responses. We performed studies in multiple human xenograft and syngeneic tumor models, including orthotopic ovarian cancer models and a syngeneic Kras-mutant p53-null lung adenocarcinoma model; histology validated in vivo imaging results. As a proof-of-principle, we tested cellular response to platinum (Pt)-based DNA-damaging agents.
Results: Using a novel combination of orthotopic imaging setups, fluorescent reporters for TAMs and cancer cell pharmacodynamic response, along with automated 3D image segmentation and single-cell quantification, here we demonstrate that in vivo imaging can quantify correlations between local TAM concentrations and the simultaneously observed behavior of thousands of cancer cells. As a proof-of-principle, we measure relationships between single-cell DNA damage and local TAM levels in response to Pt-based chemotherapeutic treatment, and find that highly-localized concentrations of tumor-associated immune cells can influence therapeutic response at the single-cell level (and at cellular length-scale gradients). Importantly, we find these relationships to be highly probabilistic (rather than deterministic), and automated classification of thousands of cells across multiple tumors enables robust quantification of the inherent stochasticity involved in the pharmacodynamic responses. We find that single-cell heterogeneity and its explanation by microenvironmental factors such as local TAM concentration and vascular proximity can be heavily dependent on the therapeutic treatment and tumor model.
Conclusions: This work presents in vivo imaging technology that should be useful for studying interactions between cancer cells and TAMs in orthotopic sites including intraperitoneal ovarian cancer and can be extended to other cell interactions (for instance by using fluorescent genetic reporters of various lymphocyte populations). Interaction and co-localization between tumor cells and associated immune cells can influence therapeutic response at the single-cell level, and such information will likely be useful for understanding heterogeneous drug responses to chemotherapeutics and immunotherapeutics alike.
Citation Format: Miles A. Miller, Mikael Pittet, Ralph Weissleder. In vivo imaging of innate immune cells to measure drug response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B133.
- ©2016 American Association for Cancer Research.