Combination of the c-Met Inhibitor Tivantinib and Zoledronic Acid Prevents Tumor Bone Engraftment and Inhibits Progression of Established Bone Metastases in a Breast Xenograft Model

Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by in vivo non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells in vitro. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases.     

Biological basis of radiochemotherapy

The tumor biological and radiobiological aspects, the mechanism of actions and general considerations of clinical application of radiochemotherapy are discussed. The aims of radiochemotherapy are to prolong the patient's survival by improving local tumor control and decreasing distant metastases. The goal of radiochemotherapy is to enhance the therapeutic effect of radiation with tolerable and controllable local and systemic side effects. The mechanism of action of the most frequently used drugs are also discussed.     

ErbB3 silencing reduces osteosarcoma cell proliferation and tumor growth in vivo

Osteosarcoma is the most common primary bone tumor in children and adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of osteosarcoma treatment. The identification of the molecular signals that contribute to the aberrant osteosarcoma cell growth may provide clues to develop new therapeutic strategies for chemoresistant osteosarcoma. Here we show that the expression of ErbB3 is increased in human osteosarcoma cells in vitro. Tissue microarray analysis of tissue cores from osteosarcoma patients further showed that the ErbB3 protein expression is higher in bone tumors compared to normal bone tissue, and is further increased in patients with recurrent disease or soft tissue metastasis. In murine osteosarcoma cells, silencing ErbB3 using shRNA decreased cell replication, cell migration and invasion, indicating that ErbB3 contributes to tumor cell growth and invasiveness. Furthermore, ErbB3 silencing markedly reduced tumor growth in a murine allograft model in vivo. Immunohistochemal analysis showed that the reduced tumor growth induced by ErbB3 silencing in this model resulted from decreased cell osteosarcoma cell proliferation, supporting a role of ErbB3 in bone tumor growth in vivo. Taken together, the results reveal that ErbB3 expression in human osteosarcoma correlates with tumor grade. Furthermore, silencing ErbB3 in a murine osteosarcoma model results in decreased cell growth and invasiveness in vitro, and reduced tumor growth in vivo, which supports the potential therapeutic interest of targeting ErbB3 in osteosarcoma.     

Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.     

Therapeutic Effect of α-Emitting 224Ra-Labeled Calcium Carbonate Microparticles in Mice with Intraperitoneal Ovarian Cancer

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter ²²⁴Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of ²²⁴Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with ²²⁴Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with ²²⁴Ra-microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma.     

Aptamer-Dendrimer Bioconjugates for Targeted Delivery of miR-34a Expressing Plasmid and Antitumor Effects in Non-Small Cell Lung Cancer Cells

Metastasis and drug resistance are major barriers for the treatment of non-small cell lung cancer (NSCLC). To explore new therapeutic options, we successfully encapsulated MicroRNA-34a (miR-34a), a potent endogenous tumor suppressor in NSCLC into S6 aptamer-conjugated dendrimer to form lung cancer-targeted gene delivery nanoparticles (PAM-Ap/pMiR-34a NPs). PAM-Ap/pMiR-34a NPs had a diameter of 100–200 nm and Zeta potential of ~30 mV at applied N/P ratio. The aptamer conjugation significantly improved cellular uptake as well as gene transfection efficiency of PAM-Ap/pMiR-34a NPs in cultured NSCLC cells. We showed that PAM-Ap/pMiR-34a NPs enhanced the regulation of targeted genes, BCL-2 and p53 in vitro. In addition, we revealed PAM-Ap/pMiR-34a NPs significantly inhibited cell growth, migration, invasion and induced apoptosis of lung cancer cells compared with non-targeted NPs. The method provided a novel therapeutic strategy for the experimental treatment of NSCLC.     

Growth Pattern Analysis of Murine Lung Neoplasms by Advanced Semi-Automated Quantification of Micro-CT Images

Computed tomography (CT) is a non-invasive imaging modality used to monitor human lung cancers. Typically, tumor volumes are calculated using manual or semi-automated methods that require substantial user input, and an exponential growth model is used to predict tumor growth. However, these measurement methodologies are time-consuming and can lack consistency. In addition, the availability of datasets with sequential images of the same tumor that are needed to characterize in vivo growth patterns for human lung cancers is limited due to treatment interventions and radiation exposure associated with multiple scans. In this paper, we performed micro-CT imaging of mouse lung cancers induced by overexpression of ribonucleotide reductase, a key enzyme in nucleotide biosynthesis, and developed an advanced semi-automated algorithm for efficient and accurate tumor volume measurement. Tumor volumes determined by the algorithm were first validated by comparison with results from manual methods for volume determination as well as direct physical measurements. A longitudinal study was then performed to investigate in vivo murine lung tumor growth patterns. Individual mice were imaged at least three times, with at least three weeks between scans. The tumors analyzed exhibited an exponential growth pattern, with an average doubling time of 57.08 days. The accuracy of the algorithm in the longitudinal study was also confirmed by comparing its output with manual measurements. These results suggest an exponential growth model for lung neoplasms and establish a new advanced semi-automated algorithm to measure lung tumor volume in mice that can aid efforts to improve lung cancer diagnosis and the evaluation of therapeutic responses.     

