Introduction: : Poor penetration of anticancer therapies 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 must penetrate the extracellular matrix (ECM) that compromises up to 60% of the tumor mass. Collagen is the most abundant protein 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. The turnover of collagen matrix 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 T 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 Jurkat and primary human T cells that guide their way through a dense collagen layer by the degrading matrix protein.
Materials and
Methods: : Liposomes were prepared using the hydration/extrusion method. To conjugate proteins to liposomes, a stoichiometric amount of his-tagged protein was added to preformed liposomes. Size distribution of liposomes was measured using ZetaSizer (Malvern). Human immortalized Jurkat T cells were used for liposome fusion with the cell membrane. After fusion, the cells were analyzed by confocal microscopy (LSM 900, Zeiss) and flow cytometry (Guava EasyCyte HT, Millipore Cytek). For the transmigration assay, transwell inserts were coated with Type I collagen. 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. All human blood samples were collected from fully consented donors. After the blood was treated with liposomes, primary T cells were isolated using Ficoll-Paque density gradient media (Cytiva) and Dynabeads (Invitrogen) to test their fusion with liposomes and for subsequent migration studies as well. To establish a cancer model, 30,000 4T1-luc cells were injected in the right fourth mammary gland of female BALB/cJ mice. Bioluminescence images of tumor were captured using an IVIS Lumina III imaging system (PerkinElmer Inc) to monitor tumor growth. Mouse primary T cells isolated from 100 µL mouse blood were treated with MMP-8 liposomes before being injected to the same mouse. Tumors will be harvested to analyze total immune cells present especially CD4+ and CD8+ T cells.
Results, Conclusions, and Discussions:: We incorporated phase separation in fusogenic liposomes to reduce their cytotoxicity without compromising their fusogenicity (Figure A). The liposomes immediately fused with the membranes of Jurkat cells following the mixing of the two (Figure 1B). After the membrane fusion, the lipids of the liposomes, as represented by DiI, stayed on the membrane without being detached or internalized for at least 48 hr (Figure 1B). Proteins conjugated on the liposomes, as represented by GFP, can be efficiently anchored in the membrane of Jurkat cells (Figure 1C). The fusogenic liposomes can also efficiently fuse with primary T cells isolated from human and mouse blood. In the transmigration assay, >45% more Jurkat cells modified with MMP-8 were able to migrate to the bottom well than those without MMP-8. The MMP-8 proteins added to Jurkat cells at least partly stayed on the cell membrane after the migration (Figure 1D). After staining with fluorescent antibody, ~10 µm pores were found on the collagen layers through which MMP-8-modified Jurkat cells just passed, suggesting collagen degradation occurred during the migration. More interestingly, the second batch of Jurkat cells can migrate through the collagen layers with ~10 pores much faster than fresh collagen layers. These results support the idea that MMP-8 anchored on cell membrane can facilitate cell transmigration through collagen by degrading the major ECM protein. A small population of leader cells with exogenous MMP-8 could be enough to promote infiltration of more extensive T cells. We have established a triple-negative breast cancer model which is known to have a high content of collagen to test the potential of MMP-8 liposomes for the enhancement of the infiltration of T cells. We demonstrated a novel liposome/plasma membrane fusion technology to add exogenous MMP-8 to the membrane of a small number of isolated T cells. The surface-modified T cells will be transfused back to the blood circulation to excavate tunnels in the ECM of tumor mass to promote T cell infiltration. This technology could significantly improve the outcomes of a wide range of therapies including small molecules and those based on nanoparticles and viable cells.
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