Sarah M. Tam

Complete regression of local cancer using temperature-sensitive liposomes combined with ultrasound-mediated hyperthermia

The development of treatment protocols that result in a complete response to chemotherapy has been hampered by free drug toxicity and the low bioavailability of nano-formulated drugs. Here, we explore the application of temperature-sensitive liposomes that have been formulated to enhance stability in circulation. We formed a pH-sensitive complex between doxorubicin (Dox) and copper (CuDox) in the core of lysolipid-containing temperature-sensitive liposomes (LTSLs). The complex remains associated at neutral pH but dissociates to free Dox in lower pH environments. The resulting CuDox-LTSLs were injected intravenously into a syngeneic murine breast cancer model (6 mg Dox/kg body weight) and intravascular release of the drug was triggered by ultrasound. The entire tumor was insonified for 5 min prior to drug administration and 20 min post drug injection. A single-dose administration of CuDox-LTSLs combined with insonation suppressed tumor growth. Moreover, after twice per week treatment over a period of 28 days, a complete response was achieved in which the NDL tumor cells and the tumor interstitium could no longer be detected. All mice treated with ultrasound combined with CuDox-LTSLs survived, and tumor was undetectable 8 months post treatment. Iron and copper-laden macrophages were observed at early time points following treatment with this temperature sensitive formulation. Systemic toxicity indicators, such as cardiac hypertrophy, leukopenia, and weight and hair loss were not detected with CuDox-LTSLs after the 28-day therapy.

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.

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.

Magnetic resonance imaging assessment of effective ablated volume following high intensity focused ultrasound.

Under magnetic resonance (MR) guidance, high intensity focused ultrasound (HIFU) is capable of precise and accurate delivery of thermal dose to tissues. Given the excellent soft tissue imaging capabilities of MRI, but the lack of data on the correlation of MRI findings to histology following HIFU, we sought to examine tumor response to HIFU ablation to determine whether there was a correlation between histological findings and common MR imaging protocols in the assessment of the extent of thermal damage. Female FVB mice (n = 34), bearing bilateral neu deletion tumors, were unilaterally insonated under MR guidance, with the contralateral tumor as a control. Between one and five spots (focal size 0.5 × 0.5 × 2.5 mm3) were insonated per tumor with each spot receiving approximately 74.2 J of acoustic energy over a period of 7 seconds. Animals were then imaged on a 7T MR scanner with several protocols. T1 weighted images (with and without gadolinium contrast) were collected in addition to a series of T2 weighted and diffusion weighted images (for later reconstruction into T2 and apparent diffusion coefficient maps), immediately following ablation and at 6, 24, and 48 hours post treatment. Animals were sacrificed at each time point and both insonated/treated and contralateral tumors removed and stained for NADH-diaphorase, caspase 3, or with hematoxylin and eosin (H&E). We found the area of non-enhancement on contrast enhanced T1 weighted imaging immediately post ablation correlated with the region of tissue receiving a thermal dose CEM43 ≥ 240 min. Moreover, while both tumor T2 and apparent diffusion coefficient values changed from pre-ablation values, contrast enhanced T1 weighted images appeared to be more senstive to changes in tissue viability following HIFU 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.

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

Abstract 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 ± 0.011 s− 1 versus 0.116 ± 0.047 s− 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 ± 0.030 s− 1 versus 0.747 ± 0.24 s− 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 30 min 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. Graphical abstract Figure. No Caption available.

