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Purpose: Noninvasive and quantitative tracking of CD8+ T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64Cu, to assess the sensitivity of PET imaging of normal and diseased tissue.Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64Cu was developed. The accumulation of 64Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8+ T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6–8/group studied with imaging and IHC or flow cytometry).Results: We demonstrate the ability of immunoPET to detect small differences in CD8+ T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), 64Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8+ T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols.Conclusions: 64Cu-169cDb imaging can spatially map the distribution of CD8+ T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8+ T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976–87. ©2018 AACR.

Supplementary Figures and Methods

Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. Following 250 kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh–Plesset equation for a clean bubble or the Marmottant model of a lipid-shelled microbubble. The expansion ratio reached 30-fold with 250 kHz at a peak negative pressure of 400 kPa, as compared to a measured expansion ratio of 1.6 fold for 1 MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100 kPa) for the 250 kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75–150 kPa. For pressures above 150 kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250 kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

Purpose: Noninvasive and quantitative tracking of CD8+ T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64Cu, to assess the sensitivity of PET imaging of normal and diseased tissue. Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64Cu was developed. The accumulation of 64Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8+ T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6–8/group studied with imaging and IHC or flow cytometry). Results: We demonstrate the ability of immunoPET to detect small differences in CD8+ T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), 64Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8+ T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols. Conclusions: 64Cu-169cDb imaging can spatially map the distribution of CD8+ T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8+ T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976–87. ©2018 AACR.

Focused ultrasound (FUS) to open the blood brain barrier (BBB) is under evaluation for the delivery of therapeutic agents to the brain. Regarding delivery of genes through the BBB, adeno-associated viruses (AAVs) have generated interest due to their ability to specifically and efficiently transfect the brain, which was recently demonstrated in mouse models. In this work, we investigated the use of 1.5 MHz FUS-mediated BBB opening in a mouse model after systemic injection of AAV-like polymer nanoparticles designed to have a 20-nm size and a targeting peptide similar to AAV-PHP.eB. Radiolabeling of the nanoparticles allowed in vivo tracking of the accumulation in the brain following the FUS treatment with positron emission tomography imaging (PET). Significant accumulation was measured in the treated hemisphere with a 1.8-fold enhancement compared to the contra-lateral hemisphere. Our experimental setup optimized for rodents (Verasonics) enables ultrasound imaging, precise targeting with electronic steering, and monitoring of the cavitation activity during the FUS treatment using passive acoustic mapping.

Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy. Methods: Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A 64Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs. Results: Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane® when evaluated at a concentration of 10 µM. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution. Conclusion: We present a scalable technique for fabricating efficacious squalenoylated-gemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional 64Cu-SqNOTA-SqGem NP.

PURPOSE: Noninvasive and quantitative tracking of CD8(+) T-cells by positron emission tomography (PET) has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with (64)Cu, to assess the sensitivity of PET imaging of normal and diseased tissue. EXPERIMENTAL DESIGN: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with (64)Cu was developed. The accumulation of (64)Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8 per group studied with imaging and immunohistochemistry or flow cytometry) after intravenous administration. Tumor-infiltrating CD8(+) T-cells in tumor bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6–8 per group studied with imaging and immunohistochemistry or flow cytometry). RESULTS: We demonstrate the ability of immunoPET to detect small differences in CD8(+) T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), (64)Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8(+) T-cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols. CONCLUSION: (64)Cu-169cDb imaging can spatially map the distribution of CD8(+) T-cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8(+) T-cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our pre-clinical evaluation.

The recent development of ultrasound localization microscopy, where individual microbubbles (contrast agents) are detected and tracked within the vasculature, provides new opportunities for imaging the vasculature of entire organs with a spatial resolution below the diffraction limit. In stationary tissue, recent studies have demonstrated a theoretical resolution on the order of microns. In this work, single microbubbles were localized in vivo in a rat kidney using a dedicated high frame rate imaging sequence. Organ motion was tracked by assuming rigid motion (translation and rotation) and appropriate correction was applied. In contrast to previous work, coherence-based non-linear phase inversion processing was used to reject tissue echoes while maintaining echoes from very slowly moving microbubbles. Blood velocity in the small vessels was estimated by tracking microbubbles, demonstrating the potential of this technique to improve vascular characterization. Previous optical studies of microbubbles in vessels of approximately 20 microns have shown that expansion is constrained, suggesting that microbubble echoes would be difficult to detect in such regions. We therefore utilized the echoes from individual MBs as microscopic sensors of slow flow associated with such vessels and demonstrate that highly correlated, wideband echoes are detected from individual microbubbles in vessels with flow rates below 2 mm/s.

