The Three-dimensional Environment of Type Ⅰ Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells

LONG Shiqi, WU Cuifang, ZENG Zhu


To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional (3D) culture environments of the gels on the morphology, free migration ability, and cell killing function of natural killer (NK) cells.Methods Type Ⅰ collagen was isolated from the tails of Sprague Dawley (SD) rats and collagen gels with different levels of stiffnesses were prepared accordingly. The microstructure of the collagen gels was observed by laser confocal microscopy. The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer. NK-92MI cells were cultured in collagen gels with different levels of stiffness. The morphology of NK-92MI cells was observed by inverted microscope. High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance. NK-92MI cells were cultured with type Ⅰ collagen gels with different levels of stiffness for 24 h and 48 h and, then, co-cultured with human colorectal DLD-1, a human adenocarcinoma epithelial cell line. CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells.Results Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰ collagen gel with the respective stiffness of (10.970±2.10) Pa and (114.50±3.40) Pa were successfully prepared. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape (P<0.05), the mean area of the cells was reduced ([69.88±26.97] μm2 vs. [46.59±21.62] μm2, P<0.05), the roundness of the cells decreased (0.82±0.12 vs. 0.78±0.18, P<0.05), cell migration speed decreased ([2.50±0.91] μm/min vs. [1.70±0.72] μm/min, P<0.001) and the migration distance was shortened ([147.10±53.74] μm vs. [98.03± 40.95] μm, P<0.0001), with all the differences being statistically significant. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation, with the proliferation rate being (46.39±12.79)% vs. (65.87±4.45)% (P<0.05) and reduce the cell killing ability. Comparison of the cells cultured for 48 h led to similar results, with the proliferation rates being (31.36±2.88)% vs. (74.57±2.16)% (P<0.05), and the differences were all statistically significant.Conclusion The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology, migration ability, and killing function of NK-92MI cells. This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells, as well as laying the theoretical foundation for optimizing immunotherapy protocols.

Keywords: Collagen type Ⅰ,  Extracellular matrix,  Mechanics,  Killer cells,  Immune response 

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DU H, BARTLESON J M, BUTENKO S, et al. Tuning immunity through tissue mechanotransduction. Nat Rev Immunol,2023,23(3): 174–188. doi: 10.1038/s41577-022-00761-w.

ZHANG X, KIM T H, THAULAND T J, et al. Unraveling the mechanobiology of immune cells. Curr Opin Biotechnol,2020,66: 236–245. doi: 10.1016/j.copbio.2020.09.004.

ZHOU H, WANG M, ZHANG Y, et al. Functions and clinical significance of mechanical tumor microenvironment: cancer cell sensing, mechanobiology and metastasis. Cancer Commun (Lond),2022,42(5): 374–400. doi: 10.1002/cac2.12294.

HU B, XIN Y, HU G, et al. Fluid shear stress enhances natural killer cell's cytotoxicity toward circulating tumor cells through NKG2D-mediated mechanosensing. APL Bioeng,2023,7(3): 036108. doi: 10.1063/ 5.0156628.

MORDECHAY L, Le SAUX G, EDRI A, et al. Mechanical regulation of the cytotoxic activity of natural killer cells. ACS Biomater Sci Eng,2020, 7(1): 122–132. doi: 10.1021/acsbiomaterials.0c01121.

SARASWATHIBHATLA A, INDANA D, CHAUDHURI O. Cell-extracellular matrix mechanotransduction in 3D. Nat Rev Mol Cell Biol, 2023,24(7): 495–516. doi: 10.1038/s41580-023-00583-1.

JIN J, TOGO S, KADOYA K, et al. Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-β1. Respir Res,2019, 20(1): 1–14. doi: 10.1186/s12931-019-1093-z.

GORELIK R, GAUTREAU A. Quantitative and unbiased analysis of directional persistence in cell migration. Nat Protoc,2014,9(8): 1931–1943. doi: 10.1038/nprot.2014.131.

GHAEDRAHMATI F, ESMAEIL N, ABBASPOUR M. Targeting immune checkpoints: how to use natural killer cells for fighting against solid tumors. Cancer Commun (Lond),2023,43(2): 177–213. doi: 10. 1002/cac2.12394.

PIERSMA B, HAYWARD M K, WEAVER V M. Fibrosis and cancer: a strained relationship. Biochim Biophys Acta Rev Cancer,2020,1873(2): 188356. doi: 10.1016/j.bbcan.2020.188356.

GUPTA M, SARANGI B R, DESCHAMPS J, et al. Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing. Nat Commun,2015,6(1): 7525. doi: 10.1038/ncomms8525.

MACE E M, ORANGE J S. Lytic immune synapse function requires filamentous actin deconstruction by Coronin 1A. Proc Natl Acad Sci U S A,2014,111(18): 6708–6713. doi: 10.1073/pnas.1314975111.

ABLERZ K M, ARANDA-ESPINOZA H, HAYENGA H N. Substrate elasticity regulates the behavior of human monocyte-derived macrophages. Eur Biophys J,2016,45(4): 301–309. doi: 10.1007/s00249-015-1096-8.

HERRERA M, MEZHEYEUSKI A, VILLABONA L, et al. Prognostic interactions between FAP+ fibroblasts and CD8a+ T cells in colon cancer. Cancers,2020,12(11): 3238. doi: 10.3390/cancers12113238.

ZHOU X, ZHAO R, SCHWARZ K, et al. Bystander cells enhance NK cytotoxic efficiency by reducing search time. Sci Rep,2017,7: 44357. doi: 10.1038/srep44357.

ZHANG Q F, YIN W W, XIA Y, et al. Liver-infiltrating CD11b− CD27− NK subsets account for NK-cell dysfunction in patients with hepatocellular carcinoma and are associated with tumor progression. Cell Mol Immunol,2017,14(10): 819–829. doi: 10.1038/cmi.2016.28.

JENSEN C, MADSEN D H, HANSEN M, et al. Non-invasive biomarkers derived from the extracellular matrix associate with response to immune checkpoint blockade (anti-CTLA-4) in metastatic melanoma patients. J Immunother Cancer,2018,6(1): 1–10. doi: 10.1186/s40425-018-0474-z.

SRIDHARAN R, CAVANAGH B, CAMERON A R, et al. Material stiffness influences the polarization state, function and migration mode of macrophages. Acta Biomater,2019,89: 47–59. doi: 10.1016/j.actbio.2019. 02.048.

HU W, WANG Y, CHEN J, et al. Regulation of biomaterial implantation-induced fibrin deposition to immunological functions of dendritic cells. Mater Today Bio,2022,14: 100224. doi: 10.1016/j.mtbio. 2022.100224.

WU C, TENG L, WANG C, et al. Engineering hydrogels for modulation of dendritic cell function. Gels,2023,9(2): 116. doi: 10.3390/gels9020116.

BEN-SHMUEL A, SABAG B, BIBER G, et al. The role of the cytoskeleton in regulating the natural killer cell immune response in health and disease: from signaling dynamics to function. Front Cell Dev Biol,2021,9: 609532. doi: 10.3389/fcell.2021.609532.


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