Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most hypoxic, lethal, and treatment resistant cancers. Due to its desmoplasias along with the hypoxic state and scarce infiltration of T cells, PDAC is considered a cold tumor. Using a proteomic approach, we previously identified that the expression of endoplasmic reticulum oxidoreductase 1 alpha (ERO1α) is induced by hypoxia and correlates with reduced survival in PDAC. Furthermore, we observed an increased kynurenine formation under hypoxia, suggesting a role of indoleamine 2,3-dioxygenases (IDO1) in PDAC. Since ERO1α and IDO are involved in driving immune suppression through influencing the generation of immature myeloid cells, we sought to investigate whether inhibition of these pathways modulates the myeloid cell composition in PDAC. Using MiaPaCa2 as a PDAC spheroid model with hypoxic core grown in serum-free defined media and immune cell infiltration, we assessed the functional role of ERO1a with IDO1 in modulating monocyte infiltration and differentiation into pro- and anti-inflammatory phenotypes, followed by characterizing the immunomodulatory factors secreted using tandem mass spectrometry. Inhibition of ERO1a and IDO1 significantly improved U937 monocyte infiltration (p-value = ****) and differentiation into dendritic cells (p-value = ****). Secretome analysis of proteins identified with high false discovery rate (q-value<0.01) revealed a downregulation of hypoxia and pancreatic cancer pathways in PDAC spheroids. In the presence of monocytes, upregulation of immune-related pathways for antigen presentation (15-fold) and myeloid cell maintenance signaling (2-fold) were observed. Moreover, specific immune-modulatory factors involved in immune infiltration and migration including interleukin 8, lymphocyte cytosolic protein 1 and transgelin-2 were upregulated at 27-, 20-, and 14-fold respectively. Taken together, these results indicate that inhibition of ERO1a and IDO1 drives an inflamed tumor microenvironment associated with improved monocyte infiltration and differentiation into dendritic cells to potentially enable T cell infiltration and killing of PDAC tumors. Citation Format: Apple Tay, Andreas Lundqvist, Siu Kwan Sze. Inhibition of ERO1a and IDO1 improves dendritic cell infiltration into pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4466.