Free-floating patient-derived organotypic tumor spheroids (PDOTS) from non-small cell lung cancer (NSCLC) tumors: a versatile tool for personalized testing of chemotherapeutic drugs

Lilian Ismail , Komal Zahid, Anna Polyanskaya, Aya Al Othman, Ningfei Shen, Xiaoli Qi, Rushan Sulimanov, Yuri Esakov, Vladimir Makarov, Gleb Filkov, Alexander Trofimenko, Alexandre Mezentsev, Mikhail Durymanov

Abstract


Background and purpose: Patient-derived tumor 3D multicellular cultures are a novel non-small cell lung cancer (NSCLC) model for studying tumor biology and precision medicine, which recapitulates tumor morphology and gene expression profile. However, practical application is challenged by issues such as low establishment rates, long-term production difficulties, and the absence of immune microenvironment components. To address these issues, this study aimed to evaluate the efficacy of a novel method for generating free-floating patient-derived organotypic tumor spheroids (PDOTS) using a stimuli-responsive extracellular matrix (ECM)-mimicking gel.

Experimental approach: Free-floating PDOTS were established from 18 NSCLC tumors and characterized by their morphology, marker expression, and extracellular matrix composition. Cell composition in PDOTS and their parental tumors was analyzed by flow cytometry, while RT-PCR was used to assess the expression of genes encoding signaling molecules. Finally, drug response and the expression of drug resistance genes were evaluated in the NSCLC PDOTS.

Findings/Results: The PDOTS were successfully generated with a success rate exceeding 90%, forming spheroids within one week. These PDOTS preserved the parental tumor's morphology and included stromal and immune cells. Notably, 58% of the PDOTS maintained cytokine and growth factor expression profiles closely mimicked those of the original tumors. Furthermore, the PDOTS demonstrated varied responses to anticancer drugs, potentially influenced by differential expression of drug resistance-associated genes.

Conclusion and implications: The high establishment rate and rapid production timeline of free-floating PDOTS using a stimuli-responsive ECM-mimicking gel make this approach a promising tool for advancing cancer biology research and evaluating therapeutic strategies with greater accuracy.

 

 


Keywords


Cytokine; Drug resistance; Non-small cell lung cancer; Patient-derived organotypic tumor spheroids; Personalized medicine.

Full Text:

PDF

References


Garinet S, Wang P, Mansuet-Lupo A, Fournel L, Wislez M, Blons H. Updated prognostic factors in localized NSCLC. Cancers (Basel). 2022;14(6):1400,1-20.DOI: 10.3390/cancers14061400.

Mezentsev A, Durymanov M, Makarov VA. A Comprehensive review of protein biomarkers for invasive lung cancer. Curr Oncol. 2024;31(9):4818-4854.DOI: 10.3390/curroncol31090360.

Poddubskaya E, Bondarenko A, Boroda A, Zotova E, Glusker A, Sletina S, et al. Transcriptomics-guided personalized prescription of targeted therapeutics for metastatic ALK-positive lung cancer case following recurrence on ALK inhibitors. Front Oncol. 2019;9:1026,1-8.DOI: 10.3389/fonc.2019.01026.

Bepler G, Williams C, Schell MJ, Chen W, Zheng Z, Simon G, et al. Randomized international phase III trial of ERCC1 and RRM1 expression–based chemotherapy versus gemcitabine/carboplatin in advanced non–small-cell lung cancer. J Clin Oncol. 2013;31(19):2404-2412.DOI: 10.1200/JCO.2012.46.9783.

Bailey H, Lee A, Eccles L, Yuan Y, Burlison H, Forshaw C, et al. Treatment patterns and outcomes of patients with metastatic non-small cell lung cancer in five european countries:a real-world evidence survey. BMC Cancer. 2023;23(1):603,1-20.DOI: 10.1186/s12885-023-11074-z.

Rozenberg JM, Filkov GI, Trofimenko AV, Karpulevich EA, Parshin VD, Royuk VV, et al. Biomedical applications of non-small cell lung cancer spheroids. Front Oncol. 2021;11:791069,1-13.DOI: 10.3389/fonc.2021.791069.

Durymanov M. Tumor spheroids, tumor organoids, tumor explants, and tumoroids: what are the differences between them? Biochemistry (Mosc.). 2025;9(2):200-213.DOI: 10.1134/S0006297924604234.

