Laura Graham

Gemcitabine directly inhibits myeloid derived suppressor cells in BALB/c mice bearing 4T1 mammary carcinoma and augments expansion of T cells from tumor-bearing mice.

Myeloid derived suppressor cells (MDSCs) accumulate in 4T1 mammary carcinoma bearing mice and present a barrier to the success of adoptive immunotherapy (AIT) by suppressing T cell immunity. In this study, we investigated the inhibition of MDSCs by gemcitabine (GEM), a chemotherapy agent that may have favorable immunologic effects. BALB/c mice were inoculated with 4T1 mammary carcinoma cells and treated with GEM either once a week starting 5 days after tumor inoculation (EARLY GEM) or as a single dose at days 20-25 (LATE GEM). Splenic mononuclear cells were isolated, activated in vitro, expanded, and stimulated with tumor antigen. T cells were then used for AIT to treat tumor-bearing mice. EARLY GEM treatment of 4T1 tumor-bearing mice significantly inhibited tumor growth, reduced splenomegaly, and significantly decreased MDSC proportion in the spleen. Support for a direct effect was demonstrated through suppression of MDSCs in spleens, bone marrow, and blood harvested 24 and 48 h after LATE GEM treatment, despite no significant decrease in tumor burden. Interestingly, treatment of tumor-bearing mice with GEM augmented in vitro expansion of splenic T cells and boosted IFN-gamma secretion in response to stimulation by tumor antigen. However, despite GEM-mediated inhibition of MDSC suppression, splenic T cells from mice with advanced tumors were ineffective in vivo against established tumors. This study provides support for direct inhibition of MDSCs and direct reduction of tumor burden by GEM in 4T1 tumor-bearing mice. GEM treatment of mice with advanced tumors improves T cell function and growth in vitro.

Human T cells express CD25 and Foxp3 upon activation and exhibit effector/memory phenotypes without any regulatory/suppressor function

BackgroundFoxp3 has been suggested to be a standard marker for murine Tregs whereas its role as marker for human Tregs is controversial. While some reports have shown that human Foxp3+ T cells had no regulatory function others have shown their role in the inhibition of T cell proliferation.MethodsT cell activation was performed by means of brayostatin-1/ionomycin (B/I), mixed lymphocyte reaction (MLR), and CD3/CD28 activation. T cell proliferation was performed using BrdU and CFSE staining. Flow cytometry was performed to determine Foxp3 expression, cell proliferation, viabilities and phenotype analyses of T cells.ResultsBoth CD4+ and CD8+ T cells expressed Foxp3 upon activation in vitro. Expression of Foxp3 remained more stable in CD4+CD25+ T cells compared to that in CD8+CD25+ T cells. The CD4+CD25+Foxp3+ T cells expressed CD44 and CD62L, showing their effector and memory phenotypes. Both FoxP3- responder T cells and CD4+FoxP3+ T cells underwent proliferation upon CD3/CD28 activation.ConclusionExpression of Foxp3 does not necessarily convey regulatory function in human CD4+CD25+ T cells. Increased FoxP3 on CD44+ effector and CD44+CD62L+ memory T cells upon stimulation suggest the activation-induced regulation of FoxP3 expression.

Cutting Edge: Mast Cells Critically Augment Myeloid-Derived Suppressor Cell Activity

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (KitWsh/Wsh) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells

Adoptive immunotherapy (AIT) using ex vivo-expanded HER-2/neu-specific T cells has shown initial promising results against disseminated tumor cells in the bone marrow. However, it has failed to promote objective responses against primary tumors. We report for the first time that alternating gamma chain cytokines (IL-2, IL-7 and IL-15) ex vivo can expand the neu-specific lymphocytes that can kill breast tumors in vitro. However, the anti-tumor efficacy of these neu-specific T cells was compromised by the increased levels of myeloid-derived suppressor cells (MDSC) during the premalignant stage in FVBN202 transgenic mouse model of breast carcinoma. Combination of AIT with the depletion of MDSC, in vivo, resulted in the regression of neu positive primary tumors. Importantly, neu-specific antibody responses were restored only when AIT was combined with the depletion of MDSC. In vitro studies determined that MDSC caused inhibition of T cell proliferation in a contact-dependent manner. Together, these results suggest that combination of AIT with depletion or inhibition of MDSC could lead to the regression of mammary tumors.

Acute brain death alters left ventricular myocardial gene expression

OBJECTIVES The depressed myocardial function observed in brain dead organ donors has been attributed to massive sympathetic discharge and catecholamine cardiotoxicity. Because elevated catecholamines are associated with altered myocardial gene expression, we investigated whether acute brain death from increased intracranial pressure alters the expression of myocardial gene products important in contractility. METHODS A balloon expansion model was used to increase intracranial pressure in rabbits (n = 22). At timed intervals after brain death, mean arterial pressure, heart rate, electrocardiograms, histologic myocardial injury, and systemic catecholamines were assessed. Messenger RNA levels encoding myofilaments, adrenergic receptors, sarcoplasmic reticulum proteins, transcription factors, and stress-induced programs were measured with blot hybridization of total left ventricular RNA. RESULTS Increased intracranial pressure induced an immediate pressor response that temporally coincided with diffuse electrocardiographic ST segment changes. Systemic epinephrine and norepinephrine levels concurrently increased (5- to 8-fold within 1 minute), then fell below baseline within 2 hours, and remained depressed at 4 hours. By 1 hour, histologic injury was evident. Four hours after the induction of increased intracranial pressure, levels of messenger RNA-encoding skeletal and cardiac alpha-actins, egr-1, and heat shock protein 70 were significantly increased. Sham-operated animals did not exhibit these changes. CONCLUSIONS Select changes in myocardial gene expression occur in response to increased intracranial pressure and implicate ventricular remodeling in the myocardial dysfunction associated with acute brain death.

