Many malignant tumors, including breast cancer, lung cancer, colon cancer, pancreatic cancer, lymphomas, sarcomas, neuroblastomas and (among many others) are associated with a pro-inflammatory tumor microenvironment, characterized by infiltration of leukocytes whereas increases in some leukocyte subsets parallels disease progression and worse clinical outcomes (1-4). Tumor-associated macrophages (TAMs) play a key role in this context: M2-TAM phenotypes promote tumor growth and enhance pulmonary metastasis by high-level expression of epidermal growth factor (EGF) and activation of EGF-regulated signaling pathways critical for invasive tumor growth and metastatic dissemination. Conversely, M1-TAM phenotypes mediate tumor cell phagocytosis and tumor growth inhibition directly or indirectly. New therapeutic drugs that target TAM are currently being developed and are starting to enter the clinic. Thus, it becomes increasingly important to identify patients whose tumors are heavily infiltrated by TAM, in order to stratify these patients to TAM-directed immunotherapies and monitor treatment response (4-6). Unfortunately, existing clinical imaging technologies do not provide a good method for evaluating response to immunotherapies because most immune modulating therapeutics do not cause changes in tumor size, at least not in the immediate post-treatment time period, but rather changes in the immune cell composition of the tumor (7-9). Therefore, as novel immunotherapies are being integrated with classical chemotherapy (7-9), it becomes increasingly important to monitor the immune cell signature of malignant tumors. To serve this goal, an imaging test is advantageous over invasive biopsy because it is non-invasive, covers the whole tumor and can repeatedly interrogate treatment effects on the complex cross talk between innate and adaptive immune responses in vivo, in patients.

Historically, molecular imaging tests have focused on imaging cancer cells, tumor microvascular characteristics and the extracellular matrix surrounding cancer (10-12). The tumor microenvironment has not been a major focus for the development of novel imaging technologies. In the past, our team has pioneered a number of novel imaging approaches for cancer MR imaging (13-19), PET imaging (20-26), and immune cell imaging (13,14,23,27,28), including translational MR imaging tests for in vivo detection of TAM with the FDA-approved iron supplement ferumoxytol (Feraheme) (29,30). Ferumoxytol is a colloid-based USPIO with a hydrodynamic diameter of 28–32 nm, consisting of an iron oxide core with a size of 6.4 –7.2 nm and a carboxymethyldextran coat. At 20 MHz and 39°C, ferumoxytol has an r1 relaxivity of 38 mM–1s–1 and an r2 relaxivity of 83 mM–1s–1 and can therefore be used as a contrast agent for MRI. In fact, ferumoxytol is the only FDA-approved iron oxide nanoparticle compound to date, which can be offered to patients for non-invasive TAM imaging through “off label” use as a contrast agent. Unlike larger SPIO (> 50 nm), intravenously injected ferumoxytol nanoparticles (< 50 nm) transiently escape RES-phagocytosis in liver, spleen and bone marrow, which leads to a prolonged blood half-life. Ferumoxytol nanoparticles then slowly “leak” across hyperpermeable tumor microvessels into the tumor interstitium, where they are slowly phagocytosed by TAM, a process that takes several hours (29). At 24 hours p.i., the nanoparticles are compartmentalized intracellulary in TAM (Fig. 1). Following intravenous administration, ferumoxytol nanoparticles show an initial perfusion effect of tumor tissue, followed by transendothelial leak and phagocytosis by TAM via the “enhanced permeability and retention (EPR) effect”, which results in a marked hypointense signal effect on delayed T1- and T2-weighted MR images (29). This signal effect can be used to noninvasively track the degree of macrophage infiltration in a tumor before and after TAM-modulating immunotherapies. Our clinical MR imaging studies after intravenous injection of iron oxide nanoparticles demonstrate unique enhancement properties of glioblastomas, lymphomas and sarcomas in the presence of TAM-mediated inflammation, providing a novel, immediately clinically applicable imaging test to noninvasively track innate immune responses from macrophages in patients (30-32). Intracellular nanoparticles in TAMs exert a T2-signal effect on delayed MR scans, several hours to days after nanoparticle administration, with minimal or no hyperintense T1 effect due to lack of interactions with protons. This “decoupling” of T1- and T2-signal effects is different compared to the additive T1- and T2-signal effects of free iron oxides in vessels or tumor necrosis and can be used as a non-invasive imaging indicator for iron-labeled TAMs (33). Recent developments in macrophage-targeted PET-tracers may improve the specificity of iron oxide nanoparticle-based MR imaging through integrated PET/MR approaches. These novel TAM imaging concepts could represent a significant breakthrough for clinicians as a new means for risk stratification and as a new gold-standard imaging test for tracking treatment response in future TAM-directed immunotherapy trials, including planned first-in-man anti-CD47 immunotherapy trials in 2016.

Summary: in vivo imaging of distinct immune cell populations in malignant tumors should permeate our understanding of immune cell responses to cancer therapies, enable us to overcome the bottleneck of monitoring antitumor immune responses in vivo, improve patient stratification to tailored immune modulating therapies and aid in assigning non-responders to alternative treatment options. For example, patients with cancers with marked TAM infiltration could be directed to novel immune reprogramming therapies, while patients with node-negative tumors without significant leukocyte infiltration could be spared. TAM imaging approaches could also facilitate the development, monitoring and regulatory approval of new classes of immune-based drugs. Since clinical trials of new therapeutic drugs and combination therapies are expensive and take years to complete, novel TAM imaging approaches provide significant immediate value and impact. The ability to non-invasively and repetitively image several distinct immune cell populations in the tumor microenvironment will open opportunities for new discoveries in the area of cancer immunology and immunotherapy, and ultimately, help to improve long term treatment outcomes.

Figure 1: In vivo biodistribution and MR signal effects of intravenously administered superparamagnetic iron oxide nanoparticles (SPIO) with hydrodynamic diameters in the order of 20-100 nm: After intravenous injection, SPIO initially distribute in the blood pool, where they exert a strong T1- and T2-effect. Due to their large size, nanoparticles do not extravasate across intact vascular endothelia in most normal organs. However, the nanoparticles leak across hyperpermeable sinus in organs of the reticuloendothelial system (RES), such as bone marrow, liver, spleen and lymph nodes, which leads to a hypointense signal effect on T2- and T2*-weighted MR images. Nanoparticle extravasation occurs at a relatively slower rate into the interstitium of malignant tumors, where SPIO are phagocytosed by tumor-associated macrophages (TAM). Therefore, tumors in RES organs can be depicted as T2-hyperintense lesions on relatively early postcontrast scans, but do show T2-enhancement on delayed postcontrast scans. Intracellular SPIO in TAM exert a hypo-intense MR signal effect on both T1- and T2-weighted MR images while extracellular SPIO in tumor necrosis demonstrate T1-hyperintense and T2-hypointense signal. Within macrophages, SPIO undergo a slow metabolization. Baseline MR signal intensities are regained after several weeks