Breast cancers from black women exhibit higher numbers of immunosuppressive macrophages with proliferative activity and of crown-like structures associated with lower survival compared to non-black Latinas and Caucasians.

Racial disparities in breast cancer incidence and outcome are a major health care challenge. Patients in the black race group more likely present with an early onset and more aggressive disease. The occurrence of high numbers of macrophages is associated with tumor progression and poor prognosis in solid malignancies. Macrophages are observed in adipose tissues surrounding dead adipocytes in "crown-like structures" (CLS). Here we investigated whether the numbers of CD163+ tumor-associated macrophages (TAMs) and/or CD163+ CLS are associated with patient survival and whether there are significant differences across blacks, non-black Latinas, and Caucasians. Our findings confirm that race is statistically significantly associated with the numbers of TAMs and CLS in breast cancer, and demonstrate that the highest numbers of CD163+ TAM/CLS are found in black breast cancer patients. Our results reveal that the density of CD206 (M2) macrophages is a significant predictor of progression-free survival univariately and is also significant after adjusting for race and for HER2, respectively. We examined whether the high numbers of TAMs detected in tumors from black women were associated with macrophage proliferation, using the Ki-67 nuclear proliferation marker. Our results reveal that TAMs actively divide when in contact with tumor cells. There is a higher ratio of proliferating macrophages in tumors from black patients. These findings suggest that interventions based on targeting TAMs may not only benefit breast cancer patients in general but also serve as an approach to remedy racial disparity resulting in better prognosis patients from minority racial groups.

Obesity induced a leptin-Notch signaling axis in breast cancer.

To investigate whether obesity induces a leptin-Notch signaling axis in breast cancer (BC), leptin-induced Notch was determined in human MCF-7 and MDA-MB231 and mouse E0771 cells and in E0771-BC hosted by syngeneic lean and diet-induced obesity (DIO) C57BL/6J female mice. Lean and DIO mice were treated for 3 weeks with leptin inhibitor (PEG-LPrA2) 1 week after the inoculation of E0771 cells. Leptin induced Notch1, 3 and 4 in BC cells, but Notch2 expression showed opposite pattern in MCF-7 compared to MDA-MB231 cells. Notch loss-of-function (DAPT and dominant negative [R218H] RBP-Jk [CSL/CBF1]) showed that a functional leptin-Notch signaling axis was involved in the proliferation and migration of E0771 cells. E0771-BC onset was affected by obesity (lean mice7/10 [70%] vs. DIO mice: 11/12 [92%]; Pearson χ(2) : p = 0.06]). PEG-LPrA2 significantly reduced BC growth (untreated: 19/42; [45%] vs. treated: 8/42 [19%]; Pearson χ(2) : p = 0.008). PEG-LPrA2 did not influence the caloric intake of mice but increased carcass and/or body weights of lean and DIO mice inoculated with E0771 cells, which could be related to the improvement of health conditions (less aggressive disease). Importantly, BC from obese mice had higher levels of Notch3, JAG1 and survivin than lean mice. Inhibition of leptin signaling reduced protein levels of Notch (NICD1, NICD4, Notch3, JAG1 and survivin) and significantly decreased mRNA expression of Notch receptors, ligands and targets. PEG-LPrA's effects were more prominent in DIO mice. Present data suggest that leptin induces Notch, which could be involved in the reported higher incidence and aggressiveness and, poor prognosis of BC in obese patients.

Oncogenic role and therapeutic target of leptin signaling in breast cancer and cancer stem cells.