Development of Novel Patient-Derived Preclinical Models from Malignant Effusions in Patients with Tyrosine Kinase Inhibitor–Resistant Clear Cell Renal Cell Carcinoma

PURPOSE: Although targeting angiogenesis with tyrosine kinase inhibitors (TKIs) has become standard of care in the treatment of clear cell renal cell carcinoma (RCC), resistance mechanism are not fully understood, and there is a need to develop new therapeutic options overcoming them. METHODS AND MATERIALS: To develop a preclinical model that predicts clinical activity of novel agents in 19 RCC patients, we established patient-derived cell (PDC) and xenograft (PDX) models derived from malignant effusions or surgical specimen. RESULTS: Successful PDCs, defined as cells that maintained growth following two passages, were established in 5 of 15 malignant effusions and 1 of 4 surgical specimens. One PDC, clinically refractory to TKIs, was implanted and engrafted in mice, resulting in a comparable histology to the primary tumor. The PDC-PDX model also showed similar genomic features when tested using targeted sequencing of cancer-related genes. When we examined the drug effects of the PDX model, the tumor cells showed resistance to TKIs and everolimus in vitro. CONCLUSION: The results suggest that the PDC-PDX preclinical model we developed using malignant effusions can be a useful preclinical model to interrogate sensitivity to targeted agents based on genomic alterations.     

Large Shionogi tumors lose their responsiveness to flutamide treatment

Cancer of the prostate is the most frequent cancer and the second leading cause of cancer death in men in North America. The growth of Shionogi carcinoma-115 (SC-115) cells is highly sensitive to androgens, and this cell line is a well known experimental model of prostate cancer. The transplantable Shionogi carcinoma tumor was used to assess the influence of tumor size on the response to flutamide treatment. Two weeks after subcutaneous inoculation of tumor fragments in Shionogi mice, six groups of animals bearing SC-115 tumors ranging from 0.1 to 1.8 cm in diameter were treated with flutamide (1 mg, twice daily). The castrated mice received an androstenedione (Δ⁴-dione) implant to mimic the human situation, where the adrenals produce precursor steroids which are transformed into androgens in peripheral intracrine tissues. After 16 days, treatment with flutamide inhibited tumor growth by 32 to 57% in the four groups of mice having tumors ranging from 0.1 to 1.0 cm in diameter at day 0, whereas no significant inhibitory effect was observed in larger tumors. The same treatment, however, caused potent inhibitory effects on other androgen-sensitive parameters, namely prostatic and seminal vesicle weight and kidney ornithine decarboxylase (ODC) activity, the effect on these parameters being similar in all groups of animals, irrespective of tumor size. Furthermore, when those larger tumors unresponsive to antiandrogenic treatment were cut into small fragments and inoculated into new groups of mice, the same treatment with flutamide efficiently inhibited tumor growth, treatment being started at tumor sizes of 0.1 to 0.3 cm in diameter. The present data clearly demonstrate that small tumors are highly sensitive to androgen deprivation, while loss of response develops with increasing tumor size, thus indicating that, for optimal efficacy, androgen blockade should be given at the early stages of prostate cancer.     

Functional evaluation of therapeutic response for a mouse model of medulloblastoma

Medulloblastoma is an aggressive childhood cerebellar tumor. We recently reported a mouse model with conditional deletion of Patched1 gene that recapitulates many characteristics of the human medulloblastoma. Qualitative symptoms observed in the mouse model include irregular stride length, impaired cranial nerve function and decreased motor coordination and performance. In our current study, several quantitative behavioral assays including a mouse rotarod, a forced air challenge, a screen inversion test, a horizontal wire test, and stride length analysis were evaluated to determine the most sensitive and cost-effective functional assay for impaired neuromotor behavior associated with disease progression. Magnetic resonance imaging (MRI) was used to confirm and monitor tumor growth and as an anatomical biomarker for therapeutic response. Wild type mice or medulloblastoma-prone, conditional Patched1 knockout mice were observed by behavioral assays and MRI from postnatal weeks 3–6. Bortezomib treatment was administered during this period and therapeutic response was assessed using cerebellar volumes at the end of treatment. Of the behavioral tests assessed in this study, stride length analysis was best able to detect differences between tumor-prone mice and wild type mice as early as postnatal day 37 (P = 0.003). Significant differences between stride lengths of bortezomib treated and control tumor-bearing mice could be detected as early as postnatal day 42 (P = 0.020). Cerebellar volumes measured by MRI at the end of treatment validated the therapeutic effects seen by behavioral tests (P = 0.03). These findings suggest that stride length analysis may serve as one of the more sensitive and cost-effective method for assessing new therapeutic compounds in this and other preclinical model of brain tumors.     