The recent development of ultrafast ultrasound localization microscopy (Errico et al., Nature, 2015) provides new opportunities for imaging the vasculature with sub-diffraction resolution. However, the large number of images to be recorded requires acquisitions lasting several minutes. Therefore, physiological motion far larger than the achievable resolution presents challenges for moving tissues. In this work, single microbubbles (MB) were localized in vivo in a rat kidney using a dedicated imaging sequence allowing MB tracking and motion correction. Blood velocity in small vessels (

Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy.

Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

Using individual microbubbles (MBs) to image vasculature with a spatial resolution below the diffraction limit has the potential to greatly improve the in vivo characterization of healthy and diseased tissue [1–2]. Recent studies have demonstrated a theoretical resolution on the order of microns in stationary tissue using standard imaging arrays [3]. However, the application of this technique to abdominal imaging brings new challenges due to the presence of physiological motion which is far larger than the achievable resolution. In this work, single MBs were localized in vivo in a rat kidney using a dedicated high frame rate (300 Hz) contrast pulse sequence (CPS) with spatial compounding (−5°, 0°, 5°). A stack of 60000 frames was acquired, providing a B-mode image to track tissue motion and a CPS image to track MB position at each time point. Acquisition was accomplished with a standard imaging array (CL15-7, ATL) driven at 6.9 MHz and a programmable ultrasound system (Verasonics). 3.4 million positions were detected and a density map of the MB positions was obtained after compensation for cardiac motion and changes in kidney position. Blood velocity was also estimated by tracking selected MBs over time.

There is an urgent need to develop nanocarriers for the treatment of glioblastoma multiforme (GBM). Using co-registered positron emission tomography (PET) and magnetic resonance (MR) images, here we performed systematic studies to investigate how a nanocarrier's size affects the pharmacokinetics and biodistribution in rodents with a GBM xenograft. In particular, highly stable, long-circulating three-helix micelles (3HM), based on a coiled-coil protein tertiary structure, were evaluated as an alternative to larger nanocarriers. While the circulation half-life of the 3HM was similar to 110-nm PEGylated liposomes (t1/2=15.5 and 16.5h, respectively), the 20-nm micelles greatly enhanced accumulation within a U87MG xenograft in nu/nu rats after intravenous injection. After accounting for tumor blood volume, the extravasated nanoparticles were quantified from the PET images, yielding ~0.77%ID/cm(3) for the micelles and 0.45%ID/cm(3) for the liposomes. For GBM lesions with a volume greater than 100mm(3), 3HM accumulation was enhanced both within the detectable tumor and in the surrounding brain parenchyma. Further, the nanoparticle accumulation was shown to extend to the margins of the GBM xenograft. In summary, 3HM provides an attractive nanovehicle for carrying treatment to GBM.

Despite major therapeutic advances in the management of patients with metastatic melanoma, brain metastases remain an intractable problem. It has proven difficult to identify and target single genes or pathways critical to brain metastasis formation. Here, we explore a more global and preventive treatment approach to melanoma brain metastases within an established xenograft model of human melanoma. Mice were injected intracardially and followed for six weeks with whole-body BLI and brain MRI. Tumor-bearing brains, adrenals, ovaries, and femurs were harvested and enzymatically dissociated before three tumor cell samples from each organ were flow-sorted and subjected to RNA sequencing. We were able to define a 108-gene brain metastasis gene signature, and queried the Connectivity Map database for candidate drugs, i.e. drugs that induce an opposite gene expression profile in various cancer cell lines. We found the cholesterol analogue beta-sitosterol to effectively inhibit brain metastases and improve survival, both in established and preventive therapeutic settings. Beta-sitosterol extensively suppressed the MAPK pathway, and effectively reduced mitochondrial respiration through Complex I inhibition. This novel mechanistic synergy may open new avenues of systemic therapy against metastatic melanoma, as increased mitochondrial respiration is a key mediator of resistance to MAPK-targeted drugs.

