Holly C. Britton

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.

Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs.

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.

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.

Abstract 4312: Preneoplastic activity of dietary poly unsaturated fatty acid (PUFA) regulation of organ and tissue microenvironments in an iso-caloric pair-fed mouse model

Diets containing omega-3 (ω3) PUFAs have health benefits due to their anti-inflammatory activity and lower risk of chronic conditions including cardiac, autoimmune and neoplastic diseases. This contrasts with ω6 PUFA containing Western diets, which are pro-inflammatory. Balancing the dietary ω6:ω3 ratio has been suggested to have cancer preventive and potentially therapeutic activity; however, the majority of these studies have not differentiated dietary PUFA content from obesity. Herein we examined the effects of the ω6:ω3 ratio in iso-caloric diets that were pair fed. These diets had 35.5% of calories from fat with an ω6:ω3 ratio in the ω6 and ω3 based diets of 42:1 and 1:1 respectively (confirmed by gas chromatography-mass spectrometry) using the liquid, Lieber DeCarli diet and fish oil substituting for 70% of olive oil. The ω3 diet contained eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid at a 3:1 ratio, while the ω6 diet contained linoleic acid as the predominant ω6 PUFA and a low level of the ω3 PUFA, linolenic acid. After establishing female BALB/c mice on these diets, weight gain and diet consumption were monitored for 12 weeks, the mice sacrificed and the dietary effects on mammary gland and hepatic histopathology, leukocyte phenotypes and organ and tissue lipidomics determined. In association with pair feeding there were no differences in diet consumed (ω-3 diet used as baseline) and weight gain between cohorts. The cohort on the ω6 diet had depressed numbers of marrow progenitor cells and increased splenic subcapsular extramedullary hematopoiesis consistent with an inflammatory response, increased hepatocyte lipidosis, hypertrophy and multinucleation and increased hepatic vascularity with thicker intima. The ω-6 dietary cohort also had mammary fat pads (MFPs) with increased adipocytes in the tubular epithelium, stromal cellularity and epithelial tissue density. These mammary gland and hepatic histopathologic changes and subclinical inflammation are consistent with pre-neoplastic lesions and were accompanied by organ specific lipodemic changes. This included significant increases in arachidonic acid (AA) in the plasma, spleen and liver, but not MFPs, of the ω-6 cohort, and a significant increase in EPA and DHA levels in the plasma, spleen, MFPs and liver of the ω-3 cohort. In addition, the liver and MFPs had a significant increase in the ω3 PUFA, docosapentaenoic acid. In summary, our studies demonstrate that the dietary ratio of ω6:ω3, independent of obesity can regulate mammary gland and hepatocyte proliferation and subclinical inflammation in association with significant, organ specific increases in AA levels contributing to microenvironmental preneoplastic hyperplasia. Citation Format: Saraswoti Khadge, Paul Black, Concetta DiRusso, Geoff Thiele, Lynell W. Klassen, J Graham Sharp, Timothy R. McGuire, Michael J. Duryee, Holly C. Britton, Alicia Dafferner, Jordan Beck, James E. Talmadge. Preneoplastic activity of dietary poly unsaturated fatty acid (PUFA) regulation of organ and tissue microenvironments in an iso-caloric pair-fed mouse 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 4312.

Abstract 1167: Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets

Fatty diets can induce low-grade inflammation that we report is increased by chronic alcohol consumption (CAC). CAC as 16.6% of total calories when administered in combination with the Lieber-DeCarli high-fat diet increases inflammation included hepatic and splenic extramedullary hematopoiesis (EMH) as assessed by flow cytometry, immunohistochemistry and a colony forming unit-granulocyte macrophage (CFU-GM) assay. Further, an increased number of hepatic myeloid derived suppressor cells (MDSCs) CD11b+Gr1+ cells that were predominantly Ly6cbr are observed. The increase in MDSCs is associated with an increased number of hepatic non-parenchymal cells including adipocytes (Oil Red O). Consistent with the increased number of hepatic MDSCs and EMH is a decrease in bone marrow cellularity and progenitor cells measured by flow cytometry (Lin-CD11b-Gr1-Sca-1+) and CFU-GM/femur. Unexpectedly, we observed demineralization and osteolytic lesions by micro computed tomography (micro CT) in all bones examined including femur, tibia, fibula and vertebral column that was associated with osteoclast activity (Trap+). Osteolysis was most notable in the fibula and vertebral spurs associated with osteoclast channels, and the demineralization appeared to be associated with areas of active myelopoiesis. The low grade, chronic inflammation associated with the Lieber-DeCarli fatty diet and CAC accelerated the induction of orthotopic 4T1 mammary tumors, resulting in extensive bone osteolysis, demineralization and increased metastases at aberrant sites including splenic, cardiac, hepatic, and extensive lymph node foci in addition to peritoneal and pleural effusions. The latter were haemorrhagic with a predominant nucleated cell infiltrate composed of bands, segs and myelocytes, supporting EMH. These results support the suggestion that a high-fat diet and CAC together increase tumor induction, metastasis and pathology in association with MDSC mobilization, expansion and EMH resulting in increased numbers of osteoclasts, associated bone demineralization and suppression of T-cell frequency and function. These results support the development of combination therapy strategies incorporating multiple molecular therapeutics that inhibit OCs, MDSCs and their associated mediators. Citation Format: Anand Dusad, Saraswoti Khadge, Geoffrey M. Thiele, Michael J. Duryee, Holly C. Britton, Lynell W. Klassen, Alicia J. Dafferner, Tracy Farrell, Timothy R. McGuire, Carlos D. Hunter, Karen C. Easterling, Karen J. O9Kane, John Graham Sharp, James E. Talmadge. Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2014-1167