Surina, Tanggis, Suzuki T, Hisata S, Fujita K, Fujiwara S, et al. Patient-derived spheroids and patient-derived organoids simulate evolutions of lung cancer. Heliyon. 2023;9(3):e13829,1-14.DOI: 10.1016/j.heliyon.2023.e13829.

Lee SH, Hu W, Matulay JT, Silva MV, Owczarek TB, Kim K, et al. Tumor evolution and drug response in patient-derived organoid models of bladder cancer. Cell. 2018;173(2):515-528.DOI: 10.1016/j.cell.2018.03.017.

Sachs N, Papaspyropoulos A, Zomer-van Ommen DD, Heo I, Böttinger L, Klay D, et al. Long-term expanding human airway organoids for disease modeling. EMBO J. 2019;38(4):e100300,1-20.DOI: 10.15252/embj.2018100300.

Di Liello R, Ciaramella V, Barra G, Venditti M, Della Corte CM, Papaccio F, et al. Ex vivo lung cancer spheroids resemble treatment response of a patient with NSCLC to chemotherapy and immunotherapy:case report and translational study. ESMO Open. 2019;4(4):e000536,1-6.DOI: 10.1136/esmoopen-2019-000536.

Neal JT, Li X, Zhu J, Giangarra V, Grzeskowiak CL, Ju J, et al. Organoid modeling of the tumor immune microenvironment. Cell. 2018;175(7):1972-1988.DOI: 10.1016/j.cell.2018.11.021.

Gunti S, Hoke ATK, Vu KP, London NR. Organoid and spheroid tumor models: techniques and applications. Cancers (Basel). 2021;13:874,1-17.DOI: 10.3390/cancers13040874.

Ivanova E, Kuraguchi M, Xu M, Portell AJ, Taus L, Diala I, et al. Use of ex vivo patient-derived tumor organotypic spheroids to identify combination therapies for Her2 mutant non–small cell lung cancer. Clin Cancer Res. 2020;26(10):2393-2403.DOI: 10.1158/1078-0432.CCR-19-1844.

Zhang Z, Wang H, Ding Q, Xing Y, Xu Z, Lu C, et al. Establishment of patient-derived tumor spheroids for non-small cell lung cancer. PLoS One. 2018;13(3):e0194016,1-8.DOI: 10.1371/journal.pone.0194016.

Verduin M, Hoeben A, De Ruysscher D, Vooijs M. Patient-derived cancer organoids as predictors of treatment response. Front Oncol. 2021;11:641980,1-16.DOI: 10.3389/fonc.2021.641980.

Qi X, Prokhorova AV, Mezentsev AV, Shen N, Trofimenko AV, Filkov GI, et al. Comparison of EMT-related and multi-drug resistant gene expression, extracellular matrix production, and drug sensitivity in NSCLC spheroids generated by scaffold-free and scaffold-based methods. Int J Mol Sci. 2022;23(21):13306,1-12.DOI: 10.3390/ijms232113306.

Sulimanov R, Koshelev K, Makarov V, Mezentsev A, Durymanov M, Ismail L, et al. Mathematical modeling of non-small-cell lung cancer biology through the experimental data on cell composition and growth of patient-derived organoids. Life (Basel). 2023;13(11):2228,1-10.DOI: 10.3390/life13112228.

Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JH, Beasley MB, et al. The 2015 world health organization classification of lung tumors:impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol. 2015;10(9):1243-1260.DOI: 10.1097/JTO.0000000000000630.

Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991;251(5000): 1451-1455.DOI: 10.1126/science.2006419.

Durymanov MO, Rosenkranz AA, Sobolev AS. Current approaches for improving intratumoral accumulation and distribution of nanomedicines. Theranostics. 2015;5(9):1007-1020.DOI: 10.7150/thno.11742.

Dong P, Ma L, Liu L, Zhao G, Zhang S, Dong L, et al. CD86+/CD206+, diametrically polarized tumor-associated macrophages, predict hepatocellular carcinoma patient prognosis. Int J Mol Sci. 2016;17(3):320,1-12.DOI: 10.3390/ijms17030320.

Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer. 2016;16(9):582-598.DOI: 10.1038/nrc.2016.73.

Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms controlling PD-L1 expression in cancer. Mol Cell. 2019;76(3):359-370.DOI: 10.1016/j.molcel.2019.09.030.

Li H, Xu Y, Wan B, Song Y, Zhan P, Hu Y, et al. The clinicopathological and prognostic significance of PD-L1 expression assessed by immunohistochemistry in lung cancer: a meta-analysis of 50 studies with 11,383 patients. Transl Lung Cancer Res. 2019;8(4):429-449.DOI: 10.21037/tlcr.2019.08.04.

Liston DR, Davis M. Clinically relevant concentrations of anticancer drugs: a guide for nonclinical studies. Clin Cancer Res. 2017;23(14):3489-3498.DOI: 10.1158/1078-0432.CCR-16-3083.

Kaur G, Gupta SK, Singh P, Ali V, Kumar V, Verma M. Drug-metabolizing enzymes: role in drug resistance in cancer. Clin Transl Oncol. 2020;22(10):1667-1680.DOI: 10.1007/s12094-020-02325-7.

Robey RW, Pluchino KM, Hall MD, Fojo AT, Bates SE, Gottesman MM. Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat Rev Cancer. 2018;18(7):452-464.DOI: 10.1038/s41568-018-0005-8.

Sun JM, Oh DY, Lee SH, Kim DW, Im SA, Kim TY, et al. The relationship between response to previous systemic treatment and the efficacy of subsequent pemetrexed therapy in advanced non-small cell lung cancer. Lung Cancer. 2010;68(3):427-32.DOI: 10.1016/j.lungcan.2009.07.013.

Niu Q, Wang W, Li Y, Ruden DM, Wang F, Li Y, et al. Low molecular weight heparin ablates lung cancer cisplatin-resistance by inducing proteasome-mediated ABCG2 protein degradation. PLoS One. 2012;7(7):e41035,1-9.DOI: 10.1371/journal.pone.0041035.

Chen JH, Chu XP, Zhang JT, Nie Q, Tang WF, Su J, et al. Genomic characteristics and drug screening among organoids derived from non-small cell lung cancer patients. Thorac Cancer. 2020;11(8): 2279-2290.DOI: 10.1111/1759-7714.13542.

Hu Y, Sui X, Song F, Li Y, Li K, Chen Z, et la. Lung cancer organoids analyzed on microwell arrays predict drug responses of patients within a week. Nat Commun. 2021;12(1):2581,1-14.DOI: 10.1038/s41467-021-22676-1.

Koga T, Soh J, Hamada A, Miyano Y, Fujino T, Obata K, et al. Clinical relevance of patient-derived organoid of surgically resected lung cancer as an in vitro model for biomarker and drug testing. JTO Clin Res Rep. 2023;4(9):100554,1-11.DOI: 10.1016/j.jtocrr.2023.100554.

Kim SY, Kim SM, Lim S, Lee JY, Choi SJ, Yang SD, et al. Modeling clinical responses to targeted therapies by patient-derived organoids of advanced lung adenocarcinoma. Clin Cancer Res. 2021;27(15):4397-4409.DOI: 10.1158/1078-0432.CCR-20-5026.

Wang HM, Zhang CY, Peng KC, Chen ZX, Su JW, Li YF, et al. Using patient-derived organoids to predict locally advanced or metastatic lung cancer tumor response:a real-world study. Cell Rep Med. 2023;4(2):100911,1-22.DOI: 10.1016/j.xcrm.2022.100911.

Della Corte CM, Barra G, Ciaramella V, Di Liello R, Vicidomini G, Zappavigna S, et al. Antitumor activity of dual blockade of PD-L1 and MEK in NSCLC patients derived three-dimensional spheroid cultures. J Exp Clin Cancer Res. 2019;38(1):253,1-12.DOI: 10.1186/s13046-019-1257-1.

Dijkstra KK, Monkhorst K, Schipper LJ, Hartemink KJ, Smit EF, Kaing S, et al. Challenges in establishing pure lung cancer organoids limit their utility for personalized medicine. Cell Rep. 2020;31(5):107588,1-13.DOI: 10.1016/j.celrep.2020.107588.

