Int J Stem Cells.  2025 Feb;18(1):99-106. 10.15283/ijsc24021.

Lung Cancer Organoid System to Evaluate the Cytotoxicity of Natural Killer Cells

• Oh B
• Kim J
• Kim N
• Jeong Y

• KMID: 2565200
• DOI: http://doi.org/10.15283/ijsc24021

Abstract

Natural killer (NK) cells are gaining growing attention due to their promise for immunotherapy. A fast and accurate system is needed to test NK cell biology and their therapeutic application. Here, we report a lung cancer organoid-based system to evaluate NK cells’ cytotoxicity. We first established the lung cancer organoids on top of Matrigel, which allows the co-culture with NK cells. When co-cultured, NK cells moved close to and inside the lung cancer organoids. When we analyzed by flow cytometry, co-culture of NK cells induced a significantly higher ratio of cell death of lung cancer organoids, suggesting that lung cancer organoids can be employed to test the cytotoxicity of NK cells. Finally, the pre-treatment of NK cells with A83-01, a TGFβ inhibitor, significantly enhanced the cell death of lung cancer organoids by NK cells, indicating that lung cancer organoid-based system faithfully recapitulates cell line-based system in evaluating the in vitro cytotoxicity of NK cells. These data represent that cancer organoid-based NK cell co-culture system is a reliable platform for studying NK cell biology and evaluating their cytotoxicity for screening for NK cell immunotherapy.

Keyword

Organoids; Natural killer cells; Co-culture; Tumor; TGFβ

Figure

• 

  Fig. 1 Establishment of murine lung cancer organoids. (A) Schematic representation of experimental design. (B, C) Time course images (B) and diameters (C) of murine lung cancer organoids grown on top of Matrigel over 14 days. Data are presented as mean±SEM (***p<0.001). (D) Bright-field (left) and fluorescent (right) images of murine lung cancer organoids. (E) H&E staining of murine lung cancer organoids. (F) Bright-field images of lung cancer organoids on passage number 3 and 10. Scale bars=100 μm. NK: natural killer.

• 

  Fig. 2 Co-culture of murine lung cancer organoids with murine splenic natural killer (NK) cells. NK cells were pre-stained with Calcein-AM (5 μg/mL) for 1 hour before co-culture with lung cancer organoids. Immunofluorescence images of lung cancer organoids (A), NK cells (B), and overlay (C) were taken after 24 hours of co-culture. Scale bars=100 μm.

• 

  Fig. 3 Evaluation of natural killer (NK) cells’ cytotoxicity. (A, B) Comparison of cell viability measurement using zombie NIR staining and tdTomato＋ signals. Percentages of live cells in lung cancer organoids were measured by zombie NIR staining and tdTomato＋ signals in representative dot plots in flow cytometric analysis (A) and bar graph (B). (C, D) Measurement of the cell viability of lung cancer organoids with or without NK cell co-culture. NK cells were added to the lung cancer organoids grown on top of Matrigel at 1：10 ratio and co-cultured for 24 hours. Percentages of tdTomato＋ tumor organoid cells in representative dot plots in flow cytometric analysis (C) and bar graph (D) were shown. Data are presented as mean±SEM (*p<0.05). FSC-A: forward scatter-aera.

• 

  Fig. 4 A83-01, a TGFβ inhibitor, enhanced inhibitor, enhanced natural killer (NK) cells’ cytotoxicity. (A, B) Measurement of the cell viability of lung cancer organoids co-cultured with NK cells pre-treated with vehicle or A83-01 for 9 hours. Percentages of tdTomato＋ tumor organoid cells in representative dot plots in flow cytometric analysis (A) and bar graph (B) were shown. Data are presented as mean±SEM (***p<0.001). FSC-A: forward scatter-aera.

Reference

References

1. Laskowski TJ, Biederstädt A, Rezvani K. 2022; Natural killer cells in antitumour adoptive cell immunotherapy. Nat Rev Cancer. 22:557–575. DOI: 10.1038/s41568-022-00491-0. PMID: 35879429. PMCID: PMC9309992.

2. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. 2008; Functions of natural killer cells. Nat Immunol. 9:503–510. DOI: 10.1038/ni1582. PMID: 18425107.

