PO.TB04.03 · 肿瘤生物学

新鲜NSCLC的三维生物打印:Lung3Dprint概念验证研究

3D bioprinting of fresh NSCLC: The Lung3Dprint proof of concept study

编号 4872 展板 21 时间 4/21 09:00–12:00 区域 Section 28 主讲 Aurélie Cadiou, Pharm D

分会场 In Vitro Models 2: 2D, 3D, Organoids, and Spheroids

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作者与单位 Authors & Affiliations

Aurélie Cadiou¹, Clarisse Thiollier-Schmitt¹, Gabrielle Devret², Raphaële Guelminger³, Tanguy Fenouil⁴, Corinne Perrin⁵, Camille Leonce¹, Samantha Ballesta⁶, Gaëtan Pochon⁶, Nicolas Alcala⁷, Lars Petter Jordheim¹, Charles Dumontet¹, Michaël Duruisseaux¹

¹Cancer Research Center of Lyon (CRCL), Lyon, France,²Service de chirurgie thoracique, Hôpital Louis Pradel, Hospices Civils de Lyon, Lyon, France,³Hospices Civils de Lyon, Lyon, France,⁴Groupement Hospitalier Est, Institut de Pathologie Multisite - Site Est, Hospices Civils de Lyon, Bron, France,⁵Groupement Hospitalier Est, Tissu-Tumorothèque Est, CRB HCL, Hospices Civils de Lyon, Bron, France,⁶3D-ONCO platform, Cancer Research Center of Lyon (CRCL), Lyon, France,⁷International Agency for Research on Cancer, Lyon, France

摘要 Abstract

中文摘要

三维生物打印模型正在兴起,以弥合二维系统与动物模型之间的差距,提高临床前工作流程的效率和准确性。本研究旨在(i)开发一种创新方法来生成患者来源生物打印肺癌(PDBLC)模型,该模型旨在反映亲代肿瘤的异质性,以及(ii)评估这些模型作为相关的体外药物筛选工具。2025年3月至10月在里昂民用医院(Hospices Civils de Lyon)获取新鲜非小细胞肺癌(NSCLC)样本,将其解离并包埋于纤维蛋白原-海藻酸盐-明胶生物墨水中(每毫升生物墨水0.5至5×10^6个细胞)。使用Cellink BIO X™通过挤出式生物打印获得PDBLC模型,并使用凝血酶、转谷氨酰胺酶和钙进行后处理聚合。随后将PDBLC模型置于专用的肺癌细胞培养基中,并在不同时间点采用非破坏性方法(PrestoBlue™实验)和破坏性方法(流式细胞术和免疫组织化学)进行分析。流式细胞术标志物包括用于鉴定肿瘤细胞的EpCAM和CK-7/-8、用于内皮细胞的CD31、用于白细胞浸润的CD45、CD3和CD19,以及用于分析癌相关成纤维细胞的FAP、α-SMA和PDGFR-beta。肿瘤和非肿瘤细胞含量以每个物体的细胞数量及其占存活细胞总数的百分比进行量化。目前已对九个样本进行了生物打印和分析。PrestoBlue™实验能够随时间对模型的代谢活性进行非特异性监测。生物墨水中肿瘤细胞的中位百分比估计为40%(范围:11-85)。第7天和第14天存活肿瘤细胞的百分比取决于生物墨水中细胞的初始浓度,范围为5至84%。每个PDBLC模型中肿瘤细胞的绝对数量倾向于随时间稳定。存活肿瘤细胞的绝对百分比倾向于在第0天至第14天之间增加,中位倍数变化为1.7倍(范围:-0.76至7.2)。这主要是由于白细胞浸润以及内皮亚群的减少所致。生物打印后14天仍残留的白细胞亚群被确定为T淋巴细胞(CD45+ CD3+)。目前正在对PDBLC模型进行NSCLC患者所用的常规疗法和靶向疗法暴露,结果将另行呈现。三维生物打印为评估新鲜肿瘤样本提供了一种相对廉价且快速的手段。我们的初步数据表明,我们的模型不仅支持新鲜NSCLC细胞存活长达22天,而且在此期间还维持了原发肿瘤的异质性。这些三维模型作为药物筛选平台的应用正在评估之中,对转化研究和临床实践将具有相关意义。

