Veterinary Management of Harderian Gland Tumors in Cancer Rainbow (crainbow) HER2-Positive Mice.

A Cancer Rainbow mouse line that expresses 3 fluorescently labeled isoforms of the tumor-driver gene HER2 (HER2BOW) was developed recently for the study of tumorigenesis in the mammary gland. The expression of 1 of the 3 HER2 isoforms in HER2BOW mice is induced through the Cre/lox system. However, in addition to developing palpable mammary tumors, HER2BOW mice developed orbital tumors, specifically of the Harderian gland. Mice were euthanized, and histopathologic examination of the Harderian gland tumors was performed. Tumors were characterized by adenomatous hyperplasia to multinodular adenomas of the Harderian gland. Fluorescent imaging of the Harderian gland tissue confirmed the expression of HER2 in the tumors. Here we discuss monitoring and palliative approaches to allow attainment of humane experimental endpoints of mammary tumor growth in this mouse line. We describe a range of interventions, including close monitoring, topical palliative care, and surgical bilateral enucleation. Based on our data and previous reports in the literature, the overexpression of HER2 in Harderian gland tissue and subsequent tumor formation likely was driven by MMTV-Cre expression in the Harderian gland.

NIR-emissive PEG-b-TCL micelles for breast tumor imaging and minimally invasive pharmacokinetic analysis.

Motivated by the goal of developing a fully biodegradable optical contrast agent with translational clinical potential, a nanoparticle delivery vehicle was generated from the self-assembly of poly(ethylene-glycol)-block-poly(trimethylene carbonate-co-caprolactone) (PEG-b-TCL) copolymers. Cryogenic transmission electron microscopy verified that PEG-b-TCL-based micelles were formed at low solution temperatures (∼38 °C). Detailed spectroscopic experiments validated facile loading of large quantities of the high emission dipole strength, tris(porphyrin)-based fluorophore PZn3 within their cores, and the micelles displayed negligible in vitro and in vivo toxicities in model systems. The pharmacokinetics and biodistribution of PZn3-loaded PEG-b-TCL-based micelles injected intravenously were determined via ex vivo near-infrared (NIR) imaging of PZn3 emission in microcapillary tubes containing minute quantities of blood, to establish a novel method for minimally invasive pharmacokinetic monitoring. The in vivo circulatory half-life of the PEG-b-TCL-based micelles was found to be ∼19.6 h. Additionally, longitudinal in vivo imaging of orthotopically transplanted breast tumors enabled determination of micelle biodistribution that correlated to ex vivo imaging results, demonstrating accumulation predominantly within the tumors and livers of mice. The PEG-b-TCL-based micelles quickly extravasated within 4T1 orthotopic mammary carcinomas, exhibiting peak accumulation at ∼48 h following intravenous tail-vein injection. In summary, PEG-b-TCL-based micelles demonstrated favorable characteristics for further development as in vivo optical contrast agents for minimally invasive imaging of breast tumors.

Novel Manganese-Porphyrin Superoxide Dismutase-Mimetic Widens the Therapeutic Margin in a Preclinical Head and Neck Cancer Model.

PURPOSE: To test the effects of a novel Mn porphyrin oxidative stress modifier, Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (MnBuOE), for its radioprotective and radiosensitizing properties in normal tissue versus tumor, respectively. METHODS AND MATERIALS: Murine oral mucosa and salivary glands were treated with a range of radiation doses with or without MnBuOE to establish the dose-effect curves for mucositis and xerostomia. Radiation injury was quantified by intravital near-infrared imaging of cathepsin activity, assessment of salivation, and histologic analysis. To evaluate effects of MnBuOE on the tumor radiation response, we administered the drug as an adjuvant to fractionated radiation of FaDu xenografts. Again, a range of radiation therapy (RT) doses was administered to establish the radiation dose-effect curve. The 50% tumor control dose values with or without MnBuOE and dose-modifying factor were determined. RESULTS: MnBuOE protected normal tissue by reducing RT-mediated mucositis, xerostomia, and fibrosis. The dose-modifying factor for protection against xerostomia was 0.77. In contrast, MnBuOE increased tumor local control rates compared with controls. The dose-modifying factor, based on the ratio of 50% tumor control dose values, was 1.3. Immunohistochemistry showed that MnBuOE-treated tumors exhibited a significant influx of M1 tumor-associated macrophages, which provides mechanistic insight into its radiosensitizing effects in tumors. CONCLUSIONS: MnBuOE widens the therapeutic margin by decreasing the dose of radiation required to control tumor, while increasing normal tissue resistance to RT-mediated injury. This is the first study to quantitatively demonstrate the magnitude of a single drug's ability to radioprotect normal tissue while radiosensitizing tumor.

