2000648 (1 of 12) © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advmat.de COMMUNICATION A Nitric Oxide (NO) Nanoreporter for Noninvasive Real-Time Imaging of Macrophage Immunotherapy Anujan Ramesh, Sahana Kumar, Anthony Brouillard, Dipika Nandi, and Ashish Kulkarni* A. Ramesh, S. Kumar, A. Brouillard, Prof. A. Kulkarni Department of Chemical Engineering University of Massachusetts Amherst, MA 01003, USA E-mail: akulkarni@engin.umass.edu A. Ramesh, A. Kulkarni Department of Biomedical Engineering University of Massachusetts Amherst, MA 01003, USA D. Nandi, A. Kulkarni Department of Veterinary and Animal Sciences University of Massachusetts Amherst, MA 01003, USA A. Kulkarni Center for Bioactive Delivery Institute for Applied Life Sciences University of Massachusetts Amherst, MA 01003, USA The ORCID identifcation number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adma.202000648. DOI: 10.1002/adma.202000648 Macrophage-centered therapeutic approaches that rely on immune modu- lation of tumor associated macrophages (TAMs) from a pro-tumorigenic phenotype (M2) to an anti-tumorigenic phenotype (M1) have facilitated a paradigm shift in macrophage immunotherapy. However, limited clinical success has been achieved due to the low response rates observed in dif- ferent types of cancers. The ability to measure immune response in real time is critical in order to diferentiate responders from non-responders; however, there are currently no platforms to monitor real-time macrophage immunotherapy response. Hence, there is an immediate need to develop imaging techniques that can longitudinally monitor macrophage immuno- therapy response. Nitric oxide (NO) produced as a result of activation of macrophages to an anti-tumorigenic state is considered as a hallmark of M1 and can be a direct indication of response. In this study, a NO nanoreporter (NO-NR) is reported that enables real-time monitoring of macrophage immunotherapy drugs in vitro and in vivo. Furthermore, it is observed that sustained inhibition of colony stimulating factor 1 receptor (CSF1R) using a CSF1R inhibitor–NO-NR system leads to enhanced efcacy and better imaging signal. In conclusion, a frst-of-its-kind NO nanoreporter tool is reported that can be used as an activatable imaging agent to monitor mac- rophage immunotherapy response in real time. Cancer progression is primarily driven by interactions between infltrating immune cells and neoplastic cancer cells in the tumor microenvironment. [1] Among all the tumor infltrating immune cells, mac- rophages are of particular importance due to their relative abundant numbers in the tumor stroma. [2] Indeed, recent studies have suggested a strong cor- relation between infltration of tumor- associated macrophages (TAMs) with tumor progression. [1,3–5] Compelling evidence in recent studies have demon- strated that TAMs acquire an immuno- suppressive, protumoral M2 phenotype in contrast to the classically activated antitumoral M1 phenotype. [6] The M1–M2 dichotomy, as described by Mills et al., classifed macrophage activation profles on a spectrum mimicking the Th1- or Th2-derived responses. [7] Macrophages indeed exist in diferent functional states across the broad M1/M2 spectrum, based on the cytokines to which they are exposed. [8] However, the cancer microen- vironment is dominated by a cocktail of immunosuppressive cytokines that lead to a predominant Th2-derived environment. [9] Factors like interleukin 4 (IL-4) secreted by CD4+ cells and macrophage colony stimulating factor 1 (CSF1) secreted by tumor cells ini- tiate polarization of macrophages to the cancer promoting M2 phenotype. [10] With the onset of immunotherapy, studies involving TAM- targeted therapies have made signifcant strides. Thera- peutic strategies involving macrophage manipulation gener- ally include one of the following mechanisms: i) macrophage depletion by targeting macrophage survival pathways (e.g., CSF1–macrophage colony stimulating factor receptor (CSF1R)), ii) limiting macrophage recruitment and localization into the primary tumor site by blocking macrophage recruiting chemokines (e.g., CCL2/CCR2), and iii) macrophage repro- gramming, where immunosuppressive M2 macrophages are repolarized to M1 macrophages (TLR agonists, CSF1R kinase signaling inhibitors). [11] But, recent clinical trials have shown that single agent therapies like monoclonal antibodies or small molecule inhibitors of the CSF1R signaling axis have limited efcacy. [12] Trials involving Pexidartinib, ARRY-382, BLZ945, and MCS11 showed underwhelming results in terms of anticancer Adv. Mater. 2020, 2000648