Si LL, Lv L, Zhou WH, Hu WD. Establishment and identification of human primary lung cancer cell culture in vitro. Int J Clin Exp Pathol. 2015;8(6):6540-6546.PMID: 26261533.

Lin RZ, Chou LF, Chien CCM, Chang HY. Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and beta1-integrin. Cell Tissue Res. 2006;324(3):411-422.DOI: 10.1007/s00441-005-0148-2.

Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, et al. Folliculin controls lung alveolar enlargement and epithelial cell survival through E-cadherin, LKB1, and AMPK. Cell Rep. 2014;7(2):412-423.DOI: 10.1016/j.celrep.2014.03.025.

Manuel Iglesias J, Beloqui I, Garcia-Garcia F, Leis O, Vazquez-Martin A, Eguiara A, et al. Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin. PLoS One. 2013;8(10):e77281,1-12.DOI: 10.1371/journal.pone.0077281.

Ko YR, Kim JM, Kang Y, Kim M, Jang SJ. A method for culturing patient-derived lung cancer organoids from surgically resected tissues and biopsy samples. Organoid. 2022 2:e19,1-9.DOI: 10.51335/organoid.2022.2.e19.

Dijkstra KK, Cattaneo CM, Weeber F, Chalabi M, van de Haar J, Fanchi LF, et al. Generation of tumor-reactive t cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell. 2018;174(6):1586-1598.DOI: 10.1016/j.cell.2018.07.009.

Donnem T, Andersen S, Al-Saad S, Al-Shibli K, Busund LT, Bremnes RM. Prognostic impact of angiogenic markers in non–small-cell lung cancer is related to tumor size. Clin Lung Cancer. 2011;12(2):106-115.DOI: 10.1016/j.cllc.2011.03.005.

Ramachandran S, Verma AK, Dev K, Goyal Y, Bhatt D, Alsahli MA, et al. Role of cytokines and chemokines in NSCLC immune navigation and proliferation. Oxid Med Cell Longev. 2021;2021:e5563746,1-20.DOI: 10.1155/2021/5563746.

Bethune G, Bethune D, Ridgway N, Xu Z. Epidermal growth factor receptor (EGFR) in lung cancer:an overview and update. J Thorac Dis. 2010;2(1): 48-51.PMID: 22263017.

Gong k, Gao G, Beckley N, Zhang Y, Yang X, Sharma M, et al. Tumor necrosis factor in lung cancer:complex roles in biology and resistance to treatment. Neoplasia. 2021;23(2):189-196.DOI: 10.1016/j.neo.2020.12.006.

Garon EB, Chih-Hsin Yang J, Dubinett SM. The Role of interleukin 1β in the pathogenesis of lung cancer. JTO Clin Res Rep. 2020;1:100001,1-11.DOI: 10.1016/j.jtocrr.2020.100001.

Stutvoet TS, Kol A, de Vries EG, de Bruyn M, Fehrmann RS, Terwisscha van Scheltinga AG, et al. MAPK pathway activity plays a key role in PD-L1 expression of lung adenocarcinoma cells. J Pathol. 2019;249(1):52-64.DOI: 10.1002/path.5280.50. Szejniuk WM, Robles AI, McCulloch T, Falkmer UGI, Røe OD. Epigenetic predictive biomarkers for response or outcome to platinum-based chemotherapy in non-small cell lung cancer, current state-of-art. Pharmacogenomics J. 2019;19(1):5-14.DOI: 10.1038/s41397-018-0029-1.

Budagaga Y, Sabet Z, Zhang Y, Novotná E, Hanke I, Rozkoš T, et al. Tazemetostat synergistically combats multidrug resistance by the unique triple inhibition of ABCB1, ABCC1, and ABCG2 efflux transporters in vitro and ex vivo. Biochem Pharmacol. 2023;216:115769.DOI: 10.1016/j.bcp.2023.115769.

Zhou L, Chen W, Cao C, Shi Y, Ye W, Hu J, et al. Design and synthesis of α-naphthoflavone chimera derivatives able to eliminate cytochrome P450 (CYP)1B1-mediated drug resistance via targeted CYP1B1 degradation. Eur J Med Chem. 2020;189:112028,1-11.DOI: 10.1016/j.ejmech.2019.112028.


Refbacks

  • There are currently no refbacks.


Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.