Slobodanka Klein

Polyamines prevent DFMO-mediated inhibition of angiogenesis.

Tumor growth mainly depend on formation of new blood vessels. DFMO (alpha-difluoromethylornithine), an inhibitor of polyamine biosynthesis, inhibits tumor growth in many animal tumors. Our investigation was to evaluate the requirement of polyamines for induction of angiogenesis by tumor cells and spleen lymphocytes from tumor-bearing mice. In this regard, we have added DFMO to cell cultures. The neovascular response induced either by tumor cells or spleen lymphocytes was completely abrogated. This inhibition could be reversed by the addition of exogenous putrescine. These findings suggest that the effect of DFMO on angiogenesis is, in part, mediated by the inhibition of polyamine biosynthesis.

Mechanisms of paraneoplastic syndromes in mice bearing a spontaneous lung adenocarcinoma.

Paraneoplastic syndromes are rarely described in animal models. It may be useful to have a suitable experimental model to study the mechanisms by which they are produced. In this study, we describe a murine lung adenocarcinoma, P07, which presents hypercalcemia, leukocytosis and cachexia. We determined the presence of PTHrP in plasma as well as GM-CSF produced by P07 cells. TNF-α, which is responsible for cachexia, could neither be detected in serum nor in P07 cell supernatants. We conclude that this model, which shows paraneoplastic syndromes similar to those of lung tumor patients, should be useful to study the pathways and significance of these signs.

The Kinetics and Redox State of Nitric Oxide Determine the Biological Consequences in Lung Adenocarcinoma

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NOX) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NOX donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NOX donors. Exogenous NOX differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NOX delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NOX based on the mode of delivery and redox state. These data suggest that the development of NOX-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

Malignant myoepithelial cells are associated with the differentiated papillary structure and metastatic ability of a syngeneic murine mammary adenocarcinoma model

BackgroundThe normal duct and lobular system of the mammary gland is lined with luminal and myoepithelial cell types. Although evidence suggests that myoepithelial cells might suppress tumor growth, invasion and angiogenesis, their role remains a major enigma in breast cancer biology and few models are currently available for exploring their influence. Several years ago a spontaneous transplantable mammary adenocarcinoma (M38) arose in our BALB/c colony; it contains a malignant myoepithelial cell component and is able to metastasize to draining lymph nodes and lung.MethodsTo characterize this tumor further, primary M38 cultures were established. The low-passage LM38-LP subline contained two main cell components up to the 30th subculture, whereas the higher passage LM38-HP subline was mainly composed of small spindle-shaped cells. In addition, a large spindle cell clone (LM38-D2) was established by dilutional cloning of the low-passage MM38-LP cells. These cell lines were studied by immunocytochemistry, electron microscopy and ploidy, and syngeneic mice were inoculated subcutaneously and intravenously with the different cell lines, either singly or combined to establish their tumorigenic and metastatic capacity.ResultsThe two subpopulations of LM38-LP cultures were characterized as luminal and myoepithelium-like cells, whereas LM38-HP was mainly composed of small, spindle-shaped epithelial cells and LM38-D2 contained only large myoepithelial cells. All of them were tumorigenic when inoculated into syngeneic mice, but only LM38-LP cultures containing both conserved luminal and myoepithelial malignant cells developed aggressive papillary adenocarcinomas that spread to lung and regional lymph nodes.ConclusionThe differentiated histopathology and metastatic ability of the spontaneous transplantable M38 murine mammary tumor is associated with the presence and/or interaction of both luminal and myoepithelial tumor cell types.

Anti-tumor effect of SLPI on mammary but not colon tumor growth†

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over‐expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over‐expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over‐expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors. J. Cell. Physiol. 228: 469–475, 2013.

Dual activation of Toll-like receptors 7 and 9 impairs the efficacy of antitumor vaccines in murine models of metastatic breast cancer

PurposeSince combination of Toll-like receptor (TLR) ligands could boost antitumor immunity, we evaluated the efficacy of dendritic cell (DC) vaccines upon dual activation of TLR9 and TLR7 in breast cancer models.MethodsDCs were generated from mouse bone marrow or peripheral blood from healthy human donors and stimulated with CpG1826 (mouse TLR9 agonist), CpG2006 or IMT504 (human TLR9 agonists) and R848 (TLR7 agonist). Efficacy of antitumor vaccines was evaluated in BALB/c mice bearing metastatic mammary adenocarcinomas.ResultsCpG-DCs improved the survival of tumor-bearing mice, reduced the development of lung metastases and generated immunological memory. However, dual activation of TLRs impaired the efficacy of DC vaccines. In vitro, we found that R848 inhibited CpG-mediated maturation of murine DCs. A positive feedback loop in TLR9 mRNA expression was observed upon CpG stimulation that was inhibited in the presence of R848. Impaired activation of NF-κB was detected when TLR9 and TLR7 were simultaneously activated. Blockade of nitric oxide synthase (NOS) and indoleamine-pyrrole-2,3-dioxygenase (IDO) improved the activation of CpG-DCs. When we evaluated the effect of combined activation of TLR9 and TLR7 in human DCs, we found that R848 induced robust DC activation that was inhibited by TLR9 agonists.ConclusionsThese observations provide insight in the biology of TLR9 and TLR7 crosstalk and suggest caution in the selection of agonists for multiple TLR stimulation. Blockade of NOS and IDO could improve the maturation of antitumor DC vaccines. R848 could prove a useful adjuvant for DC vaccines in human patients.

Effects of in vivo Culture of Murine Mammary Adenocarcinoma Cells on Tumor and Metastatic Growth

We studied the effect of tumoral microenvironments on metastatic phenotypes. Therefore, murine mammary adenocarcinoma cells cultured in vivo in diffusion chambers (DC) were implanted intraperitoneally in BALB/c mice. The behavior of DC-cultured cells was compared with that of cells obtained from tumors growing subcutaneously or intraperitoneally and from primary cultures in vitro of the former. DC-cultured and control cells were inoculated into normal mice to evaluate their tumorigenicity and metastasizing ability. We found that DC-cultured cells were less tumorigenic and metastatic both in spontaneous and in experimental metastasis assays. The host response to tumor progression resulted in an early leukocytosis, probably due to the overproduction of a hematopoietic factor by the tumor cells. Finally, it was found that DC-cultured cells produced lower levels of urokinase-type plasminogen activator activity, while no differences were found in the metalloproteinase production compared to cells obtained from a tumor growing subcutaneously.

Immunomodulation by Soluble Factors from Tumor Cells Cultured in vivo in Diffusion Chambers

The delayed-type hypersensitivity (DTH) response and lymphocyte-mediated angiogenesis were determined in mice bearing in vivo cultures of mammary tumor cells in diffusion chambers (DCs). Soluble tumor products which diffuse from the DCs were able to stimulate the immune system for both the DTH reaction and angiogenic activity by spleen cells.