Defining tumor stage at diagnosis is a pivotal point for clinical decisions about patient treatment strategies. In this respect, early detection of occult metastasis invisible to current imaging ...methods would have a major impact on best care and long-term survival. Mathematical models that describe metastatic spreading might estimate the risk of metastasis when no clinical evidence is available. In this study, we adapted a top-down model to make such estimates. The model was constituted by a transport equation describing metastatic growth and endowed with a boundary condition for metastatic emission. Model predictions were compared with experimental results from orthotopic breast tumor xenograft experiments conducted in Nod/Scidγ mice. Primary tumor growth, metastatic spread and growth were monitored by 3D bioluminescence tomography. A tailored computational approach allowed the use of Monolix software for mixed-effects modeling with a partial differential equation model. Primary tumor growth was described best by Bertalanffy, West, and Gompertz models, which involve an initial exponential growth phase. All other tested models were rejected. The best metastatic model involved two parameters describing metastatic spreading and growth, respectively. Visual predictive check, analysis of residuals, and a bootstrap study validated the model. Coefficients of determination were Formula: see text for primary tumor growth and Formula: see text for metastatic growth. The data-based model development revealed several biologically significant findings. First, information on both growth and spreading can be obtained from measures of total metastatic burden. Second, the postulated link between primary tumor size and emission rate is validated. Finally, fast growing peritoneal metastases can only be described by such a complex partial differential equation model and not by ordinary differential equation models. This work advances efforts to predict metastatic spreading during the earliest stages of cancer.
Metronomic chemotherapy is usually associated with better tolerance than conventional chemotherapy, and encouraging response rates have been reported in various settings. However, clinical ...development of metronomic chemotherapy has been hampered by a number of limitations, including the vagueness of its definition and the resulting empiricism in protocol design. In this study, we developed a pharmacokinetic/pharmacodynamic mathematical model that identifies
the most effective administration schedule for gemcitabine monotherapy. This model is based upon four biological assumptions regarding the mechanisms of action of metronomic chemotherapy, resulting in a set of 6 minimally parameterized differential equations. Simulations identified daily 0.5-1 mg/kg gemcitabine as an optimal protocol to maximize antitumor efficacy. Both metronomic protocols (0.5 and 1 mg/kg/day for 28 days) were evaluated in chemoresistant neuroblastoma-bearing mice and compared with the standard MTD protocol (100 mg/kg once a week for 4 weeks). Systemic exposure to gemcitabine was 14 times lower in the metronomic groups compared with the standard group. Despite this, metronomic gemcitabine significantly inhibited tumor angiogenesis and reduced tumor perfusion and inflammation
, while standard gemcitabine did not. Furthermore, metronomic gemcitabine yielded a 40%-50% decrease in tumor mass at the end of treatment as compared with control mice (
= 0.002; ANOVA on ranks with Dunn test), while standard gemcitabine failed to significantly reduce tumor growth. Stable disease was maintained in the metronomic groups for up to 2 months after treatment completion (67%-72% reduction in tumor growth at study conclusion,
< 0.001; ANOVA on ranks with Dunn test). Collectively, our results confirmed the superiority of metronomic protocols in chemoresistant tumors
.
Nanoparticles are of rising interest in cancer research, but in vitro canonical cell monolayer models are not suitable to evaluate their efficacy when prototyping candidates. Here, we developed ...three-dimensional (3D) spheroid models to test the efficacy of trastuzumab-docetaxel immunoliposomes in breast cancer prior to further testing them in vivo.
Immunoliposomes were synthesized using the standard thin film method and maleimide linker. Two human breast cancer cell lines varying in Her2 expression were tested: Her2+ cells derived from metastatic site: mammary breast MDA-MB-453 and triple-negative MDA-MB-231 cells. 3D spheroids were developed and tested with fluorescence detection to evaluate viability. In vivo efficacy and biodistribution studies were performed on xenograft bearing nude mice using fluorescent and bioluminescent imaging.