Significant correlations between obesity and incidence of various cancers have been reported. Obesity, considered a mild inflammatory process, is characterized by a high level of secretion of several cytokines from adipose tissue. These molecules have disparate effects, which could be relevant to cancer development. Among the inflammatory molecules, leptin, mainly produced by adipose tissue and overexpressed with its receptor (Ob-R) in cancer cells is the most studied adipokine. Mutations of leptin or Ob-R genes associated with obesity or cancer are rarely found. However, leptin is an anti-apoptotic molecule in many cell types, and its central roles in obesity-related cancers are based on its pro-angiogenic, pro-inflammatory and mitogenic actions. Notably, these leptin actions are commonly reinforced through entangled crosstalk with multiple oncogenes, cytokines and growth factors. Leptin-induced signals comprise several pathways commonly triggered by many cytokines (i.e., canonical: JAK2/STAT; MAPK/ERK1/2 and PI-3K/AKT1 and, non-canonical signaling pathways: PKC, JNK and p38 MAP kinase). Each of these leptin-induced signals is essential to its biological effects on food intake, energy balance, adiposity, immune and endocrine systems, as well as oncogenesis. This review is mainly focused on the current knowledge of the oncogenic role of leptin in breast cancer. Additionally, leptin pro-angiogenic molecular mechanisms and its potential role in breast cancer stem cells will be reviewed. Strict biunivocal binding-affinity and activation of leptin/Ob-R complex makes it a unique molecular target for prevention and treatment of breast cancer, particularly in obesity contexts.

Tumor microenvironment profoundly modifies functional status of macrophages: peritoneal and tumor-associated macrophages are two very different subpopulations.

Macrophages are key players in the inflammatory response. In this study, we tested the hypothesis that although all macrophage subpopulations in tumor hosts are affected by the disease, it is the close proximity to the tumor that induces major alterations in these cells. We compared tumor-associated macrophages (TAMs) with peritoneal macrophages from mice bearing D1-DMBA-3 mammary tumors (T-PEMs). Our results show that TAMs downregulate IL-12p70 but upregulate IL-12p40, IL-23, IL-6 and IL-10. Some NFκB and C/EBP transcription factors family members are decreased in TAMs; however NFκBp50 homodimers, STAT1/pSTAT1 and STAT3/pSTAT3 are overexpressed. Furthermore, while TAMs block T-cell proliferation and are more prone to apoptosis compared to T-PEMs, both types of macrophages have an impaired phagocytic capacity. Moreover, TAMs constitutively express iNOS and produce nitric oxide but do not express arginase and are Gr-1(high) and CD11b(low). Collectively, our analysis of two spatially distinct macrophage subpopulations in tumor-bearing mice revealed that the tumor modulates them differently into two molecularly and functionally dissimilar macrophage subpopulations.

Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model.

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor-bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon-gamma (IFN-γ), CHI3L1 up-regulates pro-inflammatory mediators, C-chemokine ligand 2 (CCL2), chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase-9 (MMP-9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP-9 by macrophages. In vivo treatment of mammary tumor-bearing mice with chitin (ß-(1-4)-poly-N-acetyl D-glucosamine), a TH(1) adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN-γ and decreased CCL2, CXCL2 and MMP-9 expression. In vivo administration of chitin to mammary tumor-bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer.

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice.

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.

Involvement of protein kinase C and not of NF kappa B in the modulation of macrophage nitric oxide synthase by tumor-derived phosphatidyl serine.

Nitric oxide (NO) is one of the main cytotoxic effector molecules involved in the killing of tumor cells by macrophages. In macrophages, lipopolysaccharide (LPS) alone or in combination with IFN-gamma causes the generation of NO by an inducible form of NO synthase (iNOS). We have previously reported that macrophages from mammary tumor bearers have a downregulation of their NO production leading to a diminished cytotoxic activity. Further studies lead to the isolation and characterization of phosphatidyl serine (PS) as a NO inhibitory factor produced by mammary tumor cells. Pretreatment of macrophages with PS was shown to downregulate their cytotoxic potential and NO production upon stimulation with LPS. Activation of PS-pretreated macrophages with LPS and IFN-gamma resulted in higher levels of NO than those observed with LPS alone, but lower than those of untreated macrophages activated with LPS and IFN-gamma. These results correlated with the levels of iNOS RNA as detected by Northern blot analyses. A study of the expression and binding activity of the transcription factor NF kappa B in macrophages pretreated with PS revealed no differences with untreated macrophages. Investigation of the possible signaling pathways leading to the induction of iNOS revealed that in LPS-stimulated macrophages, increases in internal calcium concentration [Ca2+]i were not observed, while NO was normally produced even under calcium-deprived conditions. In contrast, an effective synergism of IFN-gamma with LPS in the production of NO by macrophages required an optimal increase in [Ca2+]i stimulated by IFN-gamma. This increment in [Ca2+]i was significantly reduced in PS-pretreated macrophages. Further experiments demonstrated that pretreatment of macrophages with PS did not change the normal pattern of tyrosine phosphorylation stimulated by LPS but strikingly inhibited PKC activity. Combinations of LPS and IFN-gamma did not alter the latter result, suggesting that IFN-gamma enhances LPS-induction of iNOS through a pathway other than activation of PKC. Importantly, expression of PKC isozymes in both untreated and PS-pretreated macrophages stimulated with LPS remained constant. Out data suggest that, in tumor bearers, PKC and not NF kappa B is the main target for PS to exert its NO inhibitory action on LPS-activated macrophages. An excess of PS in PS-PKC interaction may be responsible, at least in part, for this type of PKC inhibition. Furthermore, PS also appears to downregulate the rise in [Ca2+]i promoted by IFN-gamma in macrophages, reducing the synergism of this cytokine with LPS and leading to a less effective production of NO.

