Eric Scholar

Inflammatory cell infiltration of tumors: Jekyll or Hyde

Inflammatory cell infiltration of tumors contributes either positively or negatively to tumor invasion, growth, metastasis, and patient outcomes, creating a Dr. Jekyll or Mr. Hyde conundrum when examining mechanisms of action. This is due to tumor heterogeneity and the diversity of the inflammatory cell phenotypes that infiltrate primary and metastatic lesions. Tumor infiltration by macrophages is generally associated with neoangiogenesis and negative outcomes, whereas dendritic cell (DC) infiltration is typically associated with a positive clinical outcome in association with their ability to present tumor antigens (Ags) and induce Ag-specific T cell responses. Myeloid-derived suppressor cells (MDSCs) also infiltrate tumors, inhibiting immune responses and facilitating tumor growth and metastasis. In contrast, T cell infiltration of tumors provides a positive prognostic surrogate, although subset analyses suggest that not all infiltrating T cells predict a positive outcome. In general, infiltration by CD8+ T cells predicts a positive outcome, while CD4+ cells predict a negative outcome. Therefore, the analysis of cellular phenotypes and potentially spatial distribution of infiltrating cells are critical for an accurate assessment of outcome. Similarly, cellular infiltration of metastatic foci is also a critical parameter for inducing therapeutic responses, as well as establishing tumor dormancy. Current strategies for cellular, gene, and molecular therapies are focused on the manipulation of infiltrating cellular populations. Within this review, we discuss the role of tumor infiltrating, myeloid-monocytic cells, and T lymphocytes, as well as their potential for tumor control, immunosuppression, and facilitation of metastasis.

Inhbition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein

Tyrosine kinases are ubiquitous enzymes that have been shown to be involved in many cellular functions, including growth and differentiation. Recent studies have shown that they are also involved in integrin signal transduction pathways. Since integrins are known to be involved in cellular adhesion and thus in invasion and metastasis, the possible involvement of tyrosine kinases in invasion was tested. Tumor cell invasion was measured using filter inserts coated with Matrigel, a substance that closely resembles the natural basement membrane. A highly metastatic subline of BALB/c mammary carcinoma (410.4) cells was shown to invade nearly three times as much as a low metastatic subline (168.1). Genistein, an inhibitor of tyrosine kinases, was found to inhibit invasion of 410.4 cells with an EC50 of approximately 1 microM. At a concentration of 37 microM, there was almost complete inhibition of invasion by genistein, whereas the structural analog, daidzein, which does not inhibit tyrosine kinases, had only a small effect. At higher concentrations (370 microM), daidzein also caused marked inhibition. Genistein was able to inhibit invasion at concentrations having little effect on cell growth. However, for daidzein, most of the effect on invasion was apparently due to its effect on growth inhibition. The relatively specific effect of genistein to inhibit tumor invasion suggests a role for tyrosine phosphorylation in this process. Genistein or other tyrosine kinase inhibitors may be effective inhibitors of tumor invasion and metastasis.

The antimetastatic activity of enkephalin-like peptides

[Met5]Enkephalin and [Leu5]enkephalin, two naturally occurring opioid-like peptides were found to decrease the number of spontaneous pulmonary metastases in C57BL/6J mice implanted in the foot pad with the B16-BL6 melanoma. The number of such metastatic nodules were reduced more than 2-fold. This effect was noted after several different injection schedules. In addition to their effect on metastasis the growth rate of the primary tumor was also decreased by these compounds.

Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice.

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147–579) and 329 (161–523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4+ T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-γ, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.

Stimulation of tumor cell growth by vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) stimulated the growth of murine Lewis lung carcinoma cells in culture. The growth promoting effect was dependent on the concentration of VIP. Exposure to VIP for 12 hours followed by removal of the peptide resulted in sustained growth promotion for 4 to 5 days in culture. Synthetic fragments of VIP, i.e., VIP (1‐16) and VIP (22‐28), and the unrelated peptide neurotensin failed to stimulate the growth of the Lewis lung carcinoma cells. The growth‐promoting effect of VIP was also observed in a murine mammary tumor cell line and a human lung adenocarcinoma cell line.

Therapeutic activity of sunitinib for Her2/neu induced mammary cancer in FVB mice

