Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

摘要

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

摘要

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1−10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

引言

Tumor antigen-specific cytotoxic T-lymphocytes (CTLs) play a crucial role in the response to cancers through a process known as cancer "immune surveillance". Their anti-tumor functions are enhanced by immune checkpoint blockade antibodies such as CTLA-4 inhibitors and PD-1/PD-L1 inhibitors. In non-small cell lung cancer (NSCLC), anti-PD-1/PD-L1 therapies result in response rates ranging from 0%-17% in patients with PD-L1-negative tumors and 36%-100% in those expressing PD-L1. The robust responses to PD-1/PD-L1 blockade observed in melanoma and NSCLC are shown by evidence of improved overall response rate (RR), durable clinical benefits, and progression-free su....

方案

Samples were prospectively collected within the framework of the CIRCAN ("CIRculating CANcer") cohort based at the Lyon University Hospital following patient written consent. This study was integrated into the CIRCAN_ALL cohort. The study CIRCAN