Person:

Kohler, Rainer

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behavior. Model TNPs comprised of a fluorescent platinum(IV) pro-drug and a clinically-tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumor associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload, and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA damaging Pt payload gradually releases to neighboring tumor cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials.

Quantification of cell-cycle state at a single-cell level is essential to understand fundamental three-dimensional biological processes such as tissue development and cancer. Analysis of 3D in vivo images, however, is very challenging. Today’s best practice, manual annotation of select image events, generates arbitrarily sampled data distributions, unsuitable for reliable mechanistic inferences. Here, we present an integrated workflow for quantitative in vivo cell-cycle profiling. It combines image analysis and machine learning methods for automated 3D segmentation and cell-cycle state identification of individual cell-nuclei with widely varying morphologies embedded in complex tumor environments. We applied our workflow to quantify cell-cycle effects of three antimitotic cancer drugs over 8 days in HT-1080 fibrosarcoma xenografts in living mice using a dataset of 38,000 cells and compared the induced phenotypes. In contrast to 2D culture, observed mitotic arrest was relatively low, suggesting involvement of additional mechanisms in their antitumor effect in vivo.

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi–Goutières syndrome and STING-associated vasculopathy of infancy (SAVI)3. Myocardial infarction (MI) elicits inflammation5, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production. In mice, single-cell RNA-seq analysis of 4,215 leukocytes isolated from infarcted and non-infarcted hearts showed that MI provokes activation of an IRF3–interferon axis in a distinct population of interferon-inducible cells (IFNICs) that were classified as cardiac macrophages. Mice genetically deficient in cyclic GMP-AMP synthase (cGAS), its adaptor STING, IRF3, or the type I IFN receptor IFNAR exhibited impaired interferon-stimulated gene (ISG) expression and, in the case of mice deficient in IRF3 or IFNAR, improved survival after MI as compared to controls. Interruption of IRF3-dependent signaling resulted in decreased cardiac expression of inflammatory cytokines and chemokines and decreased inflammatory cell infiltration of the heart, as well as in attenuated ventricular dilation and improved cardiac function. Similarly, treatment of mice with an IFNAR-neutralizing antibody after MI ablated the interferon response and improved left ventricular dysfunction and survival. These results identify IRF3 and the type I IFN response as a potential therapeutic target for post-MI cardioprotection.