研究背景：血管新生（angiogenesis）長久以來被視為實體腫瘤生長與轉移的關鍵，臨床上常使用 bevacizumab（癌思停）、ramucirumab（欣銳擇）等抗血管新生藥物進行治療。然而，在非小細胞肺癌（non-small cell lung cancer, NSCLC）中，此類藥物對疾病控制之效果有限，推測係因腫瘤細胞發展出替代性的血液供應途徑。血管擬態（vasculogenic mimicry, VM）於 1999 年被提出，係由腫瘤細胞自身形成、不依賴血管內皮細胞之類血管通道，於組織切片中呈現醣蛋白 PAS 染色陽性、內皮細胞標記 CD31 陰性，並與腫瘤惡化、轉移及治療抗性密切相關。惟過去 VM 研究多仰賴二維（two-dimensional, 2D）Matrigel 網絡影像，無法呈現體內真實之三維（three-dimensional, 3D）管腔結構，亦缺乏可信賴之定量偵測與藥物篩選平台，此為 VM 轉譯研究之關鍵瓶頸。
研究目的：本研究旨在建立可信賴的 3D VM 偵測與藥物篩選平台，釐清 EGFR 突變狀態與 VM 形成能力之間的關聯，並於人體肺腺癌檢體中定義原位 VM 指標。
材料與方法：本研究以光片式螢光顯微鏡（light-sheet fluorescence microscopy, LSFM）建立動態 3D VM 影像平台，並先於 B16F10 黑色素瘤細胞（陽性對照細胞株）完成平台驗證。將不同 EGFR 基因型之肺癌細胞株（A549、H1299、H1650、HCC827，以及同源基因背景之 H1299-EGFR 野生型與 H1299-EGFR-L858R）培養於 3D 基質中連續監測，定量管徑、管腔形成與網絡複雜度。以高內涵影像系統評估 first-in-class 抗 VM 小分子 foslinanib 與第三代 EGFR-TKI osimertinib 單用及併用之抗 VM 活性。人體檢體方面，取自成大醫院之四例晚期肺腺癌（lung adenocarcinoma, LUAD）福馬林固定石蠟包埋組織（IRB 編號 B-ER-108-090），以 PhenoCycler-Fusion 多重免疫螢光與 GeoMx-NGS 空間轉錄體進行空間多體學分析，並以 CCLE 與 TCGA 資料庫進行外部驗證。
研究結果：本研究成功建立動態 3D VM 影像平台，可即時觀察並定量具管腔（直徑約 25–50 µm，與生理性微血管相當）之 VM 結構，並證實三維 VM 與二維網絡在結構上截然不同，傳統平面分析系統性地低估了 VM 之管腔構造。利用此平台發現，EGFR 突變之肺癌細胞株（H1650、HCC827）能形成完整之三維 VM 管腔，而 EGFR 野生型（A549、H1299）則維持平面單層而無管腔；同源基因背景之 H1299-EGFR-L858R 亦重現突變型表現型，具有三維 VM 管腔，確認此關聯係由突變本身而非細胞株特性所致。CCLE 與 TCGA 之生物資訊分析（CINP／VM 基因分數）進一步支持 EGFR 突變之 LUAD 具較高之 VM 傾向。在藥物層面，foslinanib 以劑量依賴且基因型依賴之方式選擇性瓦解三維 VM 管腔；foslinanib 與 osimertinib 併用於 EGFR-L858R 模式呈現協同（synergy）效果，於其他 EGFR 突變株則呈相加效果，顯示此併用之抗 VM 活性具情境專一性。空間多體學分析將人體 LUAD 的 VM 管道定義為 PanCK⁺／CD31⁻／VEGFR1⁺ 的結構，並證實 EGFR 突變 LUAD 的腫瘤細胞富含 VM 基因特徵。
結論：VM 在肺癌中是具管腔的三維結構，傳統的二維分析無法適當地呈現。本研究建立之動態三維影像平台可穩定定量 VM 行為並篩選抗 VM 藥物，並據以證實 EGFR 突變狀態決定三維 VM 形成能力，此關聯經同源模式、人體檢體原位指標與獨立資料庫三重驗證。研究成果指出 EGFR 突變肺腺癌具內在之 VM 傾向，並提供偵測工具與候選指標，為此一族群在傳統抗血管新生治療成效有限之現況下，開啟針對非內皮依賴性血流途徑之治療契機。本研究核心成果已獲 Translational Research（IF>5）接受刊登 (doi.org/10.1016/j.trsl.2026.07.010)，相關方法亦取得中華民國發明專利（第 I902393 號）。

Background: Neovascularization, a hallmark of cancer, encompasses sprouting angiogenesis, vasculogenic mimicry (VM), intussusception, vessel co-option, and vasculogenesis. Anti-angiogenic therapies that block VEGF signaling, such as bevacizumab and ramucirumab, have attained only modest efficacy in non-small cell lung cancer (NSCLC), in part because tumors establish alternative perfusion pathways such as VM — a de novo, endothelium-independent vascular network built by the tumor cells themselves, which is PAS-positive and CD31-negative in tissue. Research on VM has relied predominantly on two-dimensional (2D) imaging, which cannot capture the three-dimensional (3D) luminal architecture that defines the structure, and this methodological gap has constrained the development of anti-VM therapeutics.
