Introduction: Poor penetration of anticancer therapy into solid tumors significantly limits their efficacy. This phenomenon has long been observed for small-molecule chemotherapeutics, and it has been found even more pronounced for nanoscale therapies and immune cell therapies. For example, although CAR-T cell therapy has shown great success in hematological malignancies, their application to solid tumors has been hampered by the inability of the transferred T cells to infiltrate tumors. To reach tumor cells in a solid tumor, CAR-T cells have to penetrate the extracellular matrix (ECM) that compromises up to 60% of the tumor mass. Collagens are the most abundant proteins in the ECM with significantly more collagen content in tumors than in healthy tissues, resulting in an increased collagen deposition around the tumor. Previous studies have shown that the dense collagen layer disrupts the tumor infiltration of a large variety of immune cells, including T cells. The turnover of matrix collagen is partly regulated by matrix metalloproteinases (MMPs) that are capable of proteolysis of ECM components. Among the different MMPs, MMP8 is attractive as a lead protease because it has a broad range of protein targets in the ECM, including collagen. It has been recently reported that increased immune cell infiltration into tumors is associated with improved patient survival and is predictive of response to immune therapies. In this study, we demonstrated a liposome fusion technology to anchor recombinant MMP-8 in the membrane of model T cells that guides their way through a dense collagen layer by degrading matrix protein.
Materials and
Methods: Liposomes were prepared according to the lipid film hydration/extrusion method. In brief, DOTAP, DOPE, DGS-NTA (Ni) and DiI was mixed at a molar ratio of 1:1:0.1:0.1 in chloroform to form a lipid film after evaporation of the solvent under vacuum. The lipid film was then hydrated with PBS before the mixture was vortexed and extruded through a 100 nm polycarbonate membrane filter. To conjugate proteins to liposomes, a stoichiometric amount of his-tagged E-selectin, GFP or MMP-8 was added to preformed liposomes. Size distribution before and after protein conjugation was measured using ZetaSizer (Malvern). Human immortalized Jurkat T cells were used as a model of circulating immune cells for liposome fusion with the cell membrane. Cells were mixed with liposomes at a prescribed number ratio of 1:5,000 to 300,000, immediately followed by centrifugation to remove free liposomes. Cells were then analyzed by confocal microscopy (LSM 900, Zeiss) and flow cytometry (Guava EasyCyte HT, Millipore Cytek). For the transmigration assay, 6-well transwell inserts were coated with 150 uL Type I collagen at 1 - 3 mg/mL. The collagen layer was imaged with confocal microscopy before and after cell transmigration. 1 million cells were plated in each transwell well on top of the collagen layer. 0.5% and 10% serum media were placed in the top and bottom well, respectively, to create a serum gradient. The system was incubated at 37°C for 24 - 48 hr to allow the migration to complete. Cells in the top and bottom well were then collected for analysis.
Results, Conclusions, and Discussions: The increase in size distribution of the liposomes after protein conjugation supports the association of the protein to the surface of liposomes (Figure 1A). The liposomes immediately fused with the membranes of Jurkat cells following mixing of the two, as revealed by confocal microscopy and flow cytometry (Figure 1B,C). Almost all Jurkat cells were found to carry liposome lipids when incubated with >100,000 liposomes per cell. After the membrane fusion, the lipids of liposomes as represented by DiI stayed on the membrane stably for at least 48 hr. It is likely that cells do not have a mechanism to effectively remove the added foreign lipids. His-tagged GFP as a model protein was successfully anchored in the membrane of Jurkat cells (Figure 1D). Merging of the fluorescence of DiI labelling the liposomes and GFP indicates the sites of fusion and thus GFP anchoring on the cell membrane. Similar to what we previously found, Jurkat cells fused with liposomes without MMP-8 could migrate through the collagen layer, with fewer cells migrating at higher collagen concentration. A greater number of Jurkat cells were found in the bottom well when their membrane was pre-anchored with MMP-8 than samples without exogenous MMP-8. Large pores on the collagen layers were observed via confocal microscopy following cell migration, suggesting collagen degradation occurred during this migration. These results support the idea that MMP-8 anchored on the cell surface can facilitate cell transmigration through collagen by degrading the major ECM protein. We demonstrated a new liposome fusion technology to modify the surface of circulating immune cells with MMP-8 to promote their tumor infiltration. We previously demonstrated a technology to use E-selectin conjugated liposomes that specifically functionalize leukocytes in the blood to target circulating tumor cells for the prevention of cancer metastasis. In this project, E-selectin was conjugated simultaneously with MMP-8 to the liposomes for specific fusion with the membrane of circulating immune cells, while sparing that of red blood cells and endothelial cells. Animal studies are ongoing to validate the technology of E-selecting/MMP-8 liposomes and thus improve therapeutic outcomes.
