Immune modulation resulting from MR-guided high intensity focused ultrasound in a model of murine breast cancer.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1ß and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

Development of thermosensitive resiquimod-loaded liposomes for enhanced cancer immunotherapy.

Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28 days (non-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 µg for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

Immune-mediated ECM depletion improves tumour perfusion and payload delivery.

High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune-modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin-nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα-CSG fusion protein to tumour ECM in tumour-bearing mice. Intravenously injected TNFα-CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM-rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour-bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα.

Tumor-specific delivery of gemcitabine with activatable liposomes.

Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper (II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 wt% loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine­copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42 °C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis.

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis.

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was ß-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that ß-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of ß-sitosterol was linked to mitochondrial interference. Mechanistically, ß-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either ß-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, ß-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.

Combining activatable nanodelivery with immunotherapy in a murine breast cancer model.

A successful chemotherapy-immunotherapy solid-tumor protocol should accomplish the following goals: debulk large tumors, release tumor antigen for cross-presentation and cross-priming, release cancer-suppressive cytokines and enhance anti-tumor immune cell populations. Thermally-activated drug delivery particles have the potential to synergize with immunotherapeutics to accomplish these goals; activation can release chemotherapy within bulky solid tumors and can enhance response when combined with immunotherapy. We set out to determine whether a single protocol, combining locally-activated chemotherapy and agonist immunotherapy, could accomplish these goals and yield a potentially translational therapy. For effective delivery of free doxorubicin to tumors with minimal toxicity, we stabilized doxorubicin with copper in temperature-sensitive liposomes that rapidly release free drug in the vasculature of cancer lesions upon exposure to ultrasound-mediated hyperthermia. We found that in vitro exposure of tumor cells to hyperthermia and doxorubicin resulted in immunogenic cell death and the local release of type I interferons across murine cancer cell lines. Following intravenous injection, local activation of the liposomes within a single tumor released doxorubicin and enhanced cross-presentation of a model antigen at distant tumor sites. While a variety of protocols achieved a complete response in >50% of treated mice, the complete response rate was greatest (90%) when 1 week of immunotherapy priming preceded a single activatable chemotherapeutic administration. While repeated chemotherapeutic delivery reduced local viable tumor, the complete response rate and a subset of tumor immune cells were also reduced. Taken together, the results suggest that activatable chemotherapy can enhance adjuvant immunotherapy; however, in a murine model the systemic adaptive immune response was greatest with a single administration of chemotherapy.

Enhanced microbubble contrast agent oscillation following 250 kHz insonation.

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.

A Scalable Method for Squalenoylation and Assembly of Multifunctional 64Cu-Labeled Squalenoylated Gemcitabine Nanoparticles.

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.

Unimicellar hyperstars as multi-antigen cancer nanovaccines displaying clustered epitopes of immunostimulating peptides.

Unimicellar hyperstar macromolecular chimeras displaying multiple melanoma peptide antigens were prepared primarily via a combination of click chemistry and esterification reactions starting from a biodegradable hyperbranched polymer template. Solubilization of the hyperstars in aqueous solution afforded a multi-antigen unimicellar cancer nanovaccine of about 20 nm. The nanovaccine showed good biocompatibility and uptake by dendritic cells in vitro. An in vivo evaluation of the nanovaccine therapeutic efficacy against melanoma in mice implanted with B16OVA tumors revealed significantly greater T-cell recruitment and improved survival rates for mice treated with nanovaccine and adjuvant compared to non-treated mice.

Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

CD8+ T-Cell Density Imaging with 64Cu-Labeled Cys-Diabody Informs Immunotherapy Protocols.

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.

Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach.

Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).

Dynamic contrast enhanced MRI detects changes in vascular transport rate constants following treatment with thermally-sensitive liposomal doxorubicin.

Temperature-sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature-sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature-sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE-MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature-sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1=0.0478±0.011s-1 versus 0.116±0.047s-1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2=0.291±0.030s-1 versus 0.747±0.24s-1). While DCE-MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30min after treatment. In summary, DCE-MRI provides a means to detect changes that are associated with treatment by thermally-activated particles and such changes can be exploited to enhance local delivery.

Priming is key to effective incorporation of image-guided thermal ablation into immunotherapy protocols.

Focal therapies play an important role in the treatment of cancers where palliation is desired, local control is needed, or surgical resection is not feasible. Pairing immunotherapy with such focal treatments is particularly attractive; however, there is emerging evidence that focal therapy can have a positive or negative impact on the efficacy of immunotherapy. Thermal ablation is an appealing modality to pair with such protocols, as tumors can be rapidly debulked (cell death occurring within minutes to hours), tumor antigens can be released locally, and treatment can be conducted and repeated without the concerns of radiation-based therapies. In a syngeneic model of epithelial cancer, we found that 7 days of immunotherapy (TLR9 agonist and checkpoint blockade), prior to thermal ablation, reduced macrophages and myeloid-derived suppressor cells and enhanced IFN-γ-producing CD8+ T cells, the M1 macrophage fraction, and PD-L1 expression on CD45+ cells. Continued treatment with immunotherapy alone or with immunotherapy combined with ablation (primed ablation) then resulted in a complete response in 80% of treated mice at day 90, and primed ablation expanded CD8+ T cells as compared with all control groups. When the tumor burden was increased by implantation of 3 orthotopic tumors, successive primed ablation of 2 discrete lesions resulted in survival of 60% of treated mice as compared with 25% of mice treated with immunotherapy alone. Alternatively, when immunotherapy was begun immediately after thermal ablation, the abscopal effect was diminished and none of the mice within the cohort exhibited a complete response. In summary, we found that immunotherapy begun before ablation can be curative and can enhance efficacy in the presence of a high tumor burden. Two mechanisms have potential to impact the efficacy of immunotherapy when begun immediately after thermal ablation: mechanical changes in the tumor microenvironment and inflammatory-mediated changes in immune phenotype.