查看英文原文 English abstract

3D bioprinted models are emerging to bridge the gap between 2D systems and animal models, advancing the efficiency and accuracy of preclinical workflows. This work aimed to (i) develop an innovative methodology to generate patient-derived bioprinted lung cancer (PDBLC) models that aim to reflect the heterogeneity of the parent tumor, and (ii) evaluate these models as a relevant ex vivo drug screening tool. Fresh Non-Small Cell Lung Cancer (NSCLC) samples were obtained from March to October 2025 at Hospices Civils de Lyon, dissociated and embedded into a fibrinogen-alginate-gelatin bioink (0.5 to 5x10 6 cells/mL of bioink). PDBLC models were obtained by extrusion-based bioprinting using a Cellink BIO X TM , with post-conditioning polymerization using thrombin, transglutaminase, and calcium. PDBLC models were then placed in a dedicated lung cancer cell culture medium and analyzed at different time points, using a non-disruptive method (PrestoBlue TM assay) and disruptive methods (flow cytometry and immunohistochemistry). Flow cytometry markers include EpCAM and CK-7/-8 to identify tumor cells, CD31 for endothelial cells, CD45, CD3, and CD19 for leukocyte infiltrate, and FAP, ɑ-SMA and PDGFR-beta for analysis of cancer-associated fibroblasts. Tumor and non-tumor cell contents were quantified as number of cells per object and their percentage of total live cells present. Nine samples have presently been bioprinted and analyzed. PrestoBlue TM assay enabled non-specific monitoring of the model's metabolic activity over time. The median percentage of tumor cells in the bioink was estimated at 40% (range: 11 - 85). The percentage of viable tumor cells on days 7 and 14 were dependent on the initial concentration of cells in the bioink, ranging from 5 to 84%. The absolute number of tumor cells per PDBLC model tended to stabilize with time. The absolute percentage of viable tumor cells tended to increase between day 0 and day 14, with a median fold-change of 1.7-fold (range: -0.76 - 7.2). This was notably due to the decrease in the leukocyte infiltration as well as the endothelial subpopulation. Leukocyte subpopulation remaining 14 days after bioprinting was found to be T lymphocytes (CD45+ CD3+). Exposure of PDBLC models to conventional and targeted therapies used in NSCLC patients is ongoing and results will be presented. 3D bioprinting offers a relatively inexpensive and rapid means to evaluate fresh tumor samples. Our preliminary data show that our model not only supports fresh NSCLC cell survival for up to 22 days, but also maintains the primary tumor's heterogeneity over this period. The use of these 3D models as a drug-screening platform is being evaluated and would be relevant for translational research and clinical practice.

利益披露 Disclosure

A. Cadiou, None.. C. Thiollier-Schmitt, None.. G. Devret, None.. R. Guelminger, None.. T. Fenouil, None.. C. Perrin, None.. C. Leonce, None.. S. Ballesta, None.. G. Pochon, None.. L. Jordheim, None.. C. Dumontet, None. M. Duruisseaux, Pfizer g., Board of Directors, non-salaried role), ), Other, Remuneration for participation in scientific meetings. Merus ). Takeda g., Board of Directors, non-salaried role), ). Guardant g., Board of Directors, non-salaried role), ), Other, Remuneration for participation in scientific meetings. Eli Lilly ). Boehringer Ingelheim g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. Astra Zeneca g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. Roche g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. BMS g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. Abbvie g., Board of Directors, non-salaried role). MSD g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. Novartis g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. GSK ). Sanofi g., Board of Directors, non-salaried role). Amgen g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings. Regeneron g., Board of Directors, non-salaried role). Revolution Medecine g., Board of Directors, non-salaried role). Novocure g., Board of Directors, non-salaried role).

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