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One third of patients with bladder cancer present with muscle invasive bladder cancer (MIBC) which has a poor prognosis. International guidelines for the management of MIBC recommend radical cystectomy or bladder-preserving treatment based on radical radiotherapy with a form of radiosensitisation. In the UK, both conventional fractionation with 64 Gy in 32 fractions and hypofractionation with 55 Gy in 20 fractions are standard of care options with the choice varying between centres. A meta-analysis of individual patients with locally advanced bladder cancer from two UK multicentre phase 3 trials was published recently. This study evaluated the non-inferiority of a hypofractionated schedule compared to a conventional regime. This analysis confirmed the non-inferiority of the hypofractionated regimen, and noted superior locoregional control. We discuss the relevance of these findings to current practice while considering the radiobiology of hypofractionation, the role of systemic therapies and radiosensitisation, as well as the socioeconomic benefits.

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.

INTRODUCTION: Tumor populations may selectively colonize bone that is being actively remodeled. In prostate cancer patients, androgen deprivation directly inhibits tumor growth initially, whilst induced bone loss may facilitate tumor colonization of bone by androgen-insensitive cells. We have tested this hypothesis using a xenograft model of early growth of prostate cancer in bone. METHODS: PC3 cells transfected with Green fluorescent protein (GFP) were injected into castrated and non-castrated athymic mice via intrabial and intracardiac routes. In vivo tumor growth was monitored daily and animals sacrificed 6-9 days following initial GFP-based detection of tumors. Tumor bearing and contra-lateral non-tumor bearing tibias were analyzed extensively by micro-CT and histology/immunohistochemistry for the presence of tumor cells and the effects of tumor and/or castration on bone cells and bone structure evaluated. RESULTS: GFP-positive tumors in bone were visible from 12 days post-injection following intratibial injection, allowing tumors

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.

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.

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular\ud matrix (ECM) proteins play important roles in sustaining vascular tone and resistance.\ud The main goal of this study was to determine whether VSMCs adhesion to type I collagen\ud (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by\ud vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle\ud arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus\ud were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of\ud the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling\ud distance (approach and retraction). AFM probes were tipped with a 5 μm diameter\ud microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin\ud II (ANG-II; 10−6\ud ) significantly increased (p

Cancer metastasis to the skeleton causes mortality in breast cancer, however the mechanisms involved in bone metastases development remain unclear. A metastatic initiating cell (MIC) phenotype CD44...

Mammography is an important screening modality for the early detection of DCIS and breast cancer lesions. More specifically, high mammographic density is associated with an increased risk of breast cancer. However, the biological processes underlying this phenomenon remain largely unknown. Here, we re-interrogated genome-wide transcriptional profiling data obtained from low-density (LD) mammary fibroblasts (n = 6 patients) and high-density (HD) mammary fibroblasts (n = 7 patients) derived from a series of 13 female patients. We used these raw data to generate a “breast density” gene signature consisting of >1250 transcripts that were significantly increased in HD fibroblasts, relative to LD fibroblasts. We then focused on the genes that were increased by ≥ 1.5-fold (P < 0.05) and performed gene set enrichment analysis (GSEA), using the molecular signatures database (MSigDB). Our results indicate that HD fibroblasts show the upregulation and/or hyper-activation of several key cellular processes, including the stress response, inflammation, stemness, and signal transduction. The transcriptional profiles of HD fibroblasts also showed striking similarities to human tumors, including head and neck, liver, thyroid, lung, and breast cancers. This may reflect functional similarities between cancer-associated fibroblasts (CAFs) and HD fibroblasts. This is consistent with the idea that the presence of HD fibroblasts may be a hallmark of a pre-cancerous phenotype. In these biological processes, GSEA predicts that several key signaling pathways may be involved, including JNK1, iNOS, Rho GTPase(s), FGF-R, EGF-R, and PDGF-R-mediated signal transduction, thereby creating a pro-inflammatory, pro-proliferative, cytokine, and chemokine-rich microenvironment. HD fibroblasts also showed significant overlap with gene profiles derived from smooth muscle cells under stress (JNK1) and activated/infected macrophages (iNOS). Thus, HD fibroblasts may behave like activated myofibroblasts and macrophages, to create and maintain a fibrotic and inflammatory microenvironment. Finally, comparisons between the HD fibroblast gene signature and breast cancer tumor stroma revealed that JNK1 stress signaling is the single most significant biological process that is shared between these 2 data sets (with P values between 5.40E-09 and 1.02E-14), and is specifically associated with tumor recurrence. These results implicate “stromal JNK1 signaling” in the pathogenesis of human breast cancers and the transition to malignancy. Augmented TGF-β signaling also emerged as a common feature linking high breast density with tumor stroma and breast cancer recurrence (P = 5.23E-05). Similarities between the HD fibroblast gene signature, wound healing, and the cancer-associated fibroblast phenotype were also noted. Thus, this unbiased informatics analysis of high breast density provides a novel framework for additional experimental exploration and new hypothesis-driven breast cancer research, with a focus on cancer prevention and personalized medicine.

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.

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.

Background: A number of recent clinical trials have suggested that the benefits of adjuvant bone targeted treatments on risks of recurrence or death in early breast cancer are restricted to women with established menopause. We have used a model system that mimics pre- and post-menopausal status to investigate the effects of zoledronic acid on breast tumour growth in bone, demonstrating a differential anti-tumour effect in these two settings. Methodology: Tumour growth was assessed by in vivo imaging following intra-cardiac inoculation of luciferase expressing MDA-MB-231 breast cancer cells in 12-week old female Balb-c nude mice. Animals underwent ovariectomy (OVX, mimicking the post-menopausal setting) or a sham operation (mimicking the pre-menopausal setting) 7 days after tumour cell implantation (n = 10/group). In treatment experiments, OVX and sham operated animals received either saline or 100ug/kg zoledronic acid (ZOL) weekly s.c. from day 4. Effects on bone were assessed by uCT analysis, bone histomorphometry and measurement of serum bone markers. Presence of tumour cells in bone was detected by multiphoton microscopy. Results: OVX induced significant bone loss compared to sham within 2 weeks, which was completely inhibited by ZOL. Tumour growth was detected in the long bones of 29% of animals in the sham group on day 35 following tumour cell inoculation, and this was not affected by ZOL treatment. In contrast, 83% of the OVX animals had detectable tumours in the long bones, demonstrating that OVX mediated changes to the bone microenvironment that stimulated tumour growth. Crucially, this OVX-induced increase in tumour growth was completely prevented by weekly ZOL treatment, with only 17% of animals in the OVX+ZOL group having detectable bone tumours compared to 83% in the OVX+saline group. Multiphoton microscopy revealed that numerous individual tumour cells were present in long bones of animals in the OVX+ZOL group, supporting that ZOL acts by inhibiting an OVX-mediated trigger of tumour cell proliferation. Conclusions: Our data are in agreement with those reported from clinical trials of adjuvant zoledronic acid, showing an anti-cancer effect exclusively in the post-menopausal setting. This is the first demonstration that zoledronic acid prevents tumour progression in bone specifically through inhibition of OVX-induced proliferation of tumour cells resident in the bone marrow. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD07-08.