Objective Diet-induced obesity has been shown to alter immune function in mice, but distinguishing the effects of obesity from changes in diet composition is complicated. We hypothesized that immunological differences would exist between diet-induced obese (DIO) and obese-resistant (OB-Res) mice fed the same high-fat diet (HFD). Methods BALB/c mice were fed either standard chow or HFD to generate lean or DIO and OB-Res mice, respectively. Resulting mice were analyzed for serum immunologic and metabolic profiles, and cellular immune parameters. Results BALB/c mice on HFD can be categorized as DIO or OB-Res, based on body weight versus lean controls. DIO mice are physiologically distinct from OB-Res mice, whose serum Insulin, Leptin, GIP, and Eotaxin concentrations remain similar to lean controls. DIO mice have increased macrophage+ crown-like structures in white adipose tissue, although macrophage percentages were unchanged from OB-Res and lean mice. DIO mice also have decreased splenic CD4+ T cells, elevated serum GM-CSF, and increased splenic CD11c+ dendritic cells, but impaired dendritic cell stimulatory capacity (p < 0.05 versus lean controls). These parameters were unaltered in OB-Res mice versus lean controls. Conclusions Diet-induced obesity results in alterations in immune and metabolic profiles that are distinct from effects caused by HFD alone.

Abstract. Metastatic breast cancer is currently incurable, and available therapies are associated with severe toxicities. Induction of protective anti-tumor immunity is a promising therapeutic approach for disseminated breast cancer, as immune responses are (i) systemic; (ii) antigen-specific; and (iii) capable of generating long-lived "memory" populations that protect against future tumor recurrences. Pursuant with this approach, we have developed a novel heterologous prime/boost vaccination regimen that reduces spontaneous lung metastases in mice with established murine 4T1 adenocarcinoma breast tumors. In our studies, mice were orthotopically challenged with luciferase-expressing 4T1 tumor cells; luciferase expression was retained in vivo, enabling us to quantitatively track metastatic tumor growth via bioluminescent imaging. On day 6 post-challenge, mice received a therapeutic "prime" consisting of bulk tumor lysates encapsulated in poly(lactic-co-glycolic) acid (PLGA) microparticles (MPs). On day 11, mice received a "boost" composed of free tumor lysates plus a cocktail of Toll-like receptor (TLR)-stimulating adjuvants. Tumor progression was monitored in vaccinated and untreated mice for 25 days, a time at which 100% of untreated mice had detectable lung tumors. PLGA MPs injected subcutaneously trafficked to draining lymph nodes and were efficiently phagocytosed by dendritic cells (DCs) within 48 h. Our combination therapy reduced metastatic lung tumor burdens by 42% and did not induce autoimmunity. These findings illustrate that vaccines based upon MP delivery of tumor lysates can form the basis of an effective treatment for metastatic breast cancer and suggest that similar approaches may be both efficacious and well-tolerated in the clinic.

Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn and is tyrosine phosphorylated by Fyn. The presence of tyrosine phosphorylated tau in AD and the involvement of Fyn in AD has drawn attention to the tau-Fyn complex.In this study, a tau-Fyn double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled Fyn KO in novel object recognition and contextual fear conditioning tasks and resembled tau KO mice in the pole test and protection from pentylenetetrazole-induced seizures. In glutamate-induced Ca 2+ response, Fyn KO was decreased relative to WT and DKO had a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO resembled WT in its Ca 2+ response, we investigated whether microtubule-associated protein 2 (MAP2) served to compensate for tau, since the MAP2 level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca 2+ response. K E Y W O R D Scalcium, Fyn, MAP2, proximity ligation assay, tau