High fat and alcoholic diets increase hepatic EMH, osteolysis and spontaneous metastases by orthotopic mammary tumors

High-fat diets and chronic alcohol consumption (CAC) can both induce a low-grade inflammation. We report that when CAC (16.6% of total calories) is administered in combination with the Lieber-DeCarli high-fat diet an additive myeloid response is induced. This increase in inflammation included hepatic and splenic EMM as assessed by flow cytometry, immunohistochemistry and a colony forming unit-granulocyte macrophage (CFU-GM) assay. Further, an increased number of hepatic myeloid derived suppressor cells (MDSCs) were observed that were predominantly Ly6c bright. The increase in MDSCs was associated with an increased number of hepatic non-parenchymal cells. Consistent with the increased number of hepatic MDSCs and EMM was a decrease in bone marrow cellularity, MDSCs and progenitor cells (Lin-CD11b-Gr1-Sca-1+), resulting in a decrease in CFU-GM/femur. Unexpectedly, we observed demineralization and osteolytic lesions by micro computed tomography (micro CT) in all bones examined including femur, tibia, humerus and vertebral column. The low grade, chronic inflammation associated with the Lieber-DeCarli high fat diet and CAC increased the growth of orthotropic 4T1 mammary tumors, resulting in extensive bone osteolysis, demineralization, myeloplasia and lymphopenia and increased metastasis as defined by pulmonary lesions and aberrant metastatic sites including splenic, cardiac, hepatic and large and extensive lymphatic foci in addition to peritoneal effusions. The effusions were haemorrhagic with predominantly a nucleated cell infiltrate composed of bands, segs and myelocytes, supporting EMM. The osteolysis was most notable in the distal femur, proximal tibia, and vertebral spurs, associated with osteoclast channels and the demineralization appeared to be associated with areas of active myelopoiesis. We suggest that a high fat diet and CAC can increase tumor metastasis and pathology in association with myeloid progenitor mobilization and EMM resulting in increased numbers of osteoclasts, MDSCs, associated bone demineralization and suppression of T-cell frequency and function. These results support the use of combination therapy strategies that incorporate multiple molecular therapeutics that inhibit OCs, MDSCs and their associated mediators i.e. COX-2, NO.

Abstract 4363: Tumor regulation of myeloid-derived suppressor cell proliferation, circulation and apoptosis.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In response to stress, myeloid progenitor cells (MPCs) mobilize and establish sites of extramedullary hematopoiesis (EMH) within lymphoid and parenchymal organs. We report that the spleens of 4T1 mammary tumor-bearing (TB) mice are the primary site of MPC proliferation resulting in extensive EMH in association with decreased apoptosis, increased myeloid cell survival, and increased hematopoietic growth factor (GF) transcription by the tumor cells. MPC trafficking and survival differs between TB and naive mice with leukocyte arrest in the lungs of naive mice and accumulation in the splenic red pulp of TB mice with little marrow or tumor arrest or proliferation. Indeed, despite the high levels of GF transcription by tumor cells MDSC apoptosis in tumors is very high perhaps due to the high levels of NOS2 in the tumor microenvironment. Further myeloid proliferation directly correlates with the decrease in T-cell frequency, which is associated with extrathymic, but not thymic T-cell proliferation. In summary, tumor cytokines regulate a dynamic relationship between MPCs and T-cells regulating their proliferation, trafficking, accumulation, apoptosis, and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4363. doi:1538-7445.AM2012-4363