In vitro, antiproliferative efficacy was dependent upon cell type, size of the spheroids, and treatment scheduling, resulting in subsequent changes between tested conditions and in vivo results. Immunoliposomes performed better than free docetaxel + free trastuzumab and ado-trastuzumab emtansine (T-DM1). On MDA-MB-453 and MDA-MB-231 cell growth was reduced by 76% and 25%, when compared to free docetaxel + free trastuzumab and by 85% and 70% when compared to T-DM1, respectively. In vivo studies showed tumor accumulation ranging from 3% up to 15% of the total administered dose in MDA-MB-453 and MDA-MB-231 bearing mice. When compared to free docetaxel + free trastuzumab, tumor growth was reduced by 89% (MDA-MB-453) and 25% (MDA-MB-231) and reduced by 66% (MDA-MB-453) and 29% (MDA-MB-231) when compared to T-DM1, an observation in line with data collected from 3D spheroids experiments.
We demonstrated the predictivity of 3D in vitro models when developing and testing nanoparticles in experimental oncology. In vitro and in vivo data showed efficient drug delivery with higher efficacy and prolonged survival with immunoliposomes when compared to current anti-Her2 breast cancer strategies.
Prostate cancer (PCa) is the second most common cancer in men worldwide and the fifth leading cause of death by cancer. The overexpression of TCTP protein plays an important role in castration ...resistance. Over the last decade, antisense technology has emerged as a rising strategy in oncology. Using antisense oligonucleotide (ASO) to silence TCTP protein is a promising therapeutic option-however, the pharmacokinetics of ASO does not always meet the requirements of proper delivery to the tumor site. In this context, developing drug delivery systems is an attractive strategy for improving the efficacy of ASO directed against TCTP. The liposome should protect and deliver ASO at the intracellular level in order to be effective. In addition, because prostate cancer cells express Her2, using an anti-Her2 targeting antibody will increase the affinity of the liposome for the cell and optimize the intratumoral penetration of the ASO, thus improving efficacy. Here, we have designed and developed pegylated liposomes and Her2-targeting immunoliposomes. Mean diameter was below 200 nm, thus ensuring proper enhanced permeation and retention (EPR) effect. Encapsulation rate for ASO was about 40%. Using human PC-3 prostate cancer cells as a canonical model, free ASO and ASO encapsulated into either liposomes or anti-Her2 immunoliposomes were tested for efficacy in vitro using 2D and 3D spheroid models. While the encapsulated forms of ASO were always more effective than free ASO, we observed differences in efficacy of encapsulated ASO. For short exposure times (i.e., 4 h) ASO liposomes (ASO-Li) were more effective than ASO-immunoliposomes (ASO-iLi). Conversely, for longer exposure times, ASO-iLi performed better than ASO-Li. This pilot study demonstrates that it is possible to encapsulate ASO into liposomes and to yield antiproliferative efficacy against PCa. Importantly, despite mild Her2 expression in this PC-3 model, using a surface mAb as targeting agent provides further efficacy, especially when exposure is longer. Overall, the development of third-generation ASO-iLi should help to take advantage of the expression of Her2 by prostate cancer cells in order to allow greater specificity of action in vivo and thus a gain in efficacy.
PURPOSE Anticipating toxicities with gemcitabine is an ongoing story, and deregulation in cytidine deaminase (CDA) could be associated with increased risk of developing early severe toxicities on ...drug exposure. PATIENTS AND METHODS A simple test to evaluate CDA phenotypic status was first validated in an animal model investigating relationships between CDA activity and gemcitabine-related toxicities. Next, relevance of this test as a marker for toxicities was retrospectively tested in a first subset of 64 adult patients treated with gemcitabine alone, then it was tested in a larger group of 130 patients who received gemcitabine either alone or combined with other drugs and in 20 children. Additionally, search for the 435 T>C, 208 G>A and 79 A>C mutations on the CDA gene was performed. Results In mice, CDA deficiency impacted on gemcitabine pharmacokinetics and had subsequent lethal toxicities. In human, 12% of adult patients experienced early severe toxicities after gemcitabine administration. A significant difference in CDA activities was observed between patients with and without toxicities (1.2 +/- 0.8 U/mg v 4 +/- 2.6 U/mg; P < .01). Conversely, no genotype-to-phenotype relationships were found. Of note, the patients who displayed particularly reduced CDA activity all experienced strong toxicities. Gemcitabine was well tolerated in children, and no CDA deficiency was evidenced. CONCLUSION Our data suggest that CDA functional testing could be a simple and easy marker to discriminate adult patients at risk of developing severe toxicities with gemcitabine. Particularly, this study demonstrates that CDA deficiency, found in 7% of adult patients, is associated with a maximum risk of developing early severe toxicities with gemcitabine.