Abstract LB-052: Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model

We demonstrate for the first time that blocking of the programmed death-1 (PD-1) pathway in conjunction with immunogenic cell death induced by CpG-ODN and activatable nanodelivery of doxorubicin can generate curative responses in both primary and contralateral tumors. Activatable nanotherapeutics are attractive since the toxicity of chemotherapeutics can be constrained to a small region; combining such a strategy with immunotherapy is the goal of this study. We have previously shown that administration of CpG-ODN as an adjuvant, together with local release of doxorubicin from temperature sensitive liposomes (TSL) resulted in regression of directly-treated tumors, suppressed growth of contralateral tumors and reduced chemotherapeutic-mediated toxicity in a murine breast cancer model.1 Increases in cytotoxic CD8+ T lymphocytes and a reduction in regulatory T cells and myeloid-derived suppressor cells were observed in both directly treated and contralateral tumors. This combinatorial approach was curative for directly-treated tumors and overall survival was significantly extended, however, the contralateral tumor returned in all treated mice. The following is the protocol explored for the addition of anti-PD1: immune intact FVB/n mice with bilateral invasive neu deletion syngeneic transplanted tumors were treated with a combination of anti-PD1 (aPD-1, 200 μg, i.p.) and intratumoral administration of CpG-ODN (100 μg, i.t.) on days 0, 7, 14 and 0, 3, 7, 10, 17 and 24, respectively. Doxorubicin 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 and administrated i.v. at 6 mg doxorubicin/kg body weight on days 10, 17, 24. The formation of a complex between doxorubicin and copper was created to enhance the circulation and stability of TSL and to reduce systemic toxicity. To trigger drug release, hyperthermia was induced in the primary tumor with ultrasound (peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz) at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Immediately afterwards, 100 μg of CpG-ODN 1826 was administered intratumorally to the insonified tumor. Upon treatment with this combination of locally-released doxorubicin, local administration of CpG-ODN and systemic aPD-1, 100% of treated and contralateral tumors regressed by at least 80%; further, all of the directly-treated tumors and 50% of the contralateral tumors were eliminated without recurrence. Thus, a 50% complete response rate was achieved, with tumor regression observed immediately after the incorporation of the doxorubicin treatment. By contrast, administration of CpG-ODN and systemic aPD-1 alone resulted in regression of 66% of treated and contralateral tumors. *MS and AK contributed equally to this work. 1. J Control Release (2015); 220: 253-264. Citation Format: Matthew T. Silvestrini, Azadeh Kheirolomoom, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Josquin Foiret, Samantha Tucci, Neil E. Hubbard, Alexander D. Borowsky, Katherine W. Ferrara. Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-052.

Abstract 2155: Achieving complete response to locoregional disease without toxicity using temperature-sensitive liposomes and ultrasound-mediated hyperthermia.

By stabilizing doxorubicin within temperature-sensitive liposomes and repeating delivery twice per week for four weeks, extended survival and complete response were obtained in syngeneic murine breast tumors. Further, with the stabilized formulation, cardiac toxicity and leukopenia were not detected. Liposomes composed of DPPC:MPPC:DSPE-PEG2k, 86:10:4 were prepared in the presence of copper(II) gluconate and triethanolamine and extruded with 100 nm membrane filters. Cu-liposomes were loaded with Dox at 0.2 mg-drug/mg-lipid with 100% loading. We studied the efficacy of CuDox liposomes (CuDox-LTSLs) using the highly invasive neu deletion (NDL) tumor. Treatment began when the tumor diameter reached 4 mm. The fluorescence of circulating doxorubicin in blood was quenched after the digestion of liposomes with Triton X-100, indicating that the circulating drug remained associated with copper. Dox fluorescence was restored upon reducing the pH using a citrate-saline buffer. Over the 28 days of the study, CuDox-LTSLs were administrated intravenously two times per week at a therapeutic level of 6 mg-drug/kg-body weight. The entire tumor was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 42°C for 5 min prior to and 20 min post drug injection. A total of 30 mice were studied, including groups spanning drug treatment with ultrasound, ultrasound, drug treatment and no treatment. Although a single dose administration of CuDox-LTSLs combined with insonation of the entire tumor suppressed the tumor growth, complete response was achieved only upon repeated treatment over a period of 28 days. At 30 days after the last administered dose, none of the mice from the other groups survive; however, 100% of mice treated with ultrasound combined with CuDox-LTSLs survive and the remaining tumor is undetectable. In conclusion, repeated treatment of stabilized temperature sensitive doxorubicin liposomes is highly effective in the treatment of aggressive murine tumors. Acknowledgement: NIHR01CA134659 and NIHR01CA103828 Citation Format: Azadeh Kheirolomoom, Chun-Yen Lai, Sarah M. TAM, Lisa M. Even, Elizabeth S. Ingham, Brett Z. Fite, Katherine D. Watson, Katherine W. Ferrara. Achieving complete response to locoregional disease without toxicity using temperature-sensitive liposomes and ultrasound-mediated hyperthermia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2155. doi:10.1158/1538-7445.AM2013-2155

Combining ultrasound ablation with immunotherapy: Opportunities and challenges

Thermal ablation provides local control of cancer; however, pairing ablation with immunotherapy is attractive as there is emerging evidence that focal therapy enhances the efficacy of immunotherapy. The goals of local treatment are to rapidly debulk the tumor and release tumor antigen to stimulate immune recognition. We seek to evaluate combination protocols and their tumor suppressive effects.