Supplementary Material for: Characterization of the Immune Microenvironment in Inflammatory Breast Cancer Using Multiplex Immunofluorescence

<b><i>Introduction:</i></b> Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a poorly characterized immune microenvironment. <b><i>Methods:</i></b> We used a five-colour multiplex immunofluorescence panel, including CD68, CD4, CD8, CD20, and FOXP3 for immune microenvironment profiling in 93 treatment-naïve IBC samples. <b><i>Results:</i></b> Lower grade tumours were characterized by decreased CD4+ cells but increased accumulation of FOXP3+ cells. Increased CD20+ cells correlated with better response to neoadjuvant chemotherapy and increased CD4+ cells infiltration correlated with better overall survival. Pairwise analysis revealed that both ER+ and triple-negative breast cancer were characterized by co-infiltration of CD20 + cells with CD68+ and CD4+ cells, whereas co-infiltration of CD8+ and CD68+ cells was only observed in HER2+ IBC. Co-infiltration of CD20+, CD8+, CD4+, and FOXP3+ cells, and co-existence of CD68+ with FOXP3+ cells correlated with better therapeutic responses, while resistant tumours were characterized by co-accumulation of CD4+, CD8+, FOXP3+, and CD68+ cells and co-expression of CD68+ and CD20+ cells. In a Cox regression model, response to therapy was the most significant factor associated with improved patient survival. <b><i>Conclusion:</i></b> Those results reveal a complex unique pattern of distribution of immune cell subtypes in IBC and provide an important basis for detailed characterization of molecular pathways that govern the formation of IBC immune landscape and potential for immunotherapy.