Rational design of "heat seeking" drug loaded polypeptide nanoparticles that thermally target solid tumors.

This paper demonstrates the first example of targeting a solid tumor that is externally heated to 42 °C by "heat seeking" drug-loaded polypeptide nanoparticles. These nanoparticles consist of a thermally responsive elastin-like polypeptide (ELP) conjugated to multiple copies of a hydrophobic cancer drug. To rationally design drug-loaded nanoparticles that exhibit thermal responsiveness in the narrow temperature range between 37 and 42 °C, an analytical model was developed that relates ELP composition and chain length to the nanoparticle phase transition temperature. Suitable candidates were designed based on the predictions of the model and tested in vivo by intravital confocal fluorescence microscopy of solid tumors, which revealed that the nanoparticles aggregate in the vasculature of tumors heated to 42 °C and that the aggregation is reversible as the temperature reverts to 37 °C. Biodistribution studies showed that the most effective strategy to target the nanoparticles to tumors is to thermally cycle the tumors between 37 and 42 °C. These nanoparticles set the stage for the targeted delivery of a range of cancer chemotherapeutics by externally applied mild hyperthermia of solid tumors.

Magnetic fluid hyperthermia for bladder cancer: a preclinical dosimetry study.

PURPOSE: This paper describes a preclinical investigation of the feasibility of thermotherapy treatment of bladder cancer with magnetic fluid hyperthermia (MFH), performed by analysing the thermal dosimetry of nanoparticle heating in a rat bladder model. MATERIALS AND METHODS: The bladders of 25 female rats were instilled with magnetite-based nanoparticles, and hyperthermia was induced using a novel small animal magnetic field applicator (Actium Biosystems, Boulder, CO). We aimed to increase the bladder lumen temperature to 42 °C in <10 min and maintain that temperature for 60 min. Temperatures were measured within the bladder lumen and throughout the rat with seven fibre-optic probes (OpSens Technologies, Quebec, Canada). An MRI analysis was used to confirm the effectiveness of the catheterisation method to deliver and maintain various nanoparticle volumes within the bladder. Thermal dosimetry measurements recorded the temperature rise of rat tissues for a variety of nanoparticle exposure conditions. RESULTS: Thermal dosimetry data demonstrated our ability to raise and control the temperature of rat bladder lumen ≥1 °C/min to a steady state of 42 °C with minimal heating of surrounding normal tissues. MRI scans confirmed the homogenous nanoparticle distribution throughout the bladder. CONCLUSION: These data demonstrate that our MFH system with magnetite-based nanoparticles provides well-localised heating of rat bladder lumen with effective control of temperature in the bladder and minimal heating of surrounding tissues.

Catabolism of exogenous lactate reveals it as a legitimate metabolic substrate in breast cancer.

