FBS Colloquia No.360Laboratory of Single Molecule Biology

Multiplexed imaging of IL-6 signaling pathway

Cells enable contextual responses by encoding external information into nodes of the signaling network. This process can result in heterogeneous signaling responses, playing significant roles in biological processes such as differentiation and development. However, the molecular mechanism underlying this heterogeneity and methods to control the heterogeneity remain unclear. Interleukin-6 (IL-6) is a cytokine that functions pleiotropically in inflammation, immune response, and hematopoiesis. In cancer cells, IL-6 promotes tumor progression via STAT3-mediated epithelial-mesenchymal transition (EMT). To investigate the heterogeneous cellular response induced by IL-6, we measured the signaling responses and cellular states in lung cancer cells (A549) following IL-6 stimulation, using 4i, a multiplexed antibody-based imaging technology. By clustering the extracted features from the spatial distributions of more than 30 proteins using computer vision and machine learning methods, we found a negative correlation between ERK and STAT3 activation, depending on the cellular states. Furthermore, our analysis using a correlation matrix on IL-6 signaling nodes after drug-induced ERK activation suggests that phosphorylated ERK dampens phosphorylated STAT3 via JAK2 kinase activity. Our single-cell analysis suggests that JAK2 kinase activity plays a pivotal role in the heterogeneous IL-6 signaling response. Our findings shed light on potential methods to control heterogeneous signaling responses in IL-6 signaling and related biological processes.