Background: Treatment response to chemoradiotherapy and immune checkpoint blockade is limited in gastroesophageal adenocarcinoma (GEA). This is likely caused by the fact that the majority of GEAs, belonging to the chromosomally instable subgroup (70%), are often immune excluded. We hypothesize that the immune excluded state is caused by secretion of immune suppressive factors by the tumor cells, to protect themselves from immune attack. By identifying and targeting these factors we aim to increase immune infiltration and increase treatment response. Method: To better understand the drivers of immune exclusion in GEA, we optimized methods to analyze the secretome of freshly dissociated GEA tumor biopsies (n=36) by plating single cells in a 96 well plate for 48 hours and performing targeted proteomics on the supernatant (50ul) using the Olink Target 96 ImmunoOncology panel. Using the same panel, we also analyzed the secretomes of patient-derived organoids (n=7). In parallel the immune infiltrate was characterized immediately in freshly dissociated biopsies with 14 color flow cytometry, and in FFPE material through 6 opal multicolor immunohistochemistry to determine the spatial organization of the immune infiltrate. Results: We identified 62 factors in the panel that are secreted by at least 25% of the biposies. Pro- and anti-inflammatory factors were found to correlate frequently. Using ranked scores for the pro- and anti-inflammatory factors we were able to identify 7 patients with a predominantly inflamed secretome and 29 with a suppressive secretome. The inflamed secretomes are characterized by IFN-gamma, CXCL9-10-11, the presence of granzymes and higher CD8 T cell levels identified by flow cytometry, whereas tumors with dominant anti-inflammatory profile had lower CD8 higher CD4 T cell rates. Notably, the anti-inflammatory secretomes are characterized by an overall lack of pro-inflammatory factors. Among the factors that are most frequently detected (>85% of patients) are galectin-9, IL-8, VEGFA, HO-1 and CAIX, all factors with potential immune suppressive properties. These are also detected in the secretomes of the organoids, indicating direct tumor mediated suppressive effect in the GEA TME. Conclusion: The analysis revealed that GEAs secrete immune suppressive factors and that the state of the TME as influenced by the secretome is reflected in the immune infiltrate composition. We found that tumors lacking a CD8 T cell infiltrate showed an absence of pro-inflammatory factors and had a secretome predominantly made up of anti-inflammatory factors that were also secreted by organoids. Moving forward we aim to further identify GEA associated suppressive factors by using a broader panel to analyze the secretome. Additionally, functional studies will determine whether targeting these immune suppressive factors will facilitate responses to immune checkpoint blockade Citation Format: Jasper Sanders, Micaela H. Harrasser, Tessa S. van Schooten, Emma N. Bos, Benthe H. Doeve, Maarten F. Bijlsma, Hanneke W. van Laarhoven, Donald L. van der Peet, Sarah Derks. Characterizing the secretome of freshly dissociated gastroesophageal tumor biopsies to identify immune suppressive factors [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 2324.