Resection of the primary tumor improves survival in metastatic breast cancer by reducing overall tumor burden

BACKGROUND Although many retrospective studies suggest that resection of the primary tumor improves survival in metastatic breast cancer, animal studies suggest that resection induces metastasis. Moreover, there has been no critical evaluation of how well animal studies actually model metastatic breast cancer. We used our newly established orthotopic cancer implantation under direct vision model to evaluate the hypothesis that primary tumor resection improves survival in metastatic breast cancer by reducing overall tumor burden and improving immune responsiveness. METHODS Murine mammary adenocarcinoma 4T1-luc2 cells that can be visualized by bioluminescence were implanted orthotopically into BALB/c mice under direct vision. Resection of the primary tumors at days 6, 10, and 28 were compared to sham resection of the contralateral normal mammary gland and observation alone. Tumor burden was quantified by bioluminescence. Tumor-draining lymph nodes were identified by intradermal injection of lymphazurin, and primary tumors, lymph nodes, and lungs were examined pathologically. Kaplan-Meier survival analyses were performed. Splenocyte myeloid-derived suppressor cells (MDSCs) and CD4 or CD8 single positive T lymphocytes were quantified by flow cytometry. RESULTS Tumors invaded locally, metastasized to regional lymph nodes, and then metastasized to distant organs, with subsequent mortality. Surgical stress increased tumor burden only transiently without affecting survival. When primary tumor resection decreased overall tumor burden substantially, further growth of metastatic lesions did not increase the overall tumor burden compared to observation, and survival was improved, which was not the case when resection did not significantly reduce the overall tumor burden. Decreasing overall tumor burden through resection of the primary tumor resulted in decreased splenic MDSC numbers and increased CD4 and CD8 cells, suggesting the potential for an improved immunologic response to cancer. CONCLUSION Decreasing overall tumor burden through resection of the primary breast tumor decreased MDSCs, increased CD4 and CD8 cells, and improved survival.

Influence of the phosphodiesterase-5 inhibitor, sildenafil, on sensitivity to chemotherapy in breast tumor cells

Studies were performed to determine the influence of the phosphodiesterase-5 inhibitor, sildenafil, on sensitivity to Adriamycin (doxorubicin) in four human breast tumor cell lines and one murine breast tumor line. Sildenafil did not interfere with the effectiveness of Adriamycin in any of the cell lines tested. Sildenafil also failed to protect MDA-MB231 cells against the cytotoxicity of cisplatin, taxol or camptothecin. Sildenafil enhanced sensitivity to Adriamycin markedly in the p53 mutant MDA-MB231 and p53 null MCF-7/E6 cells and moderately in the MCF-7/caspase 3 and 4T1 cell lines. In the MDA-MB231 cells, sildenafil increased the extent of DNA damage induced by Adriamycin as well as the extent of apoptotic cell death. Sildenafil did not influence sensitivity to Adriamycin in bone marrow cells or macrophages. In an immunocompetent model of breast cancer (4T1 mammary carcinoma in Balb/c mice), sildenafil did not attenuate the antitumor effects of Adriamycin; furthermore, the combination of sildenafil with Adriamycin was no more toxic to the animals than Adriamycin alone. Given that sildenafil has been shown to have the potential to protect the heart against the toxicity of Adriamycin, these studies suggest that the inclusion of sildenafil with conventional chemotherapeutic protocols involving Adriamycin (and possibly cisplatin, camptothecin and/or paclitaxel) should not compromise the antitumor effectiveness of these drugs nor enhance their toxicity to the patient.

Near-Infrared Imaging of Adoptive Immune Cell Therapy in Breast Cancer Model Using Cell Membrane Labeling

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Methyl-Binding Domain Protein 2-Dependent Proliferation and Survival of Breast Cancer Cells

Methyl cytosine binding domain protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where repatterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable short hairpin RNA (shRNA)-mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak antiproliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in Bagg Albino (BALB)/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by derepression of tumor suppressor genes, including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing show MBD2 binding directly to the hyper methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG island–associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and resilencing of the MBD2-dependent tumor suppressor genes. Our data suggest that uncoupling CpG methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results show a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers. Mol Cancer Res; 9(8); 1152–62. ©2011 AACR.

Tumor escape and progression of HER-2/neu negative breast cancer under immune pressure

BackgroundEmerging data from pre-clinical and clinical studies suggest that HER-2/neu-specific T cell responses could induce HER-2/neu antigen loss in the tumor cells. These data suggest that patients with HER-2/neu negative breast cancer might have had HER-2/neu positive premalignant lesions in the past that progressed to HER-2/neu negative breast cancer under HER-2/neu-specific immune pressure.MethodsWe conducted a pilot study in patients with HER-2/neu positive and HER-2/neu negative breast cancers as well as a patient with ductal carcinoma in situ (DCIS). HER-2/neu expression was determined by FISH. HER-2/neu-specific T cell responses were determined by using IFN-γ ELISA. Expression of IFN-γ Rα in the tumors was determined by immunohistochemistry analysis of paraffin-embedded tissues.ResultsWe determined that majority of (10 of 12) patients with HER-2/neu negative breast cancer had HER-2/neu-specific IFN-γ producing T cell responses which was stronger than those in patients with HER-2/neu positive tumors. Such immune responses were associated with nuclear translocation of IFN-γ Rα in their tumor cells. Patient with DCIS also showed HER-2/neu-specific T cell responses.ConclusionThese data suggest that conducting retrospective studies in patients with HER-2/neu negative breast cancers and prospective studies in patients with HER-2/neu positive DCIS can determine whether HER-2/neu negative invasive carcinomas arise from HER-2/neu positive DCIS under the immune pressure.