Ascorbic acid (AA), or Vitamin C, is most well known as a nutritional supplement with antioxidant properties. Recently, we demonstrated that high concentrations of AA act on PMP22 gene expression and ...partially correct the Charcot-Marie-Tooth disease phenotype in a mouse model. This is due to the capacity of AA, but not other antioxidants, to down-modulate cAMP intracellular concentration by a competitive inhibition of the adenylate cyclase enzymatic activity. Because of the critical role of cAMP in intracellular signalling, we decided to explore the possibility that ascorbic acid could modulate the expression of other genes.
Using human pangenomic microarrays, we found that AA inhibited the expression of two categories of genes necessary for cell cycle progression, tRNA synthetases and translation initiation factor subunits. In in vitro assays, we demonstrated that AA induced the S-phase arrest of proliferative normal and tumor cells. Highest concentrations of AA leaded to necrotic cell death. However, quiescent cells were not susceptible to AA toxicity, suggesting the blockage of protein synthesis was mainly detrimental in metabolically-active cells. Using animal models, we found that high concentrations of AA inhibited tumor progression in nude mice grafted with HT29 cells (derived from human colon carcinoma). Consistently, expression of tRNA synthetases and ieF2 appeared to be specifically decreased in tumors upon AA treatment.
AA has an antiproliferative activity, at elevated concentration that could be obtained using IV injection. This activity has been observed in vitro as well in vivo and likely results from the inhibition of expression of genes involved in protein synthesis. Implications for a clinical use in anticancer therapies will be discussed.
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The development of nanoparticles could help to improve the efficacy/toxicity balance of drugs. This project aimed to develop liposomes and immunoliposomes using microfluidic mixing ...technology.Various formulation tests were carried out to obtain liposomes that met the established specifications. The liposomes were then characterized in terms of size, polydispersity index (PDI), docetaxel encapsulation rate and lamellarity. Antiproliferative activity was tested in human breast cancer models ranging from near-negative (MDA-MB-231), positive (MDA-MB-453) to HER2 positive. Pharmacokinetic studies were performed in C57BL/6 mice.Numerous batches of liposomes were synthesised using identical molar ratios and by varying the microfluidic parameters TFR, FRR and buffer. All synthesized liposomes have a size < 200 nm, but only Lipo-1, Lipo-6, Lipo-7, Lipo-8 have a PDI < 0.2, which meets our initial requirements. The size of the liposomes was correlated with the total FRR, for a 1:1 FRR the size is 122.2 ± 12.3 nm, whereas for a 1:3 FRR the size obtained is 163.4 ± 34.0 nm (p = 0.019. Three batches of liposomes were obtained with high docetaxel encapsulation rates > 80 %. Furthermore, in vitro studies on breast cancer cell lines demonstrated the efficacy of liposomes obtained by microfluidic mixing technique. These liposomes also showed improved pharmacokinetics compared to free docetaxel, with a longer half-life and higher AUC (3-fold and 3.5-fold increase for the immunoliposome, respectively).This suggests that switching to the microfluidic process will produce batches of liposomes with the same characteristics in terms of in vitro properties and efficacy, as well as the ability to release the encapsulated drug over time in vivo. This time-efficiency of the microfluidic technique is critical, especially in the early stages of development.