More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of a reporter gene encoding DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging modes to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50 μg luciferase plasmid DNA and 5 x 105 microbubbles followed by ultrasound treatment at 1.4 MHz, 200 kPa, 100-cycle pulse length, and 540-Hz pulse repetition frequency (PRF) for 2 min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p < 0.0001, n = 5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21 days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation–induced gene therapies.

89Zr (t1/2 = 78.4 h), a positron-emitting metal, has been exploited for PET studies of antibodies because of its relatively long decay time and facile labeling procedures. Here, we used 89Zr to evaluate the pharmacokinetics of long-circulating liposomes over 168 hours (1 week). We first developed a liposomal-labeling method using p-isothiocyanatobenzyldesferrioxamine (df-Bz-NCS) and df-PEG1k-DSPE. Df-Bz-NCS was conjugated to 1 mol% amino- and amino-PEG2k-DSPE, where the 1 mol% df-PEG1k-DSPE was incorporated when the liposomes were formulated. Incubation of 89Zr with df, df-PEG1k, and df-PEG2k liposomes for one hour resulted in greater than 68% decay-corrected yield. The loss of the 89Zr label from liposomes after incubation in 50% human serum for 48 hours ranged from ~1 to 3% across the three formulations. Tail vein administration of the three liposomal formulations in NDL tumor-bearing mice showed that the 89Zr label at the end of the PEG2k brush was retained in the tumor, liver, spleen and whole body for a longer time interval than 89Zr labels located under the PEG2k brush. The blood clearance rate of all three liposomal formulations was similar. Overall, the results indicate that the location of the 89Zr label altered the clearance rate of intracellularly-trapped radioactivity and that df-PEG1k-DSPE provides a stable chelation site for liposomal or lipid-based particle studies over extended periods of time.

Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. Invitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. Invivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7-21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis.

Activatable nanotherapeutics provide the opportunity to deliver anthracyclines to a well-controlled region of interest and therefore to locally stimulate immunogenic cell death. Such a strategy can be combined with immunotherapy for both local tumor control and creation of an abscopal effect. Our group previously reported that combining local administration of a CpG oligonucleotide with local release of doxorubicin from a temperature-sensitive liposomal nanoparticle (TSL) led to the elimination of directly treated lesions in a syngeneic murine model of mammary adenocarcinoma.1 While this therapeutic approach increased the numbers of tumor infiltrating CD8+ T lymphocytes in distant lesions and extended survival, distant tumors returned with a growth delay of approximately 14 days and T-regulatory cells were not reduced by treatment. Therefore, checkpoint blockade of the programmed death-1 (PD-1) pathway (anti-PD-1) was incorporated in the protocol. CuDox-TSL were prepared from DPPC:MPPC:DSPE-PEG2k (86:10:4) in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-drug/mg-lipid. A complex between doxorubicin and copper was created to enhance the circulation and stability of TSL and to reduce systemic toxicity. On day 21, mice with bilateral invasive neu deletion (NDL) tumors (~4 mm) were treated with i.v. administration of CuDox-TSL at 6 mg doxorubicin/kg body weight. Ultrasound (US) hyperthermia (1.1 MPa, 1.5 MHz, achieving 42°C for 5 min before and 20 min after drug injection) triggered drug release. Immediately after US, 100 µg of CpG-ODN 1826 was administered intratumorally to the insonified tumor; anti-PD-1 (200 µg, i.p.) was administrated three days later. Flow cytometry and immunohistochemistry were performed on day 28 after one treatment with each component and survival was assessed after treatment was repeated for three weeks. While control mice survived 35 days, 50% of treated mice were tumor-free after 100 days. All primary tumors and 50% of the contralateral tumors regressed and were eliminated by day 63. With the incorporation of immunotherapy, three doses of chemotherapy were sufficient to eliminate all directly treated tumors, as compared with 8 doses without immunotherapy. After a single treatment with each component, a substantial change was observed in systemic immune cells, including a significant increase in natural killer and dendritic cells in distant lymph nodes, in CD8+ T cells in tumors and associated lymph nodes and in IFNγ-secreting CD4+ T cells in the treated tumor and distant lymph nodes. The results demonstrate that activatable chemotherapy can be paired with an immune adjuvant and PD-1 blockade to generate a curative response in primary and distant tumors. Further, local chemotherapy, as potentiated by activatable liposomes does not impede a systemic CD8+ T cell response. [1] Journal of Controlled Release (2015); 220: 253-264. Citation Format: Azadeh Kheirolomoom, Matthew Silvestrini, Lisa Mahakian, Elizabeth Ingham, Josquin Foiret, Spencer Tumbale, Sarah Tam, Neil Hubbard, Alexander Borowsky, Katherine Ferrara. Protocol optimization combining activatable nanodelivery with immunotherapy in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4603. doi:10.1158/1538-7445.AM2017-4603

