Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models.

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

The Identification of Small Molecule Inhibitors That Reduce Invasion and Metastasis of Aggressive Cancers.

Transformed epithelial cells can activate programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. This process is linked to the acquisition of an invasive phenotype and the formation of distant metastases. The development of compounds that block the acquisition of an invasive phenotype or revert the invasive mesenchymal phenotype into a more differentiated epithelial phenotype represent a promising anticancer strategy. In a high-throughput assay based on E-cadherin (re)induction and the inhibition of tumor cell invasion, 44,475 low molecular weight (LMW) compounds were screened. The screening resulted in the identification of candidate compounds from the PROAM02 class. Selected LMW compounds activated E-cadherin promoter activity and inhibited cancer cell invasion in multiple metastatic human cancer cell lines. The intraperitoneal administration of selected LMW compounds reduced the tumor burden in human prostate and breast cancer in vivo mouse models. Moreover, selected LMW compounds decreased the intra-bone growth of xenografted human prostate cancer cells. This study describes the identification of the PROAM02 class of small molecules that can be exploited to reduce cancer cell invasion and metastases. Further clinical evaluation of selected candidate inhibitors is warranted to address their safety, bioavailability and antitumor efficacy in the management of patients with aggressive cancers.

A multimodal molecular imaging approach targeting urokinase plasminogen activator receptor for the diagnosis, resection and surveillance of urothelial cell carcinoma.

With a 5-year recurrence rate of 30-78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. METHODS: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. RESULTS: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. CONCLUSIONS: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.

Cationic amphiphilic drugs as potential anticancer therapy for bladder cancer.

More effective therapy for patients with either muscle-invasive or high-risk non-muscle-invasive urothelial carcinoma of the bladder (UCB) is an unmet clinical need. For this, drug repositioning of clinically approved drugs represents an interesting approach. By repurposing existing drugs, alternative anticancer therapies can be introduced in the clinic relatively fast, because the safety and dosing of these clinically approved pharmacological agents are generally well known. Cationic amphiphilic drugs (CADs) dose-dependently decreased the viability of a panel of human UCB lines in vitro. CADs induced lysosomal puncta formation, a hallmark of lysosomal leakage. Intravesical instillation of the CAD penfluridol in an orthotopic mouse xenograft model of human UCB resulted in significantly reduced intravesical tumor growth and metastatic progression. Furthermore, treatment of patient-derived ex vivo cultured human UCB tissue caused significant partial or complete antitumor responses in 97% of the explanted tumor tissues. In conclusion, penfluridol represents a promising treatment option for bladder cancer patients and warrants further clinical evaluation.

An ex vivo Tissue Culture Model for the Assessment of Individualized Drug Responses in Prostate and Bladder Cancer.

Urological malignancies, including prostate and bladder carcinoma, represent a major clinical problem due to the frequent occurrence of therapy resistance and the formation of incurable distant metastases. As a result, there is an urgent need for versatile and predictive disease models for the assessment of the individualized drug response in urological malignancies. Compound testing on ex vivo cultured patient-derived tumor tissues could represent a promising approach. In this study, we have optimized an ex vivo culture system of explanted human prostate and bladder tumors derived from clinical specimens and human cancer cell lines xenografted in mice. The explanted and cultured tumor slices remained viable and tissue architecture could be maintained for up to 10 days of culture. Treatment of ex vivo cultured human prostate and bladder cancer tissues with docetaxel and gemcitabine, respectively, resulted in a dose-dependent anti-tumor response. The dose-dependent decrease in tumor cells upon administration of the chemotherapeutic agents was preceded by an induction of apoptosis. The implementation and optimization of the tissue slice technology may facilitate the assessment of anti-tumor efficacies of existing and candidate pharmacological agents in the complex multicellular neoplastic tissues from prostate and bladder cancer patients. Our model represents a versatile "near-patient" tool to determine tumor-targeted and/or stroma-mediated anti-neoplastic responses, thus contributing to the field of personalized therapeutics.

Spontaneous bone metastases in a preclinical orthotopic model of invasive lobular carcinoma; the effect of pharmacological targeting TGFß receptor I kinase.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80-90% and 10-15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E-cadherin and p53 in a conditional K14cre;Cdh1((F/F));Trp53((F/F)) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD-208 (60 mg/kg per day), an orally available TGFß receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD-208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD-208 treatment reduced TGFß signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti-tumour effects of TGFß. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events.

Non-invasive stress-free application of glucocorticoid ligands in mice.

Most drug delivery procedures induce stress, which might interfere with the pharmacological action of the drug and behaviour. Stress is deduced from high and long-lasting elevations of the hormone corticosterone. We set out to develop a non-invasive, stress-free method of drug delivery in mice. Validation consisted of delivery of glucocorticoid ligands via oats to male C57BL/6J mice. Oat consumption induced a small increase in corticosterone concentrations after 15 min (<50 ng/ml) that returned to low resting levels at t=30 (<10 ng/ml). Gavage and intraperitoneal (i.p.) vehicle injections resulted in long-lasting corticosterone elevations (>100 ng/ml at t=30 and approximately 50 ng/ml at t=60 min after delivery). Adding corticosterone to oats resulted in threefold higher plasma corticosterone in the 15.0-mg/kg group (+/-250 ng/ml) compared to the 4.5-mg/kg group at t=30 and 90. Application of the glucocorticoid receptor antagonist RU38486 (200 mg/kg) elevated plasma corticosterone for at least 8h. Additional swimming increased corticosterone even further. Presumably, already the small oat-consumption-induced increase of corticosterone requires negative feedback via glucocorticoid receptors. In conclusion, the context-dependent and dose-controlled application of drugs via oats avoids confounding strong stress system activation and makes it suitable for studies on learning and memory processes.

Differential MR/GR activation in mice results in emotional states beneficial or impairing for cognition.

Corticosteroids regulate stress response and influence emotion, learning, and memory via two receptors in the brain, the high-affinity mineralocorticoid (MR) and low-affinity glucocorticoid receptor (GR). We test the hypothesis that MR- and GR-mediated effects interact in emotion and cognition when a novel situation is encountered that is relevant for a learning process. By adrenalectomy and additional constant corticosterone supplement we obtained four groups of male C57BL/6J mice with differential chronic MR and GR activations. Using a hole board task, we found that mice with continuous predominant MR and moderate GR activations were fast learners that displayed low anxiety and arousal together with high directed explorative behavior. Progressive corticosterone concentrations with predominant action via GR induced strong emotional arousal at the expense of cognitive performance. These findings underline the importance of a balanced MR/GR system for emotional and cognitive functioning that is critical for mental health.

Age-related changes in hypothalamic-pituitary-adrenal axis activity of male C57BL/6J mice.

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.