This study aimed at evaluating the reliability and precision of Diffuse Luminescent Imaging Tomography (DLIT) for monitoring primary tumor and metastatic spreading in breast cancer mice, and to ...develop a biomathematical model to describe the collected data. Using orthotopic mammary fat pad model of breast cancer (MDAMB231-Luc) in mice, we monitored tumor and metastatic spreading by three-dimensional (3D) bioluminescence and cross-validated it with standard bioluminescence imaging, caliper measurement and necropsy examination. DLIT imaging proved to be reproducible and reliable throughout time. It was possible to discriminate secondary lesions from the main breast cancer, without removing the primary tumor. Preferential metastatic sites were lungs, peritoneum and lymph nodes. Necropsy examinations confirmed DLIT measurements. Marked differences in growth profiles were observed, with an overestimation of the exponential phase when using a caliper as compared with bioluminescence. Our mathematical model taking into account the balance between living and necrotic cells proved to be able to reproduce the experimental data obtained with a caliper or DLIT imaging, because it could discriminate proliferative living cells from a more composite mass consisting of tumor cells, necrotic cell, or inflammatory tissues. DLIT imaging combined with mathematical modeling could be a powerful and informative tool in experimental oncology.
Recent clinical evidence revealed that the use of beta-blockers such as propranolol, prior to diagnosis or concurrently with chemotherapy, could increase relapse-free and overall survival in breast ...cancer patients. We therefore hypothesized that propranolol may be able to increase the efficacy of chemotherapy either through direct effects on cancer cells or via anti-angiogenic mechanisms. In vitro proliferation assay showed that propranolol (from 50-100 μM) induces dose-dependent anti-proliferative effects in a panel of 9 human cancer and "normal" cell lines. Matrigel assays revealed that propranolol displays potent anti-angiogenic properties at non-toxic concentrations (less than 50 μM) but exert no vascular-disrupting activity. Combining chemotherapeutic drugs, such as 5-fluorouracil (5-FU) or paclitaxel, with propranolol at the lowest effective concentration resulted in synergistic, additive or antagonistic effects on cell proliferation in vitro depending on the cell type and the dose of chemotherapy used. Interestingly, breast cancer and vascular endothelial cells were among the most responsive to these combinations. Furthermore, Matrigel assays indicated that low concentrations of propranolol (10 - 50 μM) potentiated the anti-angiogenic effects of 5-FU and paclitaxel. Using an orthotopic xenograft model of triple-negative breast cancer, based on s.c injection of luciferase-expressing MDA-MB-231 cells in the mammary fat pad of nude mice, we showed that propranolol, when used alone, induced only transient anti-tumor effects, if at all, and did not increase median survival. However, the combination of propranolol with chemotherapy resulted in more profound and sustained anti-tumor effects and significantly increased the survival benefits induced by chemotherapy alone (+19% and +79% in median survival for the combination as compared with 5-FU alone and paclitaxel alone, respectively; p less than 0.05). Collectively our results show that propranolol can potentiate the anti-angiogenic effects and anti-tumor efficacy of chemotherapy. The current study, together with retrospective clinical data, strongly suggests that the use of propranolol concurrently with chemotherapy may improve the outcome of breast cancer patients, thus providing a strong rationale for the evaluation of this drug combination in prospective clinical studies.
Developing targeted nanoparticles is a rising strategy to improve drug delivery in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal number at ...the surface remains a challenging issue, mainly due to the difficulties in measuring precisely surface coating levels when prototyping nanoparticles. We developed an original quantitative assay to measure the exact number of coated antibodies per nanoparticle. Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human IgG-kappa mimicking various amounts of antibodies, this new method was tested as part of the prototyping of docetaxel liposomes coated with trastuzumab against Her2+ breast cancer. This quantification method allowed to discriminate various batches of immunoliposomes depending on their trastuzumab density on nanoparticle surface (i.e., 330 (Immunoliposome-1), 480 (Immunoliposome-2) and 690 (Immunoliposome-3), p = 0.004, One-way ANOVA). Here we showed that optimal number of grafted antibodies on nanoparticles should be finely tuned and highest density of targeting agent is not necessarily associated with highest efficacy. Overall, this new method should help to better prototype third generation nanoparticles.
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