Associate Professor Smith College Northampton, Massachusetts, United States
Introduction: Targeted therapy and targeted drug delivery can provide optimized treatment and reduced side effects compared to traditional treatments. Mesothelin (MSLN) is a tumor associated cell-surface antigen that is overexpressed on many types of cancer cells, including ovarian, pancreatic, triple negative breast, and pleural mesothelioma, which makes MSLN a promising biomarker for therapeutic targeting. Previously, we have engineered small proteins based on the human fibronectin type III scaffold (Fn3) that selectively bind to MSLN with high affinity and specificity. The engineered variants are internalized into MSLN-positive cells and induce apoptosis. The Fn3 proteins could be coupled with chemotherapy drugs to develop protein-drug conjugates for targeted therapy. In this current work, we are developing a more detailed understanding of the internalization, intracellular trafficking, and induced signaling of MSLN-binding variant Fn3 3.4.4 (KD = 19 nM) towards informing design of protein-drug conjugates, and to more broadly provide insight for therapeutic strategies targeting MSLN.
Materials and
Methods: Immunofluorescence
HCC 1806 cells, a triple negative breast cancer line expressing MSLN, were seeded at 2 x 10^4 cells/well in chamber slides overnight. Cells were treated with 20 nM Fn3 3.4.4 with a Flag tag or primary antibody rabbit anti-MSLN (1:50 dilution, Abcam, EPR 19025-42) for various time points at 4˚C or 37˚C. Cells were fixed in 3.7% paraformaldehyde, blocked in 10% goat serum, and permeabilized with 0.2% Triton X-100. Secondary antibodies rat anti-flag AF488 (1:200, Thermo Fisher, MA1-142-A488), goat anti-rabbit AF594 (1:500, Abcam, ab150080), and rabbit anti-EEA1 AF647 to detect early endosomes (1:100, Abcam, ab196186) were incubated for 1 h in the dark. Following washes, samples were mounted in media with DAPI. Samples were imaged using a Leica TCS SP5 laser scanning confocal microscope. Quantitative analysis was performed using ImageJ/Fiji to determine colocalization of signals, such as Fn3 protein and endosomes, or Fn3 protein and MSLN.
Results, Conclusions, and Discussions: Results and Discussion
Internalization and trafficking measurements of MSLN and Fn3 3.4.4-Flag
Receptor-mediated endocytosis is a cellular process that can help deliver therapeutic molecules specifically to a desired cell type expressing the targeted receptor. When ligands bind to receptors, a complex can be formed, internalized, and subsequently trafficking to endosomes and lysosomes, or recycled back to the cell surface. Confocal microscopy results indicate binding of Fn3 3.4.4-Flag to MSLN on the cell surface with no internalization when the experiment was conducted at 4˚C, as we would expect for specific receptor-mediated endocytosis. MSLN receptors and Fn3 3.4.4 Flag protein are internalized upon binding after a 3 h incubation at 37˚C (Figure 1).
Following internalization, the colocalization and intracellular trafficking process is currently being studied to elucidate the transport pathways and transport kinetics. We hypothesized that MSLN and Fn3 3.4.4 Flag will either enter through the endosomal pathway and proceed to the lysosome and be degraded, and that some fraction of the complex may be recycled back to the surface. Cells were treated with both Fn3 3.4.4-Flag and MSLN primary antibody for 3 h at 37˚C, followed by costaining for Fn3 3.4.4-Flag and early endosomal antigen 1 (EEA1). Confocal results (Figure 2) and quantitative colocalization analysis (Table 1) indicate that Fn3 3.4.4-Flag is trafficked to early endosomes and colocalizes with MSLN at the 3 h time point.
Conclusions and Ongoing Work We have shown receptor-specific internalization and trafficking to early endosomes of an MSLN-binding engineered protein, confirming that the protein may be useful for intracellular drug delivery for cancer. We are still pursuing studies to determine whether the protein and receptor subsequently enter lysosomes or are recycled back to the cell surface with extended treatment. The trafficking will be further measured with additional incubation times and alternate compartment staining to track protein and receptor following internalization. In addition, the specific apoptotic pathway induced by the therapeutic protein has not been confirmed, and studies to identify the signaling that leads to apoptosis are ongoing.
Acknowledgements (Optional): We would like to thank Judith Wopereis, Smith College Center for Microscopy and Imaging, for confocal microscopy training and support. This work was supported by NIH/NIGMS grant 1R15GM151648-01.
