Alicia J. Dafferner

Tumor- and organ-dependent infiltration by myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) increase during tumor growth and following cytoreductive therapy resulting in immune dysfunction and tumor escape from host control. We report organ- and tumor-specific expansion of MDSCs, differences in their molecular and membrane phenotypes and T-cell suppressive activity. A significant increase in MDSCs was observed within the spleen, peripheral blood (PB), bone marrow (BM), lungs, and livers of mice bearing orthotopic 4T1, but not CI66 mammary tumors. The PB of 4T1 TB mice had the highest frequency of MDSCs (78.6±2.1%). Similarly, the non-parenchymal cells (NPCs) in the tumor tissue, livers and lungs of 4T1 tumor-bearing (TB) mice had an increased MDSCs frequency. Studies into Gr-1 and Ly-6C staining of MDSCs revealed significant increases in CD11b+Gr-1(dull)Ly-6C(high) and CD11b+Gr-1(bright)Ly-6C(low) subsets. The frequency of MDSCs inversely correlated with the CD3+ T-cell frequency in the spleen, and blood of 4T1 TB mice and was associated with a significant decrease in splenic and NPCs IFN-γ and IL-12 transcript levels, as well as significantly increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-10 (IL-10), interleukin-13 (IL-13), arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor-A (VEGF-A) transcripts. In summary, MDSCs are significantly increased not only in lymphoid organs, but also in parenchymal organs including lungs and livers of TB mice, where they may facilitate metastasis to these organ sites.

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.

Tumor regulation of myeloid-derived suppressor cell proliferation and trafficking.

A stress response can induce myeloid progenitor cell (MPC) proliferation, mobilization, and extramedullary hematopoiesis (EMH) within lymphoid and parenchymal organs. Our studies using in vivo BrdU labeling, Ki-67 IHC staining, and carboxyfluorescein succinimidyl ester (CFSE) adoptive cell transfer revealed that spleens, rather than bone marrow (BM) and peripheral blood (PB), from 4T1 mammary tumor-bearing (TB) mice were the primary site of MPC proliferation. The resultant increase in MPCs was associated with tumor hematopoietic growth factor (GF) transcription, decreased apoptosis, as well as, prolonged survival of splenic MPCs. In naïve mice, i.v. injected CFSE-labeled MDSCs (myeloid-derived suppressor cells) initially accumulated in the lungs, while in TB mice, they rapidly sequestered in the spleen. In contrast, a few of the injected MDSCs and leukocytes arrested, proliferated, or accumulated in the marrow, tumor, or PB of TB mice. However, BrdU labeling revealed a significant demargination of proliferating splenic MPCs into the PB. In tumors, despite high GF transcript levels, we found that a high frequency of MDSCs was apoptotic. In summary, tumor growth and cytokines regulate MPC proliferation, trafficking, accumulation, apoptosis, and survival.

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.

Dietary omega-3 and omega-6 polyunsaturated fatty acids modulate hepatic pathology

Recent evidence has suggested that dietary polyunsaturated fatty acids (PUFAs) modulate inflammation; however, few studies have focused on the pathobiology of PUFA using isocaloric and isolipidic diets and it is unclear if the associated pathologies are due to dietary PUFA composition, lipid metabolism or obesity, as most studies compare diets fed ad libitum. Our studies used isocaloric and isolipidic liquid diets (35% of calories from fat), with differing compositions of omega (ω)-6 or long chain (Lc) ω-3 PUFA that were pair-fed and assessed hepatic pathology, inflammation and lipid metabolism. Consistent with an isocaloric, pair-fed model we observed no significant difference in diet consumption between the groups. In contrast, the body and liver weight, total lipid level and abdominal fat deposits were significantly higher in mice fed an ω-6 diet. An analysis of the fatty acid profile in plasma and liver showed that mice on the ω-6 diet had significantly more arachidonic acid (AA) in the plasma and liver, whereas, in these mice ω-3 fatty acids such as eicosapentaenoic acid (EPA) were not detected and docosahexaenoic acid (DHA) was significantly lower. Histopathologic analyses documented that mice on the ω-6 diet had a significant increase in macrovesicular steatosis, extramedullary myelopoiesis (EMM), apoptotic hepatocytes and decreased glycogen storage in lobular hepatocytes, and hepatocyte proliferation relative to mice fed the Lc ω-3 diet. Together, these results support PUFA dietary regulation of hepatic pathology and inflammation with implications for enteral feeding regulation of steatosis and other hepatic lesions.

Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 °C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 °C for days or maybe weeks. At 45 °C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 °C. The folding states of pure human HuBChE stored at 4 and 45 °C and boiled at 100 °C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 °C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native, partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal, C191 developed in our laboratory, was found to selectively capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 °C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors.

Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure

Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 μg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody–Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.

Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 μg/mL of depY was 0.025 μg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

Abstract 245: Dietary long-chain omega-3 fatty acids reduce adipose inflammation in mammary tissue of mice fed moderate fat-isocaloric diets

Increased adipose tissue Inflammation and breast density; including ductal epithelial hyperplasia have been associated with increased risks for breast cancer. Omega 6 (ω6) and omega 3 (ω3) fatty acids (FAs); serve as substrates for pro-inflammatory and inflammation resolving mediators respectively, emphasizing the potential regulatory role for dietary intake of these FAs in inflammation. Western diets have a ω6:ω3 FA ratio of >15:1 with low levels of long-chain (LC)-ω3FA. White adipose tissue inflammatory foci, characterized by crown-like structures (CLS) consisting of dead adipocytes and adjacent macrophages in breast tissue have been related to breast cancer risk in overweight and obese women presumably by the obesity-inflammation-aromatase axis. However, a role of dietary ω6:ω3 FA in adipose inflammation, independent of obesity is not clear. Herein, we examined effects of dietary ω6:ω3 ratio on the mammary tissue microenvironment and adipose inflammation using a moderate fat, iso-caloric diets, and pair-fed model. The LieberDeCarli diet containing 21:1 ratio of ω6:ω3 FA was used as a ω6 diet, whereas encapsulated fish oil containing a 3:1 ratio of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid was used to decrease ω6:ω3 ratio to 0.7:1 in the ω3 diet. Both iso-caloric diets contained 35.5% of calories derived from fat and were pair-fed to maintain iso-intake. Female BALB/c mice were established on the ω6 and ω3 diets for 10 weeks and weight gain and diet consumption monitored. There were no differences in the volume of diet consumed and weight gain between dietary groups. At autopsy, mammary fat pads (MFP) were collected and analyzed for fatty acid composition, histopathology, epithelial proliferation and macrophage infiltration. Arachidonic acid (AA) levels in the MFPs were not different between the groups but EPA and DHA were absent in the MFPs from the ω6 diet fed mice. Whereas, (2.41+/- 0.5) mole% of EPA and (1.52+/-0.29) mole% of DHA were detected in MFP of ω3 diet fed mice. The MFP of ω6 diet fed mice had significantly increased areas of unilocular adipocytes relative to adipocytes of the ω3 group. Similarly, ω6 diet fed mice had increased connective tissue in the ductal stroma, significantly higher numbers of proliferating cells in the ductal epithelium, as well as in adipose tissue area of MFP. In addition, ω6 diet fed mice had a significant increase in the numbers of CLS in mammary adipose tissue. In summary, our studies demonstrated that despite the comparable levels of AA in MFP in both of groups, the presence of LC-ω3 FA (EPA and DHA) was able to reduce inflammation in the MFP of ω3 diet fed mice, thus regulating the MFP microenvironments by reducing macrophage infiltration and ductal epithelial proliferation in an obesity-independent manner. Citation Format: Saraswoti Khadge, Geoffrey M. Thiele, John Graham Sharp, Lynell W. Klassen, Timothy R. McGuire, Michael J. Duryee, Holly C. Britton, Alicia J. Dafferner, Jordan Beck, Paul Black, Concetta C. DiRusso, James E. Talmadge. Dietary long-chain omega-3 fatty acids reduce adipose inflammation in mammary tissue of mice fed moderate fat-isocaloric diets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 245. doi:10.1158/1538-7445.AM2017-245

Abstract 4323: Dietary omega-3 suppress mammary tumor growth, metastasis and enhances survival in an iso-caloric pair-fed mice model

Omega-6 (ω6) and omega-3 (ω3) poly unsaturated fatty acids (PUFA) are pro- and anti-inflammatory respectively. Regulating their dietary ratio provides an approach for cancer prevention and potentially therapy; however, most studies have not separated the inflammatory effects of dietary fatty acids (FA) from that of obesity. Herein we studied the effects of the ω6:ω3 ratio in iso-caloric diets containing 35.5% of calories from fat on mammary tumor growth and metastasis. The ω6:ω3 ratio in ω6 and ω3 based diets were 42:1 and 1:1 respectively (confirmed by gas chromatography-mass spectrometry) using the liquid, Lieber DeCarli diet with fish oil substituting for 70% of olive oil. The ω3 diet contained 3:1 ratio of eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) as major ω3 FA with small amount of linolenic acids. The ω6 diet contained linoleic acid as predominant ω6 FA and small level of linolenic acid as ω3 FA. The diets were pair-fed to avoid the tumor promoting effects of obesity. Two weeks after establishing female BALB/c mice on the diets, they were orthotopically injected with 4T1 mammary tumors. Outcomes included body weight, diet consumption, tumor growth, metastasis and survival. In association with pair feeding there were no significant differences in the diet consumed (ω3 diet used as baseline) and weight gain between animals; however, the time to tumor growth was significantly more rapid in mice fed the ω6 diet versus the ω3 diet cohorts. The ω6 diet fed cohort had significantly higher numbers of pulmonary and hepatic metastases and significantly shortened survival. Further, ω6 diet fed mice had an unusual metastasis profile including an increase in ovarian and renal metastases and an increase in posterior paralysis that in prior studies was associated with demineralization, osteolysis, marrow metastases and spontaneous long bone fractures in mice fed ω6-enriched diet. Histological analysis of tissues supported the observation of more frequent unusual metastasis sites (ovaries, kidneys, heart) along with systemic changes to myeloid cells infiltration in the tissues of mice fed ω6 diet. Further, ω6 and ω3 diets had identical total and fat calories and were pair fed; however, the tumors and livers of ω6 diet fed cohorts had more lipid content in adipocytes. Our results were replicative and also found to be consistent when the tumor growth and survival experiments were compared between young (22 weeks) versus old (58 weeks) mice in independent studies. In summary, our studies demonstrate that the dietary ratio of ω6:ω3 regulates tumor growth and metastasis. Citation Format: Saraswoti Khadge, Geoffrey M. Thiele, Lynell W. Klassen, Graham J. Sharp, Timothy R. McGuire, Michael J. Duryee, Holly C. Britton, Alicia J. Dafferner, Jordan Beck, Paul Black, Concetta DiRusso, James E. Talmadge. Dietary omega-3 suppress mammary tumor growth, metastasis and enhances survival in an iso-caloric pair-fed mice model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4323.