(F-214) Revealing Spatial Subcellular Signaling Networks Using Multiplexed Protein Profiling

Introduction:: Non-small cell lung cancer (NSCLC) comprises 80% of lung cancer. The subgroup of NSCLC harboring EGFR (Epidermal Growth Factor Receptor) mutations has shown resistance to chemotherapeutic agents but benefited from EGFR tyrosine kinase inhibitors (TKIs).¹ Both WNT/β-catenin and PI3K/AKT/mTOR pathways can promote cell growth and are overexpressed in NSCLC, and AKT/mTOR pathway mediates the resistance to EGFR tyrosine kinase inhibitors (TKIs). In our study, we will utilize an image-based protein multiplexing approach termed rapid multiplexed immunofluorescence (RapMIF) to quantify the expression and spatial distribution of signaling markers involved in EGFR-mutant (EGFRm) NSCLC cell cultures and tissues.²


Materials and Methods:: NSCLC A549 cells were used to visualize the crosstalk between WNT/β-catenin and AKT/mTOR pathways and to optimize the staining and data analysis pipelines. Using the iterative staining, bleaching, and imaging process, up to 25 protein-type images on the stained slides were yielded at a single-cell level (Figure 1A.). Also, EGFRm NSCLC PC9 cells served as a model to profile the subcellular signaling networks in response to EGFR TKI, Osimertinib. In addition to intensity profiling, we also captured the spatial distribution of signaling markers from the multiplex single-cell measurements using pixel clustering analysis to reveal the subcellular interactions of signaling proteins.


Results, Conclusions, and Discussions:: Results and Discussion: After performing RapMIF on PC9 cells, we converted the 23-plex signaling protein maps into 19-pixel clusters. The subcellular spatial protein-protein interaction (PPI) networks indicating the protein-protein proximity at the pixel level demonstrated that Osimertinib may upregulate the WNT pathway upon the inhibition of the AKT/β-catenin pathway (Figure 1B.). Also, the spatial intensity plot exhibited that Osimertinib can inhibit p-EGFR by preventing the dimerization, phosphorylation, and translocation of EGFR (Figure 1C.). The spatial pixel clustering and signaling networks could visualize the drug effect on the subcellular protein distribution. The subcellular pixel clustering has revealed potential crosstalk between WNT and AKT pathways in A549 cells (Figure 1D.).

Conclusions: The expression profiles and subcellular localization dynamics of two signaling pathways visualized the translocation of non-p-β-catenin and potential crosstalk between them. Also, the spatial pixel clustering maps demonstrated the cell-to-cell heterogeneity and the interactions between two pathways under the Osimertinib treatment provide further scope for studying drug resistance. RapMIF as an image-based multiplexing approach could establish a platform for designing combinatorial interventions for overcoming drug resistance. By incorporating RapMIF with PPI detection, we aim to validate tor reconstruct baseline signaling states in EGFRm NSCLC tissues and develop a predictive pipeline for patients’ signaling therapy design.


Acknowledgements (Optional): :

References (Optional): : Reference: 1. Gu, J. et al. MEK or ERK inhibition effectively abrogates emergence of acquired osimertinib resistance in the treatment of EGFR-mutant lung cancers. Cancer 126, 3788–3799 (2020).

2. Cai, S. et al. Multiplexed protein profiling reveals spatial subcellular signaling networks. iScience 25, 104980 (2022).