Abstract Autophagy, a catabolic process that maintains cellular homeostasis, is a primary therapy resistance mechanism in cancer. Emerging studies indicate that autophagy is involved in secretion through non-classical mechanisms. However, the effects of autophagy-mediated secretion on the tumor microenvironment are poorly understood. Further, while autophagy is a potential cancer therapeutic target, efforts to inhibit autophagy in clinical trials have been hampered by sub-optimal methods to quantitatively monitor tumor autophagy levels. Here, we leveraged the recent findings that autophagy is implicated in non-classical secretion to identify secreted proteins associated with autophagy in melanoma. The conditioned media of low autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu, using quantitative proteomics. These comparisons identified secreted proteins associated with high autophagy, such as IL-1β, IL-8, LIF, FAM3C, and DKK3, which have previously been implicated in tumorigenesis. These proteins were shown to be elevated in supernatants of an independent panel of high autophagy melanoma cell lines compared with low autophagy cells, suggesting a mechanistic link between autophagy level and secretion of these proteins. In addition, secretion of these proteins increased when melanoma cells with intrinsically low autophagy levels were treated with an autophagy-inducing tat-Beclin 1 peptide. Further supporting a mechanistic link, Atg7 silencing in an intrinsically high autophagy cell line resulted in significantly decreased secretion. Finally, serum collected from untreated metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from low autophagy patients. These results suggest that autophagy-related secretion affects the tumor microenvironment and that measurement of extracellular autophagy-associated secreted proteins in tumors and in plasma can serve as surrogates for intracellular autophagy dynamics in tumor cells. Citation Format: Adam Akl, Xiaowei Xu, Shengfu Piao, Ravi K. Amaravadi, David W. Speicher. Identification of secreted proteins that reflect intracellular autophagy dynamics in 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 310. doi:10.1158/1538-7445.AM2014-310