Molecular events involved in the increased expression of matrix metalloproteinase-9 by T lymphocytes of mammary tumor-bearing mice.

Matrix metalloproteinases (MMPs) are a family of extracellular proteinases whose contributions to cancer progression have been studied because of their matrix-degrading abilities and elevated expression in advanced stage tumors. Recent findings suggest a role for MMPs during the multiple stages of tumor progression including establishment and growth, migration, invasion, metastasis, and angiogenesis. MMP-9 regulation at the molecular level can be studied by measuring the effect(s) of a variety of physiological and pharmacological agents on cells. Multiple signaling molecules such as protein kinase C, pertussis toxin-sensitive guanine nucleotide-binding protein G, and protein tyrosine kinases are known to mediate the secretion of MMPs in cell lines. We previously reported an upregulation of MMP-9 in T cells of mammary tumor-bearing mice. In this study, pharmacologic inhibitors were used to dissect the signaling pathways involved in the upregulation of MMP-9 in the splenic T cells of normal and mammary tumor-bearing mice. Staurosporine, a protein kinase inhibitor, stimulated MMP-9 secretion by normal T lymphocytes, while the constitutively high levels of MMP-9 produced by tumor bearers' T cells were decreased by Genistein, a specific tyrosine kinase inhibitor, and Rottlerin, a PKC inhibitor. Using a NF-kappaB specific probe to the murine MMP-9 promoter, electromobility shift assays of nuclear proteins from normal and tumor bearers' splenic T cells revealed a pattern of higher intensity bands from the tumor bearers' nuclear extracts, indicating a greater amount of these transcription factors bound to the recognition motif. When mammary tumor bearers' T cells were cultured with the NF-kappaB inhibitors, N-p-Tosyl-L-lysine chloromethyl ketone hydrochloride and Bay 11-7082, there was a subsequent decreased production of MMP-9. These results suggest that the tumor burden may be activating various signaling pathways within splenic T lymphocytes to upregulate MMP-9 expression.

GM-CSF up-regulates the expression of CCL2 by T lymphocytes in mammary tumor-bearing mice.

MCP-1/CCL2 (monocyte chemoattractant protein-1/CC chemokine ligand 2) is a beta or CC chemokine that is expressed by a variety of cell types, including fibroblasts, endothelial, smooth muscle, and glial cells. In addition, cells involved in immunity, such as monocytes/macrophages, neutrophils, and eosinophils have also been shown to express this chemoattractant. Using a murine model of the D1-DMBA-3 mammary adenocarcinoma, we demonstrated the unique production of CCL2 by splenic T lymphocytes from tumor-bearing animals. Because this tumor produces GM-CSF, and this factor is also up-regulated in the B lymphocytes of tumor-bearing mice, we looked at the ability of GM-CSF to induce CCL2 production by T cells. Treatment of normal and tumor bearers' T cells with GM-CSF resulted in an increased secretion of this chemokine. This up-regulation was seen with or without stimulation by Concanavalin A, although these treatments were additive in their effects. The induction of CCL2 was studied at the molecular level by analyzing the effect(s) of a variety of physiological and pharmacological agents on cultured T cells. These results suggest that the tumor-derived factor GM-CSF activates various signaling pathways within splenic T cells to up-regulate CCL2 expression.

Diminished expression of transcription factors nuclear factor kappaB and CCAAT/enhancer binding protein underlies a novel tumor evasion mechanism affecting macrophages of mammary tumor-bearing mice.