Objectives: This work aimed to establish a reliable platform for assaying 3D VM behavior and screening anti-VM agents, to determine whether VM competence is governed by EGFR mutation status, and to define an in situ VM signature in human lung adenocarcinoma (LUAD).
Methods: A dynamic 3D imaging platform based on light-sheet fluorescence microscopy (LSFM) was developed and first validated in B16F10 melanoma, an established VM reference model. We cultured NSCLC cell lines with different EGFR genotypes (A549, H1299, H1650, HCC827, along with the isogenic pair H1299-EGFR-wild-type and H1299-EGFR-L858R) in 3D matrices and continuously monitored them; we quantified tubule diameter, lumen formation, and network complexity. A high-content screening platform was used to assess the first-in-class anti-VM compound foslinanib and the third-generation EGFR tyrosine kinase inhibitor osimertinib, individually and in combination. Four formalin-fixed, paraffin-embedded advanced LUAD specimens obtained at National Cheng Kung University Hospital (IRB B-ER-108-090) were analyzed by spatial multiomics using PhenoCycler-Fusion multiplexed imaging and GeoMx-NGS spatial transcriptomics, with external validation in the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA).
Results: The platform resolved patent tubules and hollow lumens 25–50 µm in diameter, dimensions comparable to small physiological microvessels, and demonstrated that 3D VM is fundamentally distinct from planar 2D networks, which systematically under-represent VM by collapsing this luminal architecture onto a single plane. EGFR-mutant lines (H1650, HCC827) consistently formed complex 3D VM with patent lumens, whereas wild-type lines (A549, H1299) remained planar; the isogenic H1299-EGFR-L858R model reproduced the mutant phenotype, establishing that the association is driven by the mutation rather than by cell-line background. Bioinformatic analysis using the Collagen-Induced Network Phenotype (CINP) VM score confirmed a higher VM propensity in EGFR-mutant LUAD across CCLE and TCGA. Pharmacologically, foslinanib disrupted 3D VM tubules in a dose- and genotype-dependent manner; the foslinanib–osimertinib combination acted synergistically in the EGFR-L858R model and additively in other EGFR-mutant lines, indicating context-dependent, genotype-linked combination activity. Spatial multiomics defined VM tunnels in human LUAD as PanCK+/CD31−/VEGFR1+ structures and confirmed enriched VM gene signatures in EGFR-mutant tumor cells.
Conclusions: Vasculogenic mimicry in lung cancer is a three-dimensional, lumen-bearing structure that conventional planar assays cannot adequately represent. The dynamic light-sheet platform established here provides a reliable, quantitative means of assaying VM behavior and screening anti-VM agents and showed that 3D VM competence is determined by EGFR mutation status — a relationship confirmed in an isogenic model, corroborated in human tissue by the in situ signature PanCK+/CD31−/VEGFR1+, and reproduced in independent CCLE and TCGA cohorts. These findings identify EGFR-mutant LUAD as intrinsically primed for vasculogenic mimicry and supply both the tools and the candidate markers needed to test whether targeting this endothelium-independent perfusion route can benefit a population for whom conventional anti-angiogenic therapy has offered limited benefit. The core dataset has been accepted for publication in Translational Research (IF > 5) (doi.org/10.1016/j.trsl.2026.07.010), and the underlying method has been granted Taiwan Patent No. I902393.

