Prevent Cell Dissemination Research Articles

Abstract 5468: Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance

Abstract Introduction: Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are lymphoplasmacytic malignancies which are characterized by continuous spread through cell trafficking in and out of the BM. We have recently shown that the spread process in these diseases is mediated by hypoxia. Moreover, despite the introduction of novel therapies, cancer patients relapse due to the development of a drug-resistant stem cell-like subpopulation, which we recently found to be, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome spread and drug resistance in MM and WM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve cancer patient outcome when combined with radiotherapy. We use TPZ for the first time in hematologic malignancies to target the drug-resistant cancer cells and sensitize them to therapy. Procedures: Cell survival was tested on MM (MM1.s, H929, OPM1, RPMI8226) and WM (BCWM.1, MWCL.1) cell lines exposed to normoxia (21% O2) or hypoxia (1% O2) for 24hrs in the presence of bortezomib and carfilzomib, different concentrations of TPZ (in order to obtain an IC50), or combination of those drugs using MTT assay. For adhesion assay, cancer cells were pre-labeled with calcein-AM, treated with TPZ, applied to unlabeled endothelial cells, non-adherent cells were washed after 1hr, and adherent cells were analyzed by a fluorescent reader. Chemotactic properties of cells were tested using a transwell chamber and the migrated cells were counted by flow cytometry. For in vivo study, 5 × 106 MM1s-Luc-GFP cells were injected i.v. into 6 SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. All mice were treated with (1) high-dose bortezomib (1.5mg/kg) only (n = 3), or (2) bortezomib and TPZ (40mg/kg; n = 3), all administered i.p. sequentially twice a week. Bone marrow and peripheral blood were collected and analyzed for the presence of GFP+ cells by flow cytometry. Summary of data: We found that hypoxia induced drug resistance to bortezomib and carfilzomib of MM and WM cells, and that TPZ was active in a dose-dependent manner only in hypoxic conditions. Combination of TPZ with bortezomib and carfilzomib, resensitized cancer cells to death in hypoxia. Addition of TPZ increased adhesion of cancer cells to endothelial cells, and decreased chemotactic properties of cancer cells in hypoxia in vitro. Moreover, post-treatment residual cancer cells were further decreased using TPZ, preventing recurrence of MM in vivo. Conclusion: We report that TPZ prevents hypoxia-induced metastasis and overcomes hypoxia-induced drug resistance in MM and WM. This is the first study to show the efficacy of TPZ in hematologic malignancies in general and in MM and WM in particular. This data provide a preclinical basis for future clinical trials testing efficacy of TPZ in MM and WM. Citation Format: Barbara Muz, Pilar de la Puente, Feda Azab, Micah Luderer, Abdel Kareem Azab. Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2015-5468

Cyr61 silencing reduces vascularization and dissemination of osteosarcoma tumors.

Osteosarcoma is the most prevalent primary pediatric cancer-related bone disease. These tumors frequently develop resistance to chemotherapy and are highly metastatic, leading to poor outcome. Thus, there is a need for new therapeutic strategies that can prevent cell dissemination. We previously showed that CYR61/CCN1 expression in osteosarcoma cells is correlated to aggressiveness both in vitro and in vivo in mouse models, as well as in patients. In this study, we found that CYR61 is a critical contributor to the vascularization of primary tumor. We demonstrate that silencing CYR61, using lentiviral transduction, leads to a significant reduction in expression level of pro-angiogenic markers such as VEGF, FGF2, PECAM and angiopoietins concomitantly to an increased expression of major anti-angiogenic markers such as thrombospondin-1 and SPARC. Matrix metalloproteinase-2 family member expression, a key pathway in osteosarcoma metastatic capacity was also downregulated when CYR61 was downregulated in osteosarcoma cells. Using a metastatic murine model, we show that CYR61 silencing in osteosarcoma cells results in reduced tumor vasculature and slows tumor growth compared with control. We also find that microvessel density correlates with lung metastasis occurrence and that CYR61 silencing in osteosarcoma cells limits the number of metastases. Taken together, our data indicate that CYR61 silencing can blunt the malignant behavior of osteosarcoma tumor cells by limiting primary tumor growth and dissemination process.

Peri-operative filtration of disseminated cytokeratin positive cells in patients with colorectal liver metastasis

Liver resection is the only potential cure for patients with colorectal liver metastasis. However, more than 30% of patients will develop tumour recurrence, probably caused by tumour cells disseminated before or during surgery. As prevention of cell dissemination is barely obtainable, alternative concepts have to be discussed. The potential of leukocyte adhesion filters for the removal of cytokeratin positive cells (CK+) from blood was studied in 18 patients undergoing liver resection for colorectal liver metastasis. Blood sampling was done via a liver venous catheter during hepatic mobilisation. Filtration was done with an in-line WBF2 filter system. To define the relation between surgery and cell release we compared patients' pre-operative and intra-operative blood and bone marrow (BM) samples with their CK expression using immunochemical staining. CK+ cells were detected in BM samples of nine of 14 patients before surgery, indicating early dissemination. In ten of 18 patients CK+ cells were detected in blood samples during hepatic mobilisation; all ten patients underwent major liver surgery (R0 resection). In those patients recurrent disease was observed more often (P < or = 0.05). In 17 of 18 patients CK+ cells were not detectable after filtration procedure, which indicated cell adhesion to the filter medium. Liver resection due to metastasis leads to frequent intra-operative tumour cell shedding. As the detection of CK+ cells is correlated with disease recurrence, modification of surgical techniques to prevent cell dissemination, and additional therapeutic concepts such as advanced filtration technology, have to be discussed.