Interactions between malignant tumors and the host immune system shape the course of cancer progression. The molecular basis of such interactions is the subject of immense interest. Proinflammatory cytokines produced by macrophages are critical mediators of immune responses that contribute to the control of the advancement of neoplasia. We have shown that the expressions of interleukin 12 (IL-12) and inducible nitric oxide synthase (iNOS) are decreased in macrophages from mammary tumor-bearing mice. In this study, we investigated the causes of IL-12 dysregulation and found deficient nuclear factor kappaB (NFkappaB) and CCAAT/enhancer binding protein (C/EBP) expression and function in tumor bearers' peritoneal macrophages. The constitutive expressions of NFkappaB p50, c-rel, p65, and C/EBPalpha and beta, as well as the lipopolysaccharide-induced nuclear translocation and DNA binding of NFkappaB components and C/EBPalpha and beta, are profoundly impaired in macrophages from mice bearing D1-DMBA-3 tumors. Because similar findings occur with the iNOS gene, it seems that it represents a novel mechanism by which tumor-derived factors interfere with the host immune defenses.

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus.

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Diminished PKC activity and decreased binding of transcription factors are involved in the impaired production of nitric oxide by macrophages from tumor-bearing mice.

In previous studies we have shown that peritoneal macrophages (PEM) from mammary tumor-bearing BALB/c mice (T-PEM) display a diminished ability to lyse tumor cells upon stimulation with LPS, a phenomenon that is associated to a lower production of nitric oxide, and that is reverted upon costimulation with IFN-gamma. The reduced lytic activity and NO production displayed by T-PEM upon LPS activation were earlier shown by us to be due to a diminished transcription of the inducible nitric oxide synthase (iNOS) gene. In the present study, we have investigated the participation of possible signaling molecules and transcription factors - PKC, NF-kappaB, C/EBP and IRF-1 - in the downregulation of NO production in LPS-activated T-PEM. It was found that PKC activity was greatly reduced in T-PEM as compared to normal macrophages, and did not respond to activation. Interestingly, the different PKC isozyme levels were not significantly altered in T-PEM, with the exception of PKC delta. Alterations in the binding activity of the transcription factors NF-kappaB and C/EBP appeared to be involved in the reduced transcription of iNOS previously observed in T-PEM after LPS activation. These results provide evidence that reductions in iNOS transcription secondary to alterations in cell signaling may be responsible for the diminished capacity of macrophages of LPS-activated tumor-bearers to produce NO and lyse tumor targets.

Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: Results of a pilot study.

Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids. Occurrence of triglycerides and free fatty acids will be examined in bAT and similar lipidomic analyses will be carried out visceral adipose tissue, highly inflamed in obesity.

Paracrine Interactions between Adipocytes and Tumor Cells Recruit and Modify Macrophages to the Mammary Tumor Microenvironment: The Role of Obesity and Inflammation in Breast Adipose Tissue.

The relationship between obesity and breast cancer (BC) has focused on serum factors. However, the mammary gland contains adipose tissue (AT) which may enable the crosstalk between adipocytes and tumor cells contributing to tumor macrophage recruitment. We hypothesize that the breast AT (bAT) is inflamed in obese females and plays a major role in breast cancer development. The effects of this interplay on macrophage chemotaxis were examined in vitro, using co-cultures of mouse macrophages, mammary tumor cells and adipocytes. Macrophages were exposed to the adipocyte and tumor paracrine factors leptin, CCL2 and lauric acid (alone or in combinations). In cell supernatants Luminex identified additional molecules with chemotactic and other pro-tumor functions. Focus on the adipokine leptin, which has been shown to have a central role in breast cancer pathogenesis, indicated it modulates macrophage phenotypes and functions. In vivo experiments demonstrate that mammary tumors from obese mice are larger and that bAT from obese tumor-bearers contains higher numbers of macrophages/CLS and hypertrophic adipocytes than bAT from lean tumor-bearers, thus confirming it is more inflamed. Also, bAT distal from the tumor is more inflamed in obese than in lean mice. Our results reveal that bAT plays a role in breast cancer development in obesity.

