Leandro Marcelo Martinez

Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

BackgroundWhile breast cancer (BC) is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), stromal cell-derived factor-1 (SDF-1), and their receptors (R) in 2 human BC cell lines, MDA-MB-231 and MCF-7.MethodsOPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses.ResultsMCF-7 cells released higher levels of OPG in conditioned media (CM) than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3.ConclusionsMCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

Comparative prognostic relevance of breast intra-tumoral microvessel density evaluated by CD105 and CD146: A pilot study of 42 cases.

UNLABELLED Angiogenesis is a key process for metastatic progression. While it has been established that the evaluation of breast tumoral microvessel density by CD105 marker is a potential prognostic parameter, its evaluation by CD146 marker has been poorly studied. AIM The purpose of this study was to compare the prognostic value of intra-tumoral microvessel density assayed by CD105 and CD146 in early breast cancer patients. METHODS 42 women with breast infiltrative ductal carcinoma (I and II-stages) were retrospectively reviewed. Intra-tumoral microvessel density was immunohistochemically examined using antibodies anti-CD105 and CD146 in paraffin-embedded tissues, and their association with classical prognostic-markers, metastatic recurrence, metastasis-free survival and overall survival was analyzed. RESULTS High microvessel density assessed by CD146 was significantly associated with a higher risk of developing metastasis (p=0.0310) and a shorter metastasis-free survival (p=0.0197). In contrast, when we used the CD105-antibody, we did not find any significant association. Finally, CD146 showed to be an independent predictive indicator for metastasis-free survival (p=0.0055). CONCLUSION Our data suggest that the intra-tumoral microvessel density evaluated by CD146 may be a more suitable predictor of metastatic development than that evaluated by CD105 in early breast cancer.

PROPOSED MECHANISMS FOR OLIGONUCLEOTIDE IMT504 INDUCED DIABETES REVERSION IN A MOUSE MODEL OF IMMUNODEPENDENT DIABETES

Type 1 diabetes (T1D) originates from autoimmune β-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced β-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased β-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate β-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.

Abstract B66: Bone marrow microenvironment of advanced breast cancer patients without bone metastasis favors the cancer cell colonization

Bone metastasis is the major cause of death for advanced breast cancer patients (BCP). It is a multistep process that includes tumor cell mobilization, intravasation, survival in the circulation, extravasation and proliferation in bone marrow (BM) or bone. Accumulating evidence suggests that BM-mesenchymal stromal cells (MStC) play a critical role in BC-cell (BCC) colonization of the BM/bone. Despite increasing knowledge, the beginning of bone metastatic process in advanced BCP without BCC in the BM/bone has been poorly studied. So, this work was performed to evaluate the levels of OPG, RANKL, TRAIL, SDF-1, PDGF-AB, stanniocalcin-1 and MIF in peripheral blood (PB) and BM-plasma and BM-conditioned media (CM) of colony forming unit-fibroblastic cultures (CFU-F, day 14) from advanced BCP (IDC, without BM/bone metastasis) and healthy volunteers (HV). Also, we investigated the expression of membrane-RANKL (mRANKL) and SDF-1 in BM-MStC and their specific receptors in primary BC-tissue from these patients and MCF-7 and MDA-MB231 cells. At the end, we evaluated the effect of BM-plasma and CFU-F-CM over the migration and proliferation of both BCC lines. Methodology: Soluble factors were studied by ELISA. SDF-1, m-RANKL, CXCR-4 and RANK expression was analyzed by immunochemistry. Migration was performed over 14hs in transwells seeded with BCC exposed to BM-plasma and CFU-F-CM. Proliferation by MTS: after arrest, BCC were incubated for 48hs with 10% of BM-plasma or 100% or 50% of these CM with or without 1.25% FBS. Results: Significant difference in the OPG, RANKL, SDF-1 and MIF values in PB-plasma was found between both groups (BCP vs HV, X±SE, pg/ml): 2,005±195.90 vs 1,100±124.10 (p=0.001), 130.20±23.63 vs ≤31.25, 117±25 vs 254±28 (p<0.05) and 4,564±591 vs 2,265±402 (p<0.05), respectively. PDGF-AB level in BCP-BM-plasma was significant higher than HV-value (X±SE, pg/ml): 4,468±746 vs 2,528±421. 100% of BM-MStC expressed SDF-1 and mRANKL in both groups, but we observed higher mRANKL expression/MStC from BCP-BM compared to HV-values (++++vs++). The primary tissue-BCC expressed CXCR-4 and RANK (++) but we did not observe expression of them in the epithelial cells of non-malignan tissue. Moreover, 50% of BCC of both lines expressed CXCR-4 and 100% RANK. In addition, the BM-plasma and the CFU-F-CM from BCP induced a higher migration increase of MCF-7 and MDA-MB231cells compared with HV-values (p<0.05 in both lines and p<0.0001 and p=0.0356, respectively). We have not observed proliferation effect by CFU-F-CM over any of the lines, but we did observe a significant higher proliferation of MDA-MB231 cells when we used the BM-plasma from BCP compared with HV (p=0.0434). Conclusions: Data suggests that the high PB-RANKL and MIF levels in these BCP could play a role in the intravasation of BCC into the blood vasculature, binding to their R (RANK and CXCR-4, respectively) expressed in them. MIF and OPG not only are pro-angiogenic factors, but also, they have an anti-apoptotic effect over BCC favoring the circulating BCC survival. In addition, PDGF-AB could be responsible of higher proliferation of MDA-MB231 cells when we used the BM-plasma from BCP compared with HV, inducing a favorable BM microenvironment to seed and proliferation of circulating BCC. Moreover, the BM-MStC from BCP could enhance the migration of circulating BCC to BM/bone and the likely association between RANK present in BCC and the mRANKL in BM-MStC could promote the tumor cell proliferation favoring the bone metastatic process. Our findings suggest that the BM microenvironment of advanced BCP without BM/bone metastasis may induce a premetastatic niche to BCC colonization. Citation Format: Leandro M. Martinez, Valeria B. Fernandez Vallone, Vivian Labovsky, Hosoon Choi, Leonardo Feldman, Raul H. Bordenave, Emilio Batagelj, Federico Dimase, Ana Rodriguez Villafane, Norma A. Chasseing. Bone marrow microenvironment of advanced breast cancer patients without bone metastasis favors the cancer cell colonization. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B66.

