Description Pancreatic cancer remains a deadly disease due to difficulties hindering its earlydiagnosis, giving way to metastasis of the tumor and resulting in poor prognosis.While there are many neoplasms of the pancreas, pancreatic invasive ductaladenocarcinoma (PDAC) is the most common, and treatment options are few,with poor overall survival.The complexities of the tumor microenvironment have been implicated in thefailure of chemotherapy, radiation therapy, and immunotherapy. The tumormicroenvironment of PDAC is especially rich with multiple interactions betweenpancreatic epithelial/cancer cells, stromal cells, immune cells, and theextracellular matrix (ECM). PDACs are characterized by a complex ECM ofdesmoplastic reaction consisting of an extensive and dense fibrotic stroma thatsurrounds and infiltrates clusters of malignant epithelial cells, together with theloss of basement membrane integrity and an abnormal vasculature.In the present study we demonstrate a tissue phenotyping workflow combiningthree complementary methods that can unravel novel insights in the complextumor microenvironment. This novel workflow delivers tissue morphologyinformation, spatial phenotyping of immune cell population on whole slides, andhigh-dimensional imaging in selected regions of interest (ROIs) by combiningH&E, multiplex immunofluorescence (mIF), and Imaging Mass Cytometry™ (IMC™). Presented at SITC 2021.