Machine learning analyses of highly-multiplexed immunofluorescence identifies distinct tumor and stromal cell populations in primary pancreatic tumors

Background: Pancreatic ductal adenocarcinoma (PDAC) is a formidable challenge for patients and clinicians. Objective: To analyze the distribution of 31 different markers in tumor and stromal portions of the tumor microenvironment (TME) and identify immune cell populations to better understand how neoplastic, non-malignant structural, and immune cells, diversify the TME and influence PDAC progression. METHODS: Whole slide imaging (WSI) and cyclic multiplexed-immunofluorescence (MxIF) was used to collect 31 different markers over the course of nine distinctive imaging series of human PDAC samples. Image registration and machine learning algorithms were developed to largely automate an imaging analysis pipeline identifying distinct cell types in the TME. Results: A random forest algorithm accurately predicted tumor and stromal-rich areas with 87% accuracy using 31 markers and 77% accuracy using only five markers. Top tumor-predictive markers guided downstream analyses to identify immune populations effectively invading into the tumor, including dendritic cells, CD4+ T cells, and multiple immunoregulatory subtypes. Conclusions: Immunoprofiling of PDAC to identify differential distribution of immune cells in the TME is critical for understanding disease progression, response and/or resistance to treatment, and the development of new treatment strategies.