3. Zhang C, Oberoi P, Oelsner S, et al. 2017; Chimeric antigen receptor-engineered NK-92 cells: an off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Front Immunol. 8:533. DOI: 10.3389/fimmu.2017.00533. PMID: 28572802. PMCID: PMC5435757.

4. Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ. GermeWTV. 2021; Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 14:73. DOI: 10.1186/s13045-021-01083-5. PMID: 33933160. PMCID: PMC8088725.

5. Marofi F, Abdul-Rasheed OF, Rahman HS, et al. 2021; CAR-NK cell in cancer immunotherapy; a promising frontier. Cancer Sci. 112:3427–3436. DOI: 10.1111/cas.14993. PMID: 34050690. PMCID: PMC8409419.

6. Dutta D, Heo I, Clevers H. 2017; Disease modeling in stem cell-derived 3D organoid systems. Trends Mol Med. 23:393–410. DOI: 10.1016/j.molmed.2017.02.007. PMID: 28341301.

7. Jeong Y. 2022; Methods to identify epithelial stem cells. Orga. 2:e24. DOI: 10.51335/organoid.2022.2.e24.

8. Siegel RL, Miller KD, Wagle NS, Jemal A. 2023; Cancer stati2023. CA Cancer J Clin. 73:17–48. DOI: 10.3322/caac.21763. PMID: 36633525.

9. Sung H, Ferlay J, Siegel RL, et al. 2021; Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 71:209–249. DOI: 10.3322/caac.21660. PMID: 33538338.

10. Jeong Y, Hoang NT, Lovejoy A, et al. 2017; Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance. Cancer Discov. 7:86–101. DOI: 10.1158/2159-8290.CD-16-0127. PMID: 27663899. PMCID: PMC5222718.

11. Jeong Y, Hellyer JA, Stehr H, et al. 2020; Role of KEAP1/NFE2L2 mutations in the chemotherapeutic response of patients with non-small cell lung cancer. Clin Cancer Res. 26:274–281. DOI: 10.1158/1078-0432.CCR-19-1237. PMID: 31548347. PMCID: PMC6942632.

12. Yokota E, Iwai M, Yukawa T, et al. 2021; Clinical application of a lung cancer organoid (tumoroid) culture system. NPJ Precis Oncol. 5:29. DOI: 10.1038/s41698-021-00166-3. PMID: 33846488. PMCID: PMC8042017.

13. Tang PM, Zhou S, Meng XM, et al. 2017; Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development. Nat Commun. 8:14677. DOI: 10.1038/ncomms14677. PMID: 28262747. PMCID: PMC5343519.

14. Du Y, Liu X, Guo SW. 2017; Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms. Hum Reprod. 32:794–810. DOI: 10.1093/humrep/dex014. PMID: 28184445.

15. Paul S, Lal G. 2017; The molecular mechanism of natural killer cells function and its importance in cancer immunotherapy. Front Immunol. 8:1124. DOI: 10.3389/fimmu.2017.01124. PMID: 28955340. PMCID: PMC5601256.

16. Benton G, Arnaoutova I, George J, Kleinman HK, Koblinski J. 2014; Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Adv Drug Deliv Rev. 79-80:3–18. DOI: 10.1016/j.addr.2014.06.005. PMID: 24997339.

17. Kim HS, Kim JY, Seol B, Song CL, Jeong JE, Cho YS. 2021; Directly reprogrammed natural killer cells for cancer immunotherapy. Nat Biomed Eng. 5:1360–1376. DOI: 10.1038/s41551-021-00768-z. PMID: 34341536.

18. Aspuria PJ, Vivona S, Bauer M, et al. 2021; An orthogonal IL-2 and IL-2Rβ system drives persistence and activation of CAR T cells and clearance of bulky lymphoma. Sci Transl Med. 13:eabg7565. DOI: 10.1126/scitranslmed.abg7565. PMID: 34936383.

19. Khawar MB, Sun H. 2021; CAR-NK cells: from natural basis to design for kill. Front Immunol. 12:707542. DOI: 10.3389/fimmu.2021.707542. PMID: 34970253. PMCID: PMC8712563.

20. Liu E, Marin D, Banerjee P, et al. 2020; Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N Engl J Med. 382:545–553. DOI: 10.1056/NEJMoa1910607. PMID: 32023374. PMCID: PMC7101242.