Lactate accumulation in tumors has been associated with metastases and poor overall survival in cancer patients. Lactate promotes angiogenesis and metastasis, providing rationale for understanding how it is processed by cells. The concentration of lactate in tumors is a balance between the amount produced, amount carried away by vasculature and if/how it is catabolized by aerobic tumor or stromal cells. We examined lactate metabolism in human normal and breast tumor cell lines and rat breast cancer: 1. at relevant concentrations, 2. under aerobic vs. hypoxic conditions, 3. under conditions of normo vs. hypoglucosis. We also compared the avidity of tumors for lactate vs. glucose and identified key lactate catabolites to reveal how breast cancer cells process it. Lactate was non-toxic at clinically relevant concentrations. It was taken up and catabolized to alanine and glutamate by all cell lines. Kinetic uptake rates of lactate in vivo surpassed that of glucose in R3230Ac mammary carcinomas. The uptake appeared specific to aerobic tumor regions, consistent with the proposed "metabolic symbiont" model; here lactate produced by hypoxic cells is used by aerobic cells. We investigated whether treatment with alpha-cyano-4-hydroxycinnamate (CHC), a MCT1 inhibitor, would kill cells in the presence of high lactate. Both 0.1 mM and 5 mM CHC prevented lactate uptake in R3230Ac cells at lactate concentrations at ≤ 20 mM but not at 40 mM. 0.1 mM CHC was well-tolerated by R3230Ac and MCF7 cells, but 5 mM CHC killed both cell lines ± lactate, indicating off-target effects. This study showed that breast cancer cells tolerate and use lactate at clinically relevant concentrations in vitro (± glucose) and in vivo. We provided additional support for the metabolic symbiont model and discovered that breast cells prevailingly take up and catabolize lactate, providing rationale for future studies on manipulation of lactate catabolism pathways for therapy.

Tumor cells upregulate normoxic HIF-1α in response to doxorubicin.

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that controls cellular homeostasis. Although its activation benefits normal tissue, HIF-1 activation in tumors is a major risk factor for angiogenesis, therapeutic resistance, and poor prognosis. HIF-1 activity is usually suppressed under normoxic conditions because of rapid oxygen-dependent degradation of HIF-1α. Here, we show that, under normoxic conditions, HIF-1α is upregulated in tumor cells in response to doxorubicin, a chemotherapeutic agent used to treat many cancers. In addition, doxorubicin enhanced VEGF secretion by normoxic tumor cells and stimulated tumor angiogenesis. Doxorubicin-induced accumulation of HIF-1α in normoxic cells was caused by increased expression and activation of STAT1, the activation of which stimulated expression of iNOS and its synthesis of nitric oxide (NO) in tumor cells. Mechanistic investigations established that blocking NO synthesis or STAT1 activation was sufficient to attenuate the HIF-1α accumulation induced by doxorubicin in normoxic cancer cells. To our knowledge, this is the first report that a chemotherapeutic drug can induce HIF-1α accumulation in normoxic cells, an efficacy-limiting activity. Our results argue that HIF-1α-targeting strategies may enhance doxorubicin efficacy. More generally, they suggest a broader perspective on the design of combination chemotherapy approaches with immediate clinical impact.

A role for the copper transporter Ctr1 in the synergistic interaction between hyperthermia and cisplatin treatment.

PURPOSE: Hyperthermia enhances cytotoxic effects of chemotherapeutic agents such as cisplatin. However, the underlying molecular mechanisms remain unclear. We hypothesised that hyperthermia increases cisplatin accumulation and efficacy by modulating function of copper transport protein 1 (Ctr1), a major regulator of cellular cisplatin uptake. We examined the significance of Ctr1 in the synergistic interaction between hyperthermia and cisplatin. We assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerisation in sensitising cells to cisplatin cytotoxicity. MATERIALS AND METHODS: Ctr1 protein levels and cisplatin sensitivities were assessed in bladder cancer cell lines with immunoblotting and clonogenic survival assays. Using Myc-tagged-Ctr1 HEK293 cells, we assessed the effect of hyperthermia on Ctr1 multimerisation with immunoblotting. The effect of hyperthermia on cisplatin sensitivity and accumulation was assessed in wild-type (WT) and Ctr1 knockout (Ctr1-/-) mouse embryonic fibroblasts (MEFs) with clonogenic assays and inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Increased Ctr1 protein expression was observed for the most cisplatin-sensitive bladder cancer cell lines and MEFs. Heat-induced increase in Ctr1 multimerisation with cisplatin was observed in Myc-tagged Ctr1 cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT more than Ctr1-/- cells (dose modifying factors 1.75 versus 1.4, respectively). WT cells accumulated more platinum versus Ctr1-/- cells; this was further increased by hyperthermia in WT cells. CONCLUSIONS: Hyperthermia enhanced cisplatin uptake and cytotoxicity in WT cells. Heat increased Ctr1 activity by increasing multimerisation, enhancing drug cytotoxicity. Furthermore, Ctr1 protein profiles of bladder tumours, as well as other tumour types, may predict their response to cisplatin and overall efficacy of treatment.

Novel approaches to treatment of hepatocellular carcinoma and hepatic metastases using thermal ablation and thermosensitive liposomes.

Because of the limitations of surgical resection, thermal ablation is commonly used for the treatment of hepatocellular carcinoma and liver metastases. Current methods of ablation can result in marginal recurrences of larger lesions and in tumors located near large vessels. This review presents a novel approach for extending treatment out to the margins where temperatures do not provide complete treatment with ablation alone, by combining thermal ablation with drug-loaded thermosensitive liposomes. A history of the development of thermosensitive liposomes is presented. Clinical trials have shown that the combination of radiofrequency ablation and doxorubicin-loaded thermosensitive liposomes is a promising treatment.

Overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream.

Miniature microwave applicator for murine bladder hyperthermia studies.

PURPOSE: Novel combinations of heat with chemotherapeutic agents are often studied in murine tumour models. Currently, no device exists to selectively heat small tumours at depth in mice. In this project we modelled, built and tested a miniature microwave heat applicator, the physical dimensions of which can be scaled to adjust the volume and depth of heating to focus on the tumour volume. Of particular interest is a device that can selectively heat murine bladder. MATERIALS AND METHODS: Using Avizo(®) segmentation software, we created a numerical mouse model based on micro-MRI scan data. The model was imported into HFSS™ (Ansys) simulation software and parametric studies were performed to optimise the dimensions of a water-loaded circular waveguide for selective power deposition inside a 0.15 mL bladder. A working prototype was constructed operating at 2.45 GHz. Heating performance was characterised by mapping fibre-optic temperature sensors along catheters inserted at depths of 0-1 mm (subcutaneous), 2-3 mm (vaginal), and 4-5 mm (rectal) below the abdominal wall, with the mid depth catheter adjacent to the bladder. Core temperature was monitored orally. RESULTS: Thermal measurements confirm the simulations which demonstrate that this applicator can provide local heating at depth in small animals. Measured temperatures in murine pelvis show well-localised bladder heating to 42-43°C while maintaining normothermic skin and core temperatures. CONCLUSIONS: Simulation techniques facilitate the design optimisation of microwave antennas for use in pre-clinical applications such as localised tumour heating in small animals. Laboratory measurements demonstrate the effectiveness of a new miniature water-coupled microwave applicator for localised heating of murine bladder.

Nanoscale Drug Delivery and Hyperthermia: The Materials Design and Preclinical and Clinical Testing of Low Temperature-Sensitive Liposomes Used in Combination with Mild Hyperthermia in the Treatment of Local Cancer.

The overall objective of liposomal drug delivery is to selectively target drug delivery to diseased tissue, while minimizing drug delivery to critical normal tissues. The purpose of this review is to provide an overview of temperature-sensitive liposomes in general and the Low Temperature-Sensitive Liposome (LTSL) in particular. We give a brief description of the material design of LTSL and highlight the likely mechanism behind temperature-triggered drug release. A complete review of the progress and results of the latest preclinical and clinical studies that demonstrate enhanced drug delivery with the combined treatment of hyperthermia and liposomes is provided as well as a clinical perspective on cancers that would benefit from hyperthermia as an adjuvant treatment for temperature-triggered chemotherapeutics. This review discusses the ideas, goals, and processes behind temperature-sensitive liposome development in the laboratory to the current use in preclinical and clinical settings.