In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles.

Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies.

Ultrasound ablation enhances drug accumulation and survival in mammary carcinoma models.

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates noninvasive image-guided conformal thermal therapy of cancer. Yet in many scenarios, the sensitive tissues surrounding the tumor constrain the margins of ablation; therefore, augmentation of MRgFUS with chemotherapy may be required to destroy remaining tumor. Here, we used 64Cu-PET-CT, MRI, autoradiography, and fluorescence imaging to track the kinetics of long-circulating liposomes in immunocompetent mammary carcinoma-bearing FVB/n and BALB/c mice. We observed a 5-fold and 50-fold enhancement of liposome and drug concentration, respectively, within MRgFUS thermal ablation-treated tumors along with dense accumulation within the surrounding tissue rim. Ultrasound-enhanced drug accumulation was rapid and durable and greatly increased total tumor drug exposure over time. In addition, we found that the small molecule gadoteridol accumulates around and within ablated tissue. We further demonstrated that dilated vasculature, loss of vascular integrity resulting in extravasation of blood cells, stromal inflammation, and loss of cell-cell adhesion and tissue architecture all contribute to the enhanced accumulation of the liposomes and small molecule probe. The locally enhanced liposome accumulation was preserved even after a multiweek protocol of doxorubicin-loaded liposomes and partial ablation. Finally, by supplementing ablation with concurrent liposomal drug therapy, a complete and durable response was obtained using protocols for which a sub-mm rim of tumor remained after ablation.

CpG expedites regression of local and systemic tumors when combined with activatable nanodelivery.

Ultrasonic activation of nanoparticles provides the opportunity to deliver a large fraction of the injected dose to insonified tumors and produce a complete local response. Here, we evaluate whether the local and systemic response to chemotherapy can be enhanced by combining such a therapy with locally-administered CpG as an immune adjuvant. In order to create stable, activatable particles, a complex between copper and doxorubicin (CuDox) was created within temperature-sensitive liposomes. Whereas insonation of the CuDox liposomes alone has been shown to produce a complete response in murine breast cancer after 8 treatments of 6 mg/kg delivered over 4 weeks, combining this treatment with CpG resolved local cancers within 3 treatments delivered over 7 days. Further, contralateral tumors regressed as a result of the combined treatment, and survival was extended in systemic disease. In both the treated and contralateral tumor site, the combined treatment increased leukocytes and CD4+ and CD8+ T-effector cells and reduced myeloid-derived suppressor cells (MDSCs). Taken together, the results suggest that this combinatorial treatment significantly enhances the systemic efficacy of locally-activated nanotherapy.

Self-assembled 20-nm (64)Cu-micelles enhance accumulation in rat glioblastoma.

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.

Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI.

Manual palpation is a common and very informative diagnostic tool based on estimation of changes in the stiffness of tissues that result from pathology. In the case of a small lesion or a lesion that is located deep within the body, it is difficult for changes in mechanical properties of tissue to be detected or evaluated via palpation. Furthermore, palpation is non-quantitative and cannot be used to localize the lesion. Magnetic Resonance-guided Focused Ultrasound (MRgFUS) can also be used to evaluate the properties of biological tissues non-invasively. In this study, an MRgFUS system combines high field (7T) MR and 3 MHz focused ultrasound to provide high resolution MR imaging and a small ultrasonic interrogation region (~0.5 x 0.5 x 2 mm), as compared with current clinical systems. MR-Acoustic Radiation Force Imaging (MR-ARFI) provides a reliable and efficient method for beam localization by detecting micron-scale displacements induced by ultrasound mechanical forces. The first aim of this study is to develop a sequence that can concurrently quantify acoustic radiation force displacements and image the resulting transient shear wave. Our motivation in combining these two measurements is to develop a technique that can rapidly provide both ARFI and shear wave velocity estimation data, making it suitable for use in interventional radiology. Secondly, we validate this sequence in vivo by estimating the displacement before and after high intensity focused ultrasound (HIFU) ablation, and we validate the shear wave velocity in vitro using tissue-mimicking gelatin and tofu phantoms. Such rapid acquisitions are especially useful in interventional radiology applications where minimizing scan time is highly desirable.

Ultrasound molecular imaging of tumor angiogenesis with a neuropilin-1-targeted microbubble.

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. In vitro, 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. In vivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6 min 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 (8 min), 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.