Mouse mammary tumor virus-Neu (MMTV/neu) transgenic mice on an FVB-background (FVB-neuN) have increased numbers of myeloid derived suppressor cells (MDSCs) and regulatory T-cells (T-regs) in the spleen during mammary tumor induction and progression. Using this transgenic tumor model, we assessed the therapeutic activity of sunitinib, a multi-targeted, tyrosine kinase (TK) inhibitor and its effects on immune-regulatory cells. Our preliminary results show that sunitinib at 40mg/kg/day, p.o. (per os), delayed the time to tumor induction and reduced the incidence and growth of tumors in FVB-neuN mice. In association with its therapeutic activity, sunitinib reduced the absolute number of splenic T-reg cells (CD4(+)CD25(+)CD62L(+)) and MDSCs (CD11b(+)Gr1(+)) that were increased during tumor progression with less activity in mice with gross tumors. A significant decrease in the absolute number of splenic T-regs, dendritic cells (DCs), MDSCs and hematopoietic progenitors (Lin(-)Sca1(+)CD90(dull)) was observed following sunitinib treatment. The frequency of splenic T-regs and hematopoietic progenitors, but not MDSCs was also reduced by sunitinib treatment. Additionally immune-regulatory cytokines and enzymes were down regulated by sunitinib treatment, including TGFbeta and NOS2 in the spleen cells of sunitinib treated mice as compared to untreated tumor bearing (TB) mice. We conclude that sunitinib has therapeutic activity, in association with the down regulation of MDSCs and T-regs and has a trend towards the normalization of the inflammatory cytokine levels induced by tumor progression and growth. Based on these results, we suggest that sunitinib reduction of immune suppressive cells is a critical part of its adjuvant immune therapeutic activity.

The effect of dietary fat on metastasis of the lewis lung carcinoma and the BALB/c mammary carcinoma

The effect of feeding mice diets high in beef tallow (high in saturated fat) or corn oil (high in polyunsaturated fat) on the production of lung metastases by the Lewis lung carcinoma and the BALB/c mammary tumor was determined. Diets were fed ad libitum, and the mice fed the high-fat (24.6%) diets consumed more calories and gained more weight than those fed the control (5%) diets. With the Lewis lung carcinoma, we found that both high-fat diets significantly increased the growth of the primary tumor in the footpad as well as the number of spontaneous metastases produced after the primary was removed; this was in comparison with results from the appropriate control diets. With the BALB/c mammary tumor, the high-fat beef tallow diet (but not the corn oil diet) significantly increased the number of lung metastases formed after tail vein injection. In addition, the group given the control corn oil diet had more metastases than the group given the control beef tallow diet. Overall, these studies showed that the consumption of high-fat/high-calorie diets increased metastasis compared to the consumption of high-fat/high-calorie diets increased metastasis compared to the consumption of low-fat diets. However, the results varied depending on the tumor model used and the type of fat.

Relationship between membrane-bound protein kinase C activity and calcium-dependent proliferation of BALBc 3T3 cells

Extracellular calcium-deprivation inhibited the proliferation of BALB/c 3T3 cells and this inhibition correlated with a loss of protein kinase C activity from the particulate fraction. Addition of calcium induced proliferation of the cells with the DNA synthetic activity returning to the control rate at 18 hours following calcium addition. The level of protein kinase C activity in the particulate fraction was monitored at various times after calcium addition and increased in parallel with the DNA synthetic activity.

Adenosine deaminase and purine nucleoside phosphorylase activity in spleen cells of aged mice

The activities of two enzymes of purine metabolism, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), were determined in spleen lymphocytes from mice of various ages. We found that in the older animals, which have depressed responses to concanavalin A and phytohemagglutinin, there is a decrease in the activity of PNP but normal activity of ADA. The decline of PNP activity was seen at 7.5 months of age and appears to be concurrent with a decline in T-cell function. These results suggest that a decrease in PNP activity may be a contributing factor in the immunodeficient state of the aged.

Comparison of the properties of phosphoribosylpyrophosphate synthetase in normal and leukemic human white blood cells

Abstract Phosphoribosylpyrophosphate synthetase (PRPP synthetase) from normal human lymphocytes and granulocytes was compared with phytohemagglutinin (PHA)-stimulated lymphocytes and leukemic white blood cells of several types with respect to enzyme activity and kinetic constants. PRPP synthetase activity was determined by measuring the production of 14CO2 in a coupled reaction with ¦14C¦orotic acid in the presence of orotidylate pyrophosphorylase and orotidylate decarboxylase. Enzyme activity was expressed both as nmoles/mg of protein/30 min and as nmoles/103 cells/30 min. In more than 50 per cent of the leukemic patients, activity was above normal values when compared on a per mg protein basis. However, when activity was compared on a per cell basis, only the acute myelocytic leukemic patients showed a change from the normal value, and in this case activity was inversely related to the percentage of blast cells in the peripheral circulation. Michaelis constants for ATP (KmATP) and ribose-5-phosphate (KmR5P) were found to be 17.6 ± 1.8 and 51.3 ± 2.4 μM, respectively, in normal lymphocytes, and 55.9 ± 8.0 and 82.5 ± 1.1 μM in normal granulocytes. The KmATP was found to decrease in all leukemic cell types, while the KmR5P showed deviation from normal values depending on the type of leukemia. The inhibition constants (KiATP and KiR5P) were also compared in all leukemic cell types and showed deviations from normal which were cell type dependent. These results suggest that sufficient alteration of the properties of PRPP synthetase from leukemic cells exists to warrant further investigation into the therapeutic implications of alterations of the properties of this enzyme.