Epidemiological and preclinical evidence supports that omega-3 dietary fatty acids (fish oil) reduce the risks of macular degeneration and cancers, but the mechanisms by which these omega-3 lipids inhibit angiogenesis and tumorigenesis are poorly understood. Here we show that epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450 epoxygenases from omega-3 fatty acid docosahexaenoic acid, inhibit VEGF- and fibroblast growth factor 2-induced angiogenesis in vivo, and suppress endothelial cell migration and protease production in vitro via a VEGF receptor 2-dependent mechanism. When EDPs (0.05 mg⋅kg −1 ⋅d −1 ) are coadministered with a low-dose soluble epoxide hydrolase inhibitor, EDPs are stabilized in circulation, causing ∼70% inhibition of primary tumor growth and metastasis. Contrary to the effects of EDPs, the corresponding metabolites derived from omega-6 arachidonic acid, epoxyeicosatrienoic acids, increase angiogenesis and tumor progression. These results designate epoxyeicosatrienoic acids and EDPs as unique endogenous mediators of an angiogenic switch to regulate tumorigenesis and implicate a unique mechanistic linkage between omega-3 and omega-6 fatty acids and cancers.

For many years, immune activation following tumor ablation has been evaluated in the treatment of systemic cancer. Ultrasound ablation is thought to promote dendritic cell maturation and T-cell immunity, and is particularly advantageous because it is non-invasive, can be controlled with high spatial precision and uses no harmful ionizing radiation.

Despite recent therapeutic advances in the management of patients with metastatic melanoma, brain metastases remain an intractable problem. It has proven difficult to identify and target single genes or pathways critical to brain metastasis formation. In this work we set out to explore a more global and preventive treatment approach to melanoma brain metastases within an established xenograft model of human melanoma. Mice were injected intracardially under ultrasound guidance and followed for six weeks with whole-body BLI and brain MRI. Tumor-bearing brains, adrenals, ovaries, and femurs were harvested and enzymatically dissociated before three tumor cell samples from each organ were flow-sorted and subjected to RNA sequencing. We developed a combined workflow of several independent computational and statistical analyses based on differential expression, and defined a brain metastasis gene signature of 54 upregulated and 54 downregulated genes. We then queried the Connectivity Map database using our 108-gene signature for candidate drugs, i.e. drugs that induce an opposite gene expression profile in various cancer cell lines. Three of the top ten drugs, already in use for other clinical conditions, showed significant efficacy in monolayer proliferation assays and colony forming assays with two brain-tropic melanoma cell lines and one brain-tropic lung cancer cell line. To examine these results in vivo, we used a highly standardized and predictive model system with automated MRI-based quantification of nanoparticle-labeled cells in the mouse brain following intracardiac injection of human melanoma cells. Treatment was started shortly after verification of comparable tumor cell exposure in the mouse brains and animals were monitored with whole-body BLI and brain MRI. Preliminary results showed promising therapeutic effects with respect to brain metastatic burden and survival, as well as acceptable toxicity profiles. We are currently validating these results on a larger scale in vivo, in other models, as well as applying systems biology and network modeling approaches to explore the fundamental mechanisms of action. Together, our results demonstrate a previously unrecognized potential for drug repurposing in the treatment of patients at risk of developing melanoma brain metastases or with clinically occult micrometastatic disease. Finding new uses for old drugs can greatly expedite translation to clinical practice; thus, further investigations are warranted to determine the safety, tolerability, and efficacy of these therapies. Citation Format: Terje Sundstrøm, Jobin K. Varughese, Francisco Azuaje, Kjell Petersen, Clifford G. Tepper, Elizabeth Ingham, Lisa Even, Sarah Tam, Kai Ove Skaftnesmo, Morten Lund-Johansen, Rolf Bjerkvig, Katherine W. Ferrara, Frits A. Thorsen. Drug repurposing to treat melanoma brain metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4036. doi:10.1158/1538-7445.AM2014-4036