IL-11-induced reduction of C/EBP transcription factor binding may contribute to the IL-12 downregulation in tumor-bearing mice.

Using a mammary tumor model syngeneic to BALB/c mice, we have characterized several tumor-derived factors. We now report that the DA-3 cell line derived from this tumor, as well as the in vivo tumor itself, express IL-11. The expression of IL-11 in the tumor is detectable at the transcriptional and translational levels, as evidenced by RT-PCR and Western blots. Using a murine IL-11 ELISA, we observed no differences in IL-11 production between normal and tumor-bearer's macrophages or T cells, with or without activation. Interestingly, elevated levels of IL-11 were found in the sera of tumor-bearers, when compared to normal animals and even higher levels of IL-11 were detected in the tumor cystic fluid. Macrophages from mice bearing large mammary tumors show an impaired production of IL-12 and NO, whereas T cells from the same animals display a deficient production of IFN-gamma. Pretreatment of normal macrophages with IL-11 resulted in no decrease in NO production, nor an impaired production of IFN-gamma was observed in normal T cells upon pretreatment with IL-11. However, pretreatment of normal macrophages with IL-11 resulted in a decreased production of IL-12, as revealed by ELISA and RT-PCR. Electromobility shift assays showed decreased binding of the transcription factor C/EBP to the IL-12p40 promoter of LPS-activated macrophages from normal animals, upon pretreatment with IL-11. In contrast, no differences were observed in the levels of NFkappaB binding under the same experimental conditions. Our results suggest that tumor-derived IL-11 may play a role in the depressed IL-12 production by macrophages, leading to the impaired immune functions observed during mammary tumorigenesis.

Alterations in macrophages and monocytes from tumor-bearing mice: evidence of local and systemic immune impairment.

Macrophages are cells of the innate immune system involved in critical activities such as maintaining tissue homeostasis and immune surveillance. Pro-inflammatory macrophages M1 are responsible for the inflammatory response, while M2 macrophages are associated with the immunosuppressive repair phase of tissue remodeling. Most cancers are associated with chronic inflammation, and a high number of macrophages in tumors have been associated with tumor progression. Much effort has been made in elucidating the mechanisms through which macrophages contribute to tumor development, yet much less is known about the initial mechanisms by which tumors modify macrophages. Our work has focused on identifying the mechanisms by which macrophages from tumor hosts are modified by tumors. We have shown that peritoneal macrophages are significantly altered in mice bearing advanced mammary tumors and are not M1 or M2 polarized, but express a mixture of both transcriptional programs. These macrophages are less differentiated and more prone to apoptosis, resulting in increased myelopoiesis as a compensation to regenerate macrophage progenitors in the marrow. Macrophages in the tumor microenvironment are also neither M1 nor M2 cells and through a display of different mechanisms are even more impaired than their peripheral counterparts. Finally, systemic blood monocytes, precursors of tissue macrophages, are also altered in tumor bearers and show a mixed program of pro- and anti-inflammatory functions. We conclude that there is evidence for local and systemic immune impairment in tumor hosts.

Macrophages as independent prognostic factors in small T1 breast cancers.

Breast cancer is the second leading cause of death by cancer in women in the United States. The occurrence of high numbers of macrophages in the tumor stroma has been associated with tumor progression and poor prognosis in breast and other solid malignancies. However, macrophage numbers in tumors have not been validated as a prognostic factor in clinical practice. The present analysis was designed as a pilot study aimed at determining whether the presence of CD68+ macrophages is an independent prognostic factor in small T1 estrogen receptor (ER)+ breast cancers across three different ethnic groups, i.e. African-American, Latina and Caucasian women. A retrospective pilot analysis of 30 T1 breast cancer cases encompassing these three ethnic groups was carried out. African-American and Latina women present with less incidence but more aggressive breast cancer disease and, therefore, proportionally higher death rates. Using immuno-histochemistry, we sought to identify whether there was any association between the presence and density of CD68+ macrophages and standard prognostic markers with overall survival in these groups. Our data revealed that overall survival did not differ significantly for the occurrence or density of CD68+ macrophages in T1 ER+ tumors. There were also no significant differences in overall survival for the occurrence of CD68+ macrophages across ethnicities, although macrophage numbers were significantly higher in tumors from African-American and Latina than in Caucasian patients. Importantly, but not surprisingly, the absence of the progesterone receptor was associated very strongly with decreased overall survival. This pilot project shows that CD68+ macrophages are not pivotal in determining tumor prognosis in early T1 breast cancers. New studies are presently being conducted to assess the value of different macrophage markers and macrophage activation profiles as prognostic factors in breast cancers of different clinical stages, using a larger number of patients among these three different ethnicities.

