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Background: One mechanism of immune escape in the tumor microenvironment is the inhibition of processes involved in tumor antigen uptake by antigen presenting cells (APC). Effective uptake of tumor cells by APC is achieved pre-clinically by labeling them with α-gal glycolipids (α-gal) and exploiting the natural anti-Gal antibody response that constitutes approximately 1% of immunoglobulins in humans. The current study involves intratumoral injection of glycolipids expressing α-gal epitopes in patients (pts) with advanced melanoma and evaluates, in HLA-A2+ treated pts, the development of immune responses to common melanoma-associated antigens (MAA). Methods: Eligibility criteria included unresectable metastatic melanoma (recurrent stage III or stage IV); at least one readily resectable cutaneous, subcutaneous, or readily palpable lymph node metastasis; and a serum anti-Gal titer above 1:50. Pts received 2 injections of alpha-gal glycolipids four weeks apart at 0.1 mg/injection (n=3; dose level 1), 1.0 mg/injection (n=3; dose level 2), or 10 mg/injection (n=3; dose level 3). Ex vivo pre-treatment and Day 57 post-treatment peripheral blood mononuclear cells (PBMC) from pts who were HLA-A2+ (8 of 9 treated pts) were stained with pooled MAA pentamers (gp100209-217, gp100154-162, Melan-A/MART-126-35, NY-ESO-1157-165, and Tyrosinase368-376) and evaluated by flow cytometry. Cells stained with cytomegalovirus (CMV) pp65495-504 or HTLV-1 Tax11-19 served as positive or negative controls, respectively. Results: MAA pentamer+ T cells were detected in 7 of 8 HLA-A2+ pts, both pre- and post-treatment. The frequency of MAA pentamer+ T cells post- versus pre-treatment increased approximately 2-fold for 2 pts [one from dose level 2 and one from dose level 3 (0.16% vs. 0.082%, and 0.033% vs. 0.017%, respectively (background subtracted))]. Similar increases were not observed for the other 5 pts with detectable MAA pentamer+ cells. Furthermore, the median fluorescence intensity (MFI) of MAA pentamer+ T cells, an indirect measure of T cell activation potential, was markedly higher post-treatment, as compared to pre-treatment, for 2 of 3 pts in dose level 1 (11227 vs. 3027, and 4045 vs. 1581). Substantial changes in MFI were not observed for the other pts.. Conclusions: Intratumoral injection of α-gal glycolipids can increase the ex vivo frequency of circulating MAA reactive T cells in some pts with advanced melanoma. Based on this finding, we are pursuing functional characterization of these pre- and post-treatment PBMC. Citation Format: Cindy L. Zuleger, Paul M. Sondel, Jacquelyn A. Hank, Erik A. Ranheim, Thomas A. McFarland, Jennifer Collins, Erin Clements, Giles Whalen, Uri Galili, Mark R. Albertini. Immune responses to common melanoma-associated antigens following intratumoral injection of alpha-gal glycolipids in patients with advanced melanoma. [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 2538. doi:10.1158/1538-7445.AM2014-2538

There is growing interest in the anticancer effect of the diabetes drug, metformin, and several clinical studies are underway worldwide. Metformin's anticancer effects are thought to be mediated by its activation of AMPK and subsequent downstream catabolic effects on protein and lipid metabolism pathways that are commonly dysregulated in cancer cells. We have used mass spectrometry techniques and stable isotopes to investigate the effects of metformin on breast cancer cell lipid metabolism. There was great variability in lipid metabolism response to 2mM metformin treatment in a panel breast cancer cell lines (MCF7, BT474, ZR-75-1, T47D MDA-MB-157, MDA-MB-468 and MDA-MB-231). Levels of stearic acid (p=0.019), palmitoleic acid (p=0.01), oleic acid (p=0.002), cis-vaccenic acid (p=0.003) increased 3-4 fold with metformin treatment in MCF7 cells but there was no significant change for MDA-MB-231 cells. 2mM Metformin decreased fatty acid β-oxidation of oleate 4-fold (p=0.03). Accumulation of de novo lipogenesis derived (p=0.004) and exogenous fatty acid (p=0.02) occurred in MCF7 cells but siRNA knockdown of AMPK did not abrogate these effects. A 3-fold decrease in the diglyceride to triglyceride ratio (p=0.02) suggested reduced lipolysis and inhibited adipose triglyceride lipase activity. Reduced incorporation of exogenous fatty acid into phospholipid was evident (p=0.002). Heterogeneity of lipid metabolism response correlated with effects on proliferation, oxygen consumption and mitochondrial morphology. Our data shows that metformin has marked effects on accumulation of fatty acid in breast cancer cells most likely due to reduced lipolysis and β-oxidation and these effects cannot be explained as a result of AMPK activation. This work will contribute to developing biomarkers for metformin's metabolic anticancer effect and our understanding of how to combine it with other therapies to exploit synthetic lethality. We have initiated a multicenter, phase II, single arm clinical study, to further characterise metformin influenced, cancer relevant metabolic pathways. We will look to assess the correlation between metabolic biomarkers, including the use of mass spectrometry and gas chromatography analysis of tissue samples, and metabolic response using 18F-FDG PET-CT scans. Citation Format: Simon Lord, Jennifer Collins, Barbara Fielding, Neel Patel, Fergus Gleeson, Adrian Harris, Fredrik Karpe. Metformin treatment leads to the accumulation of lipid in breast cancer cell lines - a potential new biomarker of response and mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1897. doi:10.1158/1538-7445.AM2013-1897