PO.IM01.03 · 免疫学
建立以CTC为驱动的工作流程用于个体化肿瘤免疫治疗中mRNA转染的moDC生产
Establishing a CTC-driven workflow for mRNA-transfected moDC production in personalized cancer immunotherapy
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：在SARS-CoV-2大流行期间取得成功后，基于RNA的疫苗如今正被探索用于肿瘤治疗。其中一种策略是使用经肿瘤mRNA转染的单核细胞来源树突状细胞（moDC），但获取肿瘤组织——尤其是转移部位的组织——是一个限制。循环肿瘤细胞（CTC）为肿瘤材料提供了一种替代来源。在此，我们开发了一套整合CTC与mRNA技术的工作流程，用于个体化肿瘤治疗。
方法：我们的首要目标是比较3种不同CTC分离平台（Parsortix [P]、RosetteSep [RS]或ScreenCell [SC]）的回收率（RR），方法是将荧光标记的乳腺癌SKBR3细胞（200、50、30个；三次重复）加标到健康供者（HD）血液中，并使用Incucyte系统计数回收的CTC数量。同时，从HD单核细胞分化出moDC，并使用Lipofectamine™ MessengerMax™（LMM）以500 ng的GFP-mRNA进行转染，以标准化CTC-mRNA转染条件。通过共聚焦显微镜在6、24、48、72和96小时评估GFP表达，以未转染和模拟转染的moDC作为对照。
结果：加标测试显示P和SC性能相似，且回收率高于RS，后者产生最低的RR（表1）。关于moDC转染，GFP表达在6小时可检测到，持续至24-48小时，72小时后下降，对照中无信号。目前正在用SC回收的SKBR3细胞测试这些条件，以评估用CTC来源mRNA转染moDC的可能性。
结论：SC和P显示出相似的RR，但SC的简便性和更低成本使其更适合与我们在巴西的合作者部署使用。moDC转染有效，LMM成功递送了目标序列供moDC加工和表达，支持将其作为一种免疫治疗方法。各富集平台的回收率（RR）：Parsortix® ScreenCell® RosetteSep™ 加标30个细胞* 30.0% ± 4 33.3% ± 9 10.8% ± 2 加标50个细胞* 44.0% ± 3 53.5% ± 5 5.0% ± 4 加标200个细胞* 59.4% ± 29 60.75% ± 8 10.9% ± 4 *加标实验均以三次重复进行
查看英文原文 English abstract
Background: After its success during SARS-CoV-2 pandemic, RNA-based vaccines are now being explored for cancer therapy. One of the strategies is the use of monocyte-derived dendritic cells (moDCs) transfected with tumor mRNA, but access to tumor tissue - especially from metastatic sites - is a limitation. Circulating tumor cells (CTCs) offer an alternative source for tumor material. Here, we developed a workflow integrating CTCs and mRNA technology for personalized cancer therapy.
Methods: Our first aim was to compare the recovery rate (RR) of 3 different CTCs isolation platforms (Parsortix [P], RosetteSep [RS], or ScreenCell [SC]) by spiking fluorescently-labeled breast cancer SKBR3 cells (200, 50, 30; triplicate) into healthy donor (HD) blood and counting the number of recovered CTCs using the Incucyte System. In parallel, moDCs were differentiated from HD monocytes and transfected with 500 ng of GFP-mRNA using Lipofectamine™ MessengerMax™ (LMM) to standardize conditions for CTC-mRNA transfection. GFP expression was assessed at 6, 24, 48, 72, and 96h by confocal microscopy, with non-transfected and mock-transfected moDCs as controls.
Results: Spiking tests showed that P and SC had similar performance and higher RR compared to RS, which yielded the lowest RR (Table 1). Regarding moDCs transfection, GFP expression was detectable at 6h, persisted through 24-48h and declined after 72h, with no signal in the controls. These conditions are currently being tested on SKBR3 cells recovered with SC, to assess the possibility to transfect moDCs with CTC-derived mRNA.
Conclusion: SC and P showed similar RR, but SC's simplicity and lower cost make it more suitable for deployment with our collaborators in Brazil. moDCs transfection was effective and LMM successfully delivered the target sequences for moDC processing and expression, supporting this as an immunotherapeutic approach. Future studies will be performed to validate the workflow also in clinical samples. Recovery rates (RR) of each enrichment platform Parsortix® ScreenCell® RosetteSep™ 30 spiked cells* 30.0% ± 4 33.3% ± 9 10.8% ± 2 50 spiked cells* 44.0% ± 3 53.5% ± 5 5.0% ± 4 200 spiked cells* 59.4% ± 29 60.75% ± 8 10.9% ± 4 *spiking experiments were conducted in triplicate
利益披露 Disclosure
G. Manga Guimaraes, None..
N. Bayou, None..
M. Serafini, None..
E. Nicolò, None..
L. Pontolillo, None..
B. Pastò, None..
N. Messali, None..
K. Sahu, None..
P. Giannakakou, None..
G. C. Evangelista, None..
O. Elemento, None..
C. Reduzzi, None..
J. Barbuto, None..
M. Cristofanilli, None.