Abstract C16: Biomarkers of proliferation, survival, and migration of human breast tumor cells: Future perspectives

Background: Despite recent major advance in the understanding of the mechanisms of breast cancer (BC) progression and in the development of novel therapeutic modalities, BC remains the second leading cause of mortality among women. Mortality is almost invariably due to metastasis. The different histological subtypes of BC and molecular maker expression (ER, PR and HER2) have strong prognostic and predictive values but are not enough to prevent that BC patients (BCP) develop a relapse and metastasis. So the aim of this work was the simultaneous evaluation of the expression of biomarkers related to BC progression and metastasis (OPG, TRAIL, TRAIL receptores (R) [R1, R2, R3 y R4], RANKL, RANK (RANKL-R), SDF-1, CXCR-4 (SDF-1-R), IL-6, IL-6-R, MCSF and M-CSF-R in BC cells together with the study of classic prognostic parameters (age, ER, PR, HER2, tumor size and histological grade) in BCP. Regarding the expression of these biomarkers in BC cells, the results are contradictory. Material and methods: This was a prospective cohort study. We included surgical biopsy samples from 19 BCP with primary infiltrative ductal carcinoma, early clinical stage (I-II) and sentinel lymph node negative. Moreover, non-malignant breast tissues (10) were analysed and used as a control. The biomarkers were evaluated by immunohistochemistry on biopsy of breast tissues. Clinicopathological information was retrieved from pathology and medical records. Statistical analysis: Fisher9s Exact Test was used to analyze associations between categorical variables in BCP and controls. Kappa coefficients were used to evaluate the degree of concordance between two categorical variables measured in the same individuals (biomarkers and classical parameters). Mann-Whitney test was used to determine differences in continuous or at least ordinal variables (biomarkers and classical parameters) between the two groups. Software: InfoStat and SPSS 18.0. The threshold for significance was set at p=0.05 Results: BC samples exhibited significant higher prevalence of the expression of R3, R4, RANK, IL-6, CXCR4 and SDF-1 than non-malignant breast tissues (p=0.0002, p=0.0003, p=0.0239, p=0.0087, p=0.0019 and p=0.0403, respectively). On the other hand, R2, found in 65% of BC samples (3/19), was associated with age (p=0.0451). R2 expression was positive in BCP with age mean of 71.5y (range 55-81y). R1, R2 and MCSF expression in BC samples was associated with HER2 [Kappa coefficients: -0.253(-0.445;-0.061); 0.354(0.036;0.672) and -0.354(-0.578;-0.129), respectively]. No statistically significant association was found between the rest of non-classical biomarkers and clinicopathological parameters. Discussion: In conclusion, R3, R4, RANK, IL-6, CXCR4 and SDF-1 expression could clearly distinguish between women with malignant and non-malignant breast tissue. R3 and R4 expression in BC cells could produce the TRAIL resistance, suggesting an anti-apoptotic effect. RANK and CXCR4 expression in BC cells suggest that they have the ability to migrate to bone through the action of RANKL and SDF-1 released by stromal cells of the tumor microenvironment. R1, R2 and M-CSF expression in BC cells were associated with worse classic prognostic parameter HER2. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these biomarkers and it association with classic prognostic parameters in tumor biopsies of BCP could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention. Citation Format: Vivian Labovsky, Leandro Martinez, Maria Calcagno, Kevin Davies, Alejandra Wernicke, Hernan Garcia-Rivello, Valeria Fernandez Vallone, Norma Chasseing. Biomarkers of proliferation, survival, and migration of human breast tumor cells: Future perspectives. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C16.