Integration of autologous dendritic cell-based immunotherapy in the standard of care treatment for patients with newly diagnosed glioblastoma: results of the HGG-2006 phase I/II trial

Purpose

Dendritic cell (DC)-based tumor vaccination has rendered promising results in relapsed high-grade glioma patients. In the HGG-2006 trial (EudraCT 2006-002881-20), feasibility, toxicity, and clinical efficacy of the full integration of DC-based tumor vaccination into standard postoperative radiochemotherapy are studied in 77 patients with newly diagnosed glioblastoma.

Patients and methods

Autologous DC are generated after leukapheresis, which is performed before the start of radiochemotherapy. Four weekly induction vaccines are administered after the 6-week course of concomitant radiochemotherapy. During maintenance chemotherapy, 4 boost vaccines are given. Feasibility and progression-free survival (PFS) at 6?months (6mo-PFS) are the primary end points. Overall survival (OS) and immune profiling, rather than monitoring, as assessed in patients’ blood samples, are the secondary end points. Analysis has been done on intent-to-treat basis.

Results

The treatment was feasible without major toxicity. The 6mo-PFS was 70.1?% from inclusion. Median OS was 18.3?months. Outcome improved significantly with lower EORTC RPA classification. Median OS was 39.7, 18.3, and 10.7?months for RPA classes III, IV, and V, respectively. Patients with a methylated MGMT promoter had significantly better PFS (p?=?0.0027) and OS (p?=?0.0082) as compared to patients with an unmethylated status. Exploratory “immunological profiles” were built to compare to clinical outcome, but no statistical significant evidence was found for these profiles to predict clinical outcome.

Conclusion

Full integration of autologous DC-based tumor vaccination into standard postoperative radiochemotherapy for newly diagnosed glioblastoma seems safe and possibly beneficial. These results were used to power the currently running phase IIb randomized clinical trial.     

Anti-tumor effects of rigosertib in high-risk neuroblastoma

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.     

MicroRNA-340 suppresses osteosarcoma tumor growth and metastasis by directly targeting ROCK1

MicroRNAs (miRNAs) play key roles in cancer development and progression. In the present study, we investigated the role of miR-340 in the progression and metastasis of osteosarcoma (OS). Our results showed that miR-340 was frequently downregulated in OS tumors and cell lines. Overexpression of miR-340 in OS cell lines significantly inhibited cell proliferation, migration, and invasion in vitro, and tumor growth and metastasis in a xenograft mouse model. ROCK1 was identified as a target of miR-340, and ectopic expression of miR-340 downregulated ROCK1 by direct binding to its 3′ untranslated region. siRNA-mediated silencing of ROCK1 phenocopied the effects of miR-340 overexpression, whereas restoration of ROCK1 in miR-340-overexpressing OS cells reversed the suppressive effects of miR-340. Together, these findings indicate that miR-340 acts as a tumor suppressor and its downregulation in tumor tissues may contribute to the progression and metastasis of OS through a mechanism involving ROCK1, suggesting miR-340 as a potential new diagnostic and therapeutic target for the treatment of OS.     

Model-Based Tumor Growth Dynamics and Therapy Response in a Mouse Model of De Novo Carcinogenesis

Tumorigenesis is a complex, multistep process that depends on numerous alterations within the cell and contribution from the surrounding stroma. The ability to model macroscopic tumor evolution with high fidelity may contribute to better predictive tools for designing tumor therapy in the clinic. However, attempts to model tumor growth have mainly been developed and validated using data from xenograft mouse models, which fail to capture important aspects of tumorigenesis including tumor-initiating events and interactions with the immune system. In the present study, we investigate tumor growth and therapy dynamics in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. We show that the rate of tumor growth and the effects of therapy are highly variable and mouse specific using a Gompertz model to describe tumor growth and a two-compartment pharmacokinetic/ pharmacodynamic model to describe the effects of therapy in mice treated with 5-FU. We show that inter-mouse growth variability is considerably larger than intra-mouse variability and that there is a correlation between tumor growth and drug kill rates. Our results show that in vivo tumor growth and regression in a double transgenic mouse model are highly variable both within and between subjects and that mathematical models can be used to capture the overall characteristics of this variability. In order for these models to become useful tools in the design of optimal therapy strategies and ultimately in clinical practice, a subject-specific modelling strategy is necessary, rather than approaches that are based on the average behavior of a given subject population which could provide erroneous results.     