Tumor-induced thymic atrophy: alteration in interferons and Jak/Stats signaling pathways.

The thymus is the major site of T cell differentiation and a key organ of the immune system. Thym atrophy has been observed in several model systems including aging, and tumor development. Previous results from our laboratory have reported that the thymic atrophy seen in mammary tumor bearers is associated with a severe depletion of CD4+CD8+ double positive immature cells and changes in the levels of cytokines expressed in the thymus microenvironment. Cytokines regulate numerous aspects of hematopoiesis via activation of the Jak/Stat pathways. In the present study we have used our mammary tumor model to investigate whether changes in the levels of cytokines in the thymus could affect the normal expression of the aforementioned pathways. RNA and protein analysis revealed an overexpression of the different members of interferons, a downregulation of most of the Jak/Stat pathways, and an increased expression of several suppressors of cytokine signaling (SOSC) in the thymuses of tumor bearers. Together, our data suggest that the impaired Jak/Stat signaling pathways observed in the whole thymus of tumor-bearing mice could be contributing to the abnormal T cell development and apoptosis observed during the tumor-induced thymic atrophy.

Decreased accumulation of immune regulatory cells is correlated to the antitumor effect of IFN-γ overexpression in the tumor.

During mammary tumorigenesis, there is a profound tumor-induced immunosuppression and a progressive thymic atrophy associated with tumor development. IFN-γ has been shown to be effective in enhancing antitumor responses in several tumor models, however, how IFN-γ exerts its anti-tumor effect is largely controversial. In the present study we have used a mammary tumor model to investigate whether the levels of IFN-γ have an important role in the tumor-induced immuno-suppression as well as in the pathogenesis of the thymic atrophy. We evaluated this possibility using DA-3 cells transfected to express IFN-γ (DA-3/IFN-γ), a system that provides constant, local production of IFN-γ within the tumor microenvironment. Overexpression of IFN-γ in the mammary tumor results in a marked delay of tumor growth, a reduction in regulatory T cells and myeloid-derived suppressor cells accumulation mostly due to down-regulation of chemokines implicated in the recruitment of immune regulatory cells, and a blockage in the tumor-associated thymus atrophy. Collectively, our data suggest that the replacement of the faulty levels of IFN-γ in the tumor results in a diminution of the tumor-induced immune suppression caused by the mammary tumor development.

Role of the proteasome in the downregulation of transcription factors NFkappaB and C/EBP in macrophages from tumor hosts.

Macrophages from mice bearing advanced mammary tumors are critically impaired in their immune functions, exhibiting reduced expression at the mRNA and protein levels of the crucial transcription factors, nuclear factor kappaB (NFkappaB) and CCAAT enhancer binding protein (C/EBP). We have previously shown that tumor-derived factors such as transforming growth factor beta (TGFbeta) and prostaglandin E2 (PGE2) modulate NFkappaB and C/EBP expression in macrophages. Transcriptional, post-transcriptional, translational and/or post-translational mechanisms may also play a role in altered levels of NFkappaB and C/EBP in macrophages from tumor hosts, contributing to impaired inflammatory response. One of the post-translational mechanisms that may tune down or recycle proteins in cells is the proteasomal pathway. Since upregulation of ubiquitin/proteasomal pathways has been described under cancer-induced cachexia, we examined the possible role of this proteolytic machinery in the decrease of NFkappaB and C/EBP proteins in macrophages from tumor hosts. Using MG-132 proteasome inhibitor to block the proteasome machinery in macrophages from normal and tumor-bearing animals we found that macrophages from tumor hosts display higher ubiquitination and proteolysis compared to those from normal mice and also that NFkappaB and C/EBP downregulation is reversed in these treated cells. Thus, proteasome degradation may contribute, at least in part, to NFkappaB and C/EBP impairment in macrophages from tumor-bearers.