Microtubule-associated protein tau, an integral component of neurofibrillary tangles, interacts with a variety of signaling molecules. Previously, our laboratory reported that nerve growth factor (NGF)induced MAPK activation in a PC12-derived cell line was potentiated by tau, with phosphorylation at T231 being required. Therefore, we sought to identify a signaling molecule involved in the NGF-induced Ras-MAPK pathway that interacted with phospho-T231-tau. Here, we report that the protein tyrosine phosphatase SHP2 (also known as PTPN11) interacted with tau, with phospho-T231 significantly enhancing the interaction. By using proximity ligation assays, we found that endogenous tau-SHP2 complexes were present in neuronal cells, where the number of tau-SHP2 complexes significantly increased when the cells were treated with NGF, with phosphorylation at T231 being required for the increase. The interaction did not require microtubule association, and an association between tau and activated SHP2 was also found. Tau-SHP2 complexes were also found in both primary mouse hippocampal cultures and adult mouse brain. Finally, SHP2 levels were upregulated in samples from patients with mild and severe Alzheimer's disease (AD), and the level of tau-SHP2 complexes were increased in AD patient samples. These findings strongly suggest a role for the tau-SHP2 interaction in NGF-stimulated neuronal development and in AD. This article has an associated First Person interview with the first author of the paper.

Statement 40The downstream effect of the interaction between microtubule-associated protein tau and Src 41 family non-receptor tyrosine kinase Fyn was investigated with a tau/Fyn double KO mouse. We 42 demonstrate that tau has a Fyn-independent role in glutamate-induced calcium response and that MAP2 can 43 compensate for tau in interacting with Fyn in dendrites. 44 Abstract 45Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn. A tau-Fyn 46 double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled 47Fyn KO in cognitive tasks and resembled tau KO mice in motor tasks and protection from 48 pentylenetetrazole-induced seizures. In Ca 2+ response, Fyn KO was decreased relative to WT and DKO had 49 a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO 50 resembled WT in its Ca 2+ response, we investigated whether MAP2 served to compensate for tau, since its 51 level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn 52 activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to 53 WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by 54 MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, 55 decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca 2+ 56 response. 57 58 59 60 61 62 63 64 suggestions, Bridget Shafit-Zagardo for MAP2c plasmid, Dan Thedens for help with MRI, Skye 595

Although existing interventions are highly efficacious for the treatment of localized breast cancers, disseminated tumors remain incurable. Therapeutic induction of systemic anti-tumor immunity is a promising approach for treating metastatic disease. We have developed a novel heterologous prime/boost vaccination protocol that significantly reduces metastatic tumor burdens in a pre-clinical murine model of breast cancer. In our studies, mice were orthotopically challenged with Luciferase-expressing 4T1 adenocarinoma cells. Six days later, mice received a priming therapy that consisted of parental 4T1 tumor lysates encapsulated in poly(lactic-co-glycolic) acid microparticles administered contralateral to the established tumor. On day eleven, mice received a boost therapy that consisted of 4T1 lysates and a cocktail of Toll-like receptor agonists. At the conclusion of the assay (day twenty-five), tumor burdens from excised lungs were quantified using bioluminescent imaging. Therapy-receiving mice showed a significant reduction in lung tumors (Student's T-test: p = 0.002, n = 37 control mice vs. 25 treated mice) with an average 46.66% decrease relative to no therapy control mice. Independent quantification of lung metastases was also performed using a standard 6-thioguanine assay. There was a strong correlation (R^2 = 0.8803) between bioluminescent intensity and 6-thioguanine colony number, demonstrating that the decrease in bioluminescence signal due to therapy is not merely explained by the in vivo loss of Luciferase expression by 4T1 cells. We are currently characterizing the vaccination-induced differences in the localized immune environment of metastatic lung tumors. Effector T cell activation/exhaustion kinetics and the influx of immunosuppressive cell populations (myeloid-derived suppressor cells and regulatory T cells) are now being explored. These studies will help elucidate the immunological mechanism(s) responsible for the diminished metastatic tumor outgrowth observed with our therapy. In conclusion, our data demonstrate that therapeutic vaccination of tumor lysates is efficacious in controlling spontaneous lung metastases in an aggressive model of murine breast cancer. Citation Format: Brett P. Gross, Amaraporn Wongrapanich, Meghan Francis, Vijaya B. Joshi, Aliasger K. Salem, Lyse A. Norian. Therapeutic microparticle-based tumor lysate vaccination reduces spontaneous lung metastases in a murine breast cancer model. [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 2885. doi:10.1158/1538-7445.AM2014-2885