Abstract: Multilayer paper-based cell culture, as an in vitro three-dimensional (3D) cell culture method, has been frequently used to research drug bioavailability, therapeutic efficacy, and dose-limiting toxicity in malignant tumors. This paper proposes a heterogenous multilayer paper stacking co-culture system to establish a model of natural killer (NK) cells moving through the endothelium layer and attacking tumor spheroids. This system consists of three layers: a bottom tumor-spheroid layer, a middle invasion layer, and a top endothelium layer. NK-92 cells were placed in the supernatant on top of the three layers. After two days of co-culture, the attack of tumor spheroids by NK cells was observed. We additionally examined the infiltration of NK-92 cells within the tumor spheroids at different Z-axis depths using a confocal microscope, and the results suggested that the system successfully realizes NK cells traveling cross the endothelium layer to form tumor-infiltrating NK cells (TINKs). The potential application of multilayer paper for co-culture models involving cancer cells and immune cells holds great promise for exploring the interaction dynamics of these two cell types.

自然杀伤细胞与肿瘤球在多层纸基堆叠支架的共培养

[1]AgarwalT, BorrelliMR, MakvandiP, et al., 2020. Paper-based cell culture: paving the pathway for liver tissue model development on a cellulose paper chip. ACS Appl Bio Mater, 3(7):3956-3974.

[2]BoyceMW, LaBoniaGJ, HummonAB, et al., 2017. Assessing chemotherapeutic effectiveness using a paper-based tumor model. Analyst, 142(15):2819-2827.

[3]BurgerMC, ZhangCC, HarterPN, et al., 2019. CAR-engineered NK cells for the treatment of glioblastoma: turning innate effectors into precision tools for cancer immunotherapy. Front Immunol, 10:2683.

[4]BursterT, GärtnerF, BulachC, et al., 2021. Regulation of MHC I molecules in glioblastoma cells and the sensitizing of NK cells. Pharmaceuticals (Basel), 14(3):236.

[5]ChoiY, PhanB, TanakaM, et al., 2020. Methods and applications of biomolecular surface coatings on individual cells. ACS Appl Bio Mater, 3(10):6556-6570.

[6]CourauT, BonnereauJ, ChicoteauJ, et al., 2019. Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment. J Immunother Cancer, 7(1):74.

[7]de AndradeLF, LuYH, LuomaA, et al., 2019. Discovery of specialized NK cell populations infiltrating human melanoma metastases. JCI Insight, 4(23):e133103.

[8]FontanaF, RaimondiM, MarzagalliM, et al., 2020. Three-dimensional cell cultures as an in vitro tool for prostate cancer modeling and drug discovery. Int J Mol Sci, 21(18):6806.

[9]FuJJ, LvXH, WangLX, et al., 2021. Cutting and bonding Parafilm^® to fast prototyping flexible hanging drop chips for 3D spheroid cultures. Cell Mol Bioeng, 14(2):187-199.

[10]Gaitán-SalvatellaI, López-VillegasEO, González-AlvaP, et al., 2021. Case report: formation of 3D osteoblast spheroid under magnetic levitation for bone tissue engineering. Front Mol Biosci, 8:672518.

[11]GuoWJ, ChenZJQ, FengZT, et al., 2022. Fabrication of concave microwells and their applications in micro-tissue engineering: a review. Micromachines (Basel), 13(9):1555.

[12]KenneyRM, BoyceMW, TruongAS, et al., 2016. Real-time imaging of cancer cell chemotaxis in paper-based scaffolds. Analyst, 141(2):661-668.

[13]LarsonTS, GlishGL, LockettMR, 2021. Spatially resolved quantification of drug metabolism and efficacy in 3D paper-based tumor mimics. Anal Chim Acta, 1186:339091.

[14]LeungEYL, EnnisDP, KennedyPR, et al., 2020. NK cells augment oncolytic adenovirus cytotoxicity in ovarian cancer. Mol Ther Oncolytics, 16:289-301.

[15]LiangJ, WangSQ, ZhangGW, et al., 2021. A new antitumor direction: tumor-specific endothelial cells. Front Oncol, 11:756334.

[16]LinDG, ChenX, LinZ, et al., 2021. Paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells. Biomed Mater, 16(2):025028.

[17]ŁuczyńskaE, AniołJ, 2013. Neoangiogenesis in prostate cancer. Contemp Oncol, 17(3):229-233.

[18]MosadeghB, LockettMR, MinnKT, et al., 2015. A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen. Biomaterials, 52:262-271.

[19]PanR, YangXY, NingK, et al., 2023a. Recapitulating the drifting and fusion of two-generation spheroids on concave agarose microwells. Int J Mol Sci, 24(15):11967.

[20]PanR, YangXY, WuSM, et al., 2023b. Using pipette tips to readily generate spheroids comprising single or multiple cell types. J Zhejiang Univ-Sci A (Appl Phys & Eng), 24(10):875-885.

[21]Romo-HerreraJM, Juarez-MorenoK, GuerriniL, et al., 2021. Paper-based plasmonic substrates as surface-enhanced Raman scattering spectroscopy platforms for cell culture applications. Mater Today Bio, 11:100125.

[22]RussickJ, TorsetC, HemeryE, et al., 2020. NK cells in the tumor microenvironment: prognostic and theranostic impact. Recent advances and trends. Semin Immunol, 48:101407.

[23]SangSB, ChengR, CaoYY, et al., 2022. Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 23(1):‍58-73.

[24]SimonKA, MosadeghB, MinnKT, et al., 2016. Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system. Biomaterials, 95:47-59.

[25]VarudkarN, OyerJL, CopikA, et al., 2021. Oncolytic parainfluenza virus combines with NK cells to mediate killing of infected and non-infected lung cancer cells within 3D spheroids: role of type I and type III interferon signaling. J Immunother Cancer, 9(6):e002373.

[26]WalterK, BourquinJ, AmiriA, et al., 2023. Probing local lateral forces of focal adhesions and cell-cell junctions of living cells by torsional force spectroscopy. Soft Matter, 19(25):4772-4779.

[27]WuSM, ChenF, YangXY, et al., 2023. Probing the interaction between metastatic breast cancer cells and osteoblasts in a thread-based breast-bone co-culture device. Lab Chip, 23(12):2838-2853.

[28]XieYY, PanR, WuSM, et al., 2023. Cell repelling agar@paper interface assisted probing of the tumor spheroids infiltrating natural killer cells. Biomater Adv, 153:213507.

[29]ZhaoDK, XuHQ, YinJ, et al., 2022. Inkjet 3D bioprinting for tissue engineering and pharmaceutics. J Zhejiang Univ-Sci A (Appl Phys & Eng), 23(12):955-973.