1. Cao Y: Future options of anti-angiogenic cancer therapy. Chin J Cancer 35:21, 2016
2. Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med 285:1182-1186, 1971
3. Fouladzadeh A, Dorraki M, Min KKM, et al: The development of tumour vascular networks. Commun Biol 4:1111, 2021
4. Garon EB, Ciuleanu TE, Arrieta O, et al: Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL): a multicentre, double-blind,randomised phase 3 trial. Lancet 384:665-73, 2014
5. Ge H, Luo H: Overview of advances in vasculogenic mimicry - a potential target for tumor therapy. Cancer Manag Res 10:2429-37, 2018
6. Gridelli C, Rossi A, Carbone DP, et al: Non-small-cell lung cancer. Nat Rev Dis Primers 1:15009, 2015
7. Grodzik M, Sawosz E, Wierzbicki M, et al: VEGF-dependent mechanism of antiangiogenic action of diamond nanoparticles in glioblastoma multiforme tumor. Nanotechnology 2012: Bio Sensors, Instruments, Medical, Environment and Energy (TechConnect Briefs) 3:218-221, 2012
8. Hendrix MJ, Seftor EA, Hess AR, et al: Vasculogenic mimicry and tumour-cell plasticity: lessons from melanoma. Nat Rev Cancer 3:411-21, 2003
9. Hendrix MJ, Seftor EA, Seftor RE, et al: Tumor cell vascular mimicry: Novel targeting opportunity in melanoma. Pharmacol Ther 159:83-92, 2016
10. Herbst RS, Morgensztern D, Boshoff C: The biology and management of non-small cell lung cancer. Nature 553:446-454, 2018
11. Hernandez de la Cruz ON, Lopez-Gonzalez JS, Garcia-Vazquez R, et al: Regulation Networks Driving Vasculogenic Mimicry in Solid Tumors. Front Oncol 9:1419, 2019
12. Kirschmann DA, Seftor EA, Hardy KM, et al: Molecular pathways: vasculogenic mimicry in tumor cells: diagnostic and therapeutic implications. Clin Cancer Res18:2726-32, 2012
13. Leslie M: Tumors' do-it-yourself blood vessels. Science 352:1381-3, 2016
14. Li Y, Sun B, Zhao X, et al: Subpopulations of uPAR+ contribute to vasculogenic mimicry and metastasis in large cell lung cancer. Exp Mol Pathol 98:136-44, 2015
15. Lim D, Do Y, Kwon BS, et al: Angiogenesis and vasculogenic mimicry as therapeutic targets in ovarian cancer. BMB Reports 53:291-298, 2020
16. Maniotis AJ, Folberg R, Hess A, et al: Vascular Channel Formation by Human Melanoma Cells in Vivo and in Vitro: Vasculogenic Mimicry. The American Journal of Pathology 155:739-752, 1999
17. Peng Z, Wang J, Shan B, et al: The long noncoding RNA LINC00312 induces lung adenocarcinoma migration and vasculogenic mimicry through directly binding YBX1. Mol Cancer 17:167, 2018
18. Potente M, Gerhardt H, Carmeliet P: Basic and therapeutic aspects of angiogenesis. Cell 146:873-87, 2011
19. Racordon D, Valdivia A, Mingo G, et al: Structural and functional identification of vasculogenic mimicry in vitro. Sci Rep 7:6985, 2017
20. Reich M, Liefeld T, Gould J, et al: GenePattern 2.0. Nat Genet 38:500-1, 2006
21. Ren K, Yao N, Wang G, et al: Vasculogenic mimicry: a new prognostic sign of human osteosarcoma. Hum Pathol 45:2120-9, 2014
22. Ricci-Vitiani L, Pallini R, Biffoni M, et al: Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 468:824-8, 2010
23. Sandler A, Gray R, Perry MC, et al: Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355:2542-50, 2006
24. Sarnella A, D’Avino G, Hill BS, et al: A Novel Inhibitor of Carbonic Anhydrases Prevents Hypoxia-Induced TNBC Cell Plasticity. International Journal of Molecular Sciences 21, 2020
25. Schurch CM, Bhate SS, Barlow GL, et al: Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front. Cell 182:1341-1359 e19, 2020
26. Shen L, Chen YL, Huang CC, et al: CVM-1118 (foslinanib), a 2-phenyl-4-quinolone derivative, promotes apoptosis and inhibits vasculogenic mimicry via targeting TRAP1. Pathol Oncol Res 29:1611038, 2023
27. Sun B, Zhang D, Zhang S, et al: Hypoxia influences vasculogenic mimicry channel formation and tumor invasion-related protein expression in melanoma. Cancer Lett 249:188-97, 2007
28. Sun H, Zhang D, Yao L: Vasculogenic mimicry and cancer stem-like cell markers are associated with poor prognosis of non-small cell lung cancer. Int J Clin Exp Pathol 9:11523-35, 2016
29. Sun T, Sun BC, Zhao XL, et al: Promotion of tumor cell metastasis and vasculogenic mimicry by way of transcription coactivation by Bcl-2 and Twist1: a study of hepatocellular carcinoma. Hepatology 54:1690-706, 2011
30. Tam SY, Wu VWC, Law HKW: Hypoxia-Induced Epithelial-Mesenchymal Transition in Cancers: HIF-1alpha and Beyond. Front Oncol 10:486, 2020
31. Valdivia A, Mingo G, Aldana V, et al: Fact or Fiction, It Is Time for a Verdict on Vasculogenic Mimicry? Front Oncol 9:680, 2019
32. Velez DO, Tsui B, Goshia T, et al: 3D collagen architecture induces a conserved migratory and transcriptional response linked to vasculogenic mimicry. Nat Commun 8:1651, 2017
33. Wang M, Zhao X, Zhu D, et al: HIF-1alpha promoted vasculogenic mimicry formation in hepatocellular carcinoma through LOXL2 up-regulation in hypoxic tumor microenvironment. J Exp Clin Cancer Res 36:60, 2017
34. Wu S, Yu L, Wang D: Aberrant expression of CD133 in non-small cell lung cancer and its relationship to vasculogenic mimicry. BMC Cancer 12:535, 2012
35. Xia Y, Cai XY, Fan JQ, et al: The role of sema4D in vasculogenic mimicry formation in non-small cell lung cancer and the underlying mechanisms. Int J Cancer, 2018
36. Xiang L, Semenza GL: Hypoxia-inducible factors promote breast cancer stem cell specification and maintenance in response to hypoxia or cytotoxic chemotherapy. Adv Cancer Res 141:175-212, 2019
37. Yao L, Zhang D, Zhao X: Dickkopf-1-promoted vasculogenic mimicry in non-small cell lung cancer is associated with EMT and development of a cancer stem-like cell phenotype. J Cell Mol Med 20:1673-85, 2016
38. Yu S, Sha H, Qin X, et al: EGFR E746-A750 deletion in lung cancer represses antitumor immunity through the exosome-mediated inhibition of dendritic cells. Oncogene 39:2643-2657, 2020
39. Zhang J, Qiao L, Liang N, et al: Vasculogenic mimicry and tumor metastasis. J BUON 21:533-541, 2016
40. Zhang X, Cui P, Ding B, et al: Netrin-1 elicits metastatic potential of non-small cell lung carcinoma cell by enhancing cell invasion, migration and vasculogenic mimicry via EMT induction. Cancer Gene Ther 25:18-26, 2018
41. Zhang X, Zhang J, Zhou H, et al: Molecular Mechanisms and Anticancer Therapeutic Strategies in Vasculogenic Mimicry. J Cancer 10:6327-40, 2019
42. Zhao B, Wu M, Hu Z, et al: Thrombin is a therapeutic target for non-small-cell lung cancer to inhibit vasculogenic mimicry formation. Signal Transduct Target Ther 5:117, 2020