Imaging CXCL12-CXCR4 Signaling in Ovarian Cancer Therapy

Chemokine CXCL12 and receptor CXCR4 have emerged as promising therapeutic targets for ovarian cancer, a disease that continues to have a dismal prognosis. CXCL12-CXCR4 signaling drives proliferation, survival, and invasion of ovarian cancer cells, leading to tumor growth and metastasis. Pleiotropic effects of CXCR4 in multiple key steps in ovarian cancer suggest that blocking this pathway will improve outcomes for patients with this disease. To quantify CXCL12-CXCR4 signaling in cell-based assays and living mouse models of ovarian cancer, we developed a click beetle red luciferase complementation reporter that detects activation of CXCR4 based on recruitment of the cytosolic adapter protein β-arrestin 2. Both in two-dimensional and three-dimensional cell cultures, we established that bioluminescence from this reporter measures CXCL12-dependent activation of CXCR4 and inhibition of this pathway with AMD3100, a clinically-approved small molecule that blocks CXCL12-CXCR4 binding. We used this imaging system to quantify CXCL12-CXCR4 signaling in a mouse model of metastatic ovarian cancer and showed that treatment with AMD3100 interrupted this pathway in vivo. Combination therapy with AMD3100 and cisplatin significantly decreased tumor burden in mice, although differences in overall survival were not significantly greater than treatment with either agent as monotherapy. These studies establish a molecular imaging reporter system for analyzing CXCL12-CXCR4 signaling in ovarian cancer, which can be used to investigate biology and therapeutic targeting of this pathway in cell-based assays and living mice.     

Gene transfer of IFN-gamma into established brain tumors represses growth by antiangiogenesis

The experiments in this paper were designed to examine the therapeutic effects of adenoviral-mediated gene transfer of IFN-gamma into a mouse model of an established metastatic brain tumor. Temperature-sensitive replication-defective adenovirus was generated for gene transfer of IFN-gamma (AdIFN) and beta-galactosidase (AdBGAL) cDNAs in vivo. In this model, treatment with AdIFN elicits prolonged survival times and brain tumor rejection. Evidence against an immune-mediated response accounting for this result include: 1) absence of a memory immune response upon challenge, 2) lack of antitumor effects at sites distal to inoculation of AdIFN, and 3) preservation of the therapeutic effects of AdIFN in scid and beige mice and in inducible NO synthase (iNOS) knockouts. High concentrations of IFN-gamma do not inhibit tumor growth in vitro making it unlikely that the antitumor effect of this treatment acts directly on the growth of the tumor cells. However, gene transfer of IFN-gamma inhibits neovascularization of the tumor in a 3LL-Matrigel assay in vivo, and AdIFN induces apoptosis of endothelial cells in vivo, supporting the idea that AdIFN represses tumor growth by inhibiting angiogenesis. The substantial non-immune-mediated therapeutic benefits of AdIFN in animals paves the way for devising novel strategies for treating human brain tumors.     

Visible light and/or UVA offer a strong amplification of the anti-tumor effect of curcumin

Curcumin, a dietary pigment from the plant Curcuma longa, inhibits cell proliferation and induces apoptosis in different cell lines. The therapeutic benefit is hampered by a very low absorption after trans-dermal or oral application. Therefore, great efforts were undertaken to enhance the effectiveness of curcumin. Recently, it was demonstrated that curcumin offers the described effects also at low concentrations (0.2–1 μg/ml) when applied in combination with UVA or visible light. The efficacy of this combination was shown in human epidermal keratinocytes and in a panel of other cell species in vitro as well as in a xenograft tumor model with A431 tumor cells injected subcutaneously in the flanks of NMRI nude mice in vivo. The treatment of keratinocytes with curcumin and light resulted in the inhibition of cell growth, and in the induction of apoptosis, whereas no toxic cell membrane damage was detectable. The treatment of tumor bearing nude mice with curcumin and visible light resulted in reduced tumor volumes, reduced proliferation rates, and the induction of apoptosis in the tumors. On the molecular level inhibition of extracellular regulated kinases 1/2 and epidermal growth factor receptor was observed which may aid to inhibition of proliferation and induction of apoptosis. This review covers the experiences of the new combination treatment of human tumors.