Kimberley J. Reeves

Zoledronic Acid Has Differential Antitumor Activity in the Pre- and Postmenopausal Bone Microenvironment In Vivo

Purpose: Clinical trials in early breast cancer have suggested that benefits of adjuvant bone-targeted treatments are restricted to women with established menopause. We developed models that mimic pre- and postmenopausal status to investigate effects of altered bone turnover on growth of disseminated breast tumor cells. Here, we report a differential antitumor effect of zoledronic acid (ZOL) in these two settings. Experimental design: Twleve-week-old female Balb/c-nude mice with disseminated MDA-MB-231 breast tumor cells in bone underwent sham operation or ovariectomy (OVX), mimicking the pre- and postmenopausal bone microenvironment, respectively. To determine the effects of bone-targeted therapy, sham/OVX animals received saline or 100 μg/kg ZOL weekly. Tumor growth was assessed by in vivo imaging and effects on bone by real-time PCR, micro-CT, histomorphometry, and measurements of bone markers. Disseminated tumor cells were detected by two-photon microscopy. Results: OVX increased bone resorption and induced growth of disseminated tumor cells in bone. Tumors were detected in 83% of animals following OVX (postmenopausal model) compared with 17% following sham operation (premenopausal model). OVX had no effect on tumors outside of bone. OVX-induced tumor growth was completely prevented by ZOL, despite the presence of disseminated tumor cells. ZOL did not affect tumor growth in bone in the sham-operated animals. ZOL increased bone volume in both groups. Conclusions: This is the first demonstration that tumor growth is driven by osteoclast-mediated mechanisms in models that mimic post- but not premenopausal bone, providing a biologic rationale for the differential antitumor effects of ZOL reported in these settings. Clin Cancer Res; 20(11); 2922–32. ©2014 AACR.

Is nitric oxide important in photodynamic therapy

Photodynamic therapy (PDT) involves the use of photochemical reactions mediated through the interaction of photosensitizing agents, light and oxygen to destroy abnormal tissue. The transfer of energy from the activated photosensitizer to available oxygen results in the formation of toxic reactive oxygen species, such as singlet oxygen and free radicals, which can damage proteins, lipids, nucleic acids, and other cellular components. PDT is now commonly used in ophthalmology, dermatology and oncology however the therapeutic response to PDT exhibits variability, ranging from highly sensitive to extremely resistant. Over the last 10 years it has been suggested that nitric oxide (NO) may play a role in PDT, with evidence that NO is produced by both tumour and normal cells in addition to controlling important functions in tumour progression. NO may also influence the outcome of cancer therapies, such as PDT. PDT induces oxidative stress, vascular-mediated damage and leukocyte recruitment, processes all sensitive to NO. This review outlines the role of nitric oxide in PDT primarily focusing on vascular damage and how this may be modulated to improve therapeutic outcome.

The role of nitric oxide in the treatment of tumours with aminolaevulinic acid-induced photodynamic therapy

Photodynamic therapy (PDT) is a local cancer treatment which induces cell death by the interaction of light with a photosensitizing drug. Previous studies indicate that nitric oxide (NO) plays a role in Photofrin-PDT, but this has not been investigated in aminolaevulinic acid (ALA)-PDT. The current study determines whether inhibition of nitric oxide synthase (NOS) activity modulates treatment responses to ALA-PDT, in tumours displaying differential levels of NO. Murine tumours with low (EMT6) or high (RIF-1) NO levels were implanted into the cremaster muscle of BALB/c or C3H/HeN mice respectively. Animals were prepared for in vivo microscopy 7-14days later. Mice received oral ALA (200mg/kg) 4h before PDT. l-NAME, l-NNA or 1400W (10mg/kg) were administered via the jugular vein 5min before PDT. NOS inhibition (l-NAME or l-NNA) combined with ALA-PDT in RIF-1 tumours demonstrated enhanced damage to both the tumour and normal microvasculature, with increased macromolecular leak and reduction in vessel diameter, whereas ALA-PDT alone had no effect. In contrast, EMT6 tumours responded to ALA-PDT alone but sensitivity was not enhanced in the presence of NOS inhibition. 1400 W combined with ALA-PDT induced similar microvascular effects to l-NAME in both tumours, but were less pronounced. The data demonstrates that NO has an important role in events likely to be critical for treatment response, sensitivity and therapeutic outcome of ALA-PDT.

Selective measurement and manipulation of adhesion forces between cancer cells and bone marrow endothelial cells using atomic force microscopy

AIMS The lack of understanding of the biology of bone cancer metastasis has limited the development of effective treatment strategies. The aim of this study was to characterize tumor cell adhesion molecules and determine active tumor cell interactions with human bone marrow endothelial (BME) cells using atomic force microscopy. MATERIALS & METHODS A single prostate (PC3) cancer cell was coupled (concanavalin A) to the atomic force microscopy cantilever then placed in contact with BME cells for cell force spectroscopy measurements. RESULTS & DISCUSSION Strong adhesive interactions between PC3 and BME cells were significantly (p < 0.05) reduced by anti-ICAM-1, anti-β1 and anti-P-selectin, but not anti-VCAM-1. The combined blocking antibodies or the therapeutic agent zoledronic acid significantly (p < 0.005) reduced the adhesive interactions by 65 and 63%, respectively, which was confirmed using a functional in vitro assay. CONCLUSION Atomic force microscopy provides a highly sensitive screening assay to determine and quantify nanoscale adhesion events between different cell types important in the metastatic cascade.

Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 μm diameter microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10−6) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10−4) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately −33% and −17%, respectively. Similarly, the NO donor (PANOate, 10−6 M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by −38% and −35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state.

Prostate cancer cells home to bone using a novel in vivo model: modulation by the integrin antagonist GLPG0187.

Micrometastasis is a barrier to the development of effective cancer therapies for prostate cancer metastasis to bone. The mechanisms remain incompletely characterised, primarily due to an inability to adequately monitor the initial metastatic events in vivo. This study aimed to establish a new model, allowing the tracking of prostate cancer cells homing to bone, and furthermore, to evaluate the response of this approach to therapeutic modulation, using the integrin antagonist GLPG0187. A single murine metatarsal was engrafted into a dorsal skinfold chamber implanted on a SCID mouse. Fluorescently‐labeled human prostate (PC3‐GFP) or oral (SCC4‐GFP) cancer cells were administered via intracardiac (i.c) injection, with simultaneous daily GLPG0187 or vehicle‐control treatment (i.p. 100 mg/kg/day) for the experimental duration. Metatarsal recordings were taken every 48 h for up to 4 weeks. Tissue was harvested and processed for microCT, multiphoton analysis, histology and immunohistochemistry. Cell viability, proliferation and migration in vitro were also quantified following treatment with GLPG0187. Metatarsals rapidly revascularised by inosculation with the host vasculature (day 5–7). PC3‐GFP cells adhered to the microvascular endothelium and/or metatarsal matrix 3 days after administration, with adhesion maintained for the experimental duration. GLPG0187 treatment significantly (p < 0.05) reduced PC3 cell number within the metatarsal in vivo and reduced migration (p < 0.05) and proliferation (p < 0.05) but not cell viability in vitro. This new model allows evaluation of the early events of tumour‐cell homing and localisation to the bone microenvironment, in addition to determining responses to therapeutic interventions.

Evaluation of fluorescent plasma markers for in vivo microscopy of the microcirculation.

This study evaluated four fluorescent-protein conjugates to monitor microcirculatory variables using the murine cremaster muscle and determined acute and long-term responses to repeated administration of FITC-BSA [conjugated at the University of Sheffield (UoS)] within a dorsal microcirculatory chamber (DMC) in rats. For analysis of the cremaster muscle, male C3H/HeN mice were anaesthetized, the cremaster muscle was exteriorized, then TRITC-BSA, TRITC-dextran, FITC-BSA, FITC-BSA (UoS) or FITC-dextran (0.25 ml/100 g) were administered systemically. The microcirculation was viewed with epi-illumination every 10 min for 120 min. For analysis of the DMC, male Wistar rats were implanted with the chamber. Three weeks later, FITC-BSA (UoS) was administered systemically, and the microcirculation response was monitored using three different protocols. In addition, in vitro stability of fluorescent conjugates was measured over 8 h. With regard to the cremaster muscle, initially no differences in interstitial fluorescence or vessel diameter were observed between the four fluorescent conjugates. By the end of the study, interstitial fluorescence from TRITC-dextran, FITC-dextran and FITC-BSA (Sigma) was significantly (p < 0.05) increased compared to FITC-BSA (UoS). With regard to the DMC, there was no interstitial fluorescence leakage after 180 min or 5 weeks despite repeated administration, but a significant (p < 0.05) leak was detected between 4 and 24 h. FITC-BSA (UoS) was the most stable fluorescent conjugate both in vitroand in vivo and was comparable with other conjugates for evaluating skeletal muscle microcirculation using fluorescent in vivo microscopy.

Hypoxia and angiogenesis: from primary tumor to bone metastasis

Abstract Breast and prostate cancer frequently metastasis to bone, causing considerable morbidity and a reduction in quality of life. Primary cancer cells must undergo a complex process to form secondary tumors in areas such as bone. This process involves cancer cell proliferation, growth and dislodgement from the primary site, surviving in the circulation, arresting at a secondary target site and migrating across the vascular barrier into the extravascular connective tissue. Subsequently, tumor cells must proliferate, induce a microvascular network in the bone microenvironment and establish as a secondary tumor. Throughout this process angiogenesis is essential, as tumors require this process to grow beyond a few millimeters and is stimulated in response to hypoxia. The vascular endothelial growth factor (VEGF) pathway plays a critical role in the angiogenic process. The use of anti-angiogenic drugs to inhibit factors critical for the tumor development such as VEGF should prove beneficial for the treatment of both primary and metastatic cancer. Bevacizumab a humanized VEGF antibody (Avastin) has been used in the treatment of metastatic breast cancer in combination with chemotherapy, however despite being the first drug of its kind to receive FDA approval, this has now been withdrawn due to side-effects associated with treatment. However currently there are a number of anti-angiogenic and anti-vascular compounds in phase I/II clinical trials for advanced breast and prostate cancer. This chapter will discuss the process of angiogenesis and the key angiogenic molecule VEGF, the involvement of hypoxia and HIF, and their role in tumor progression and metastasis. In addition the models and anti-angiogenic compounds used in pre-clinical studies of metastatic cancer will be detailed and finally, the anti-angiogenic compounds which are currently in clinical trials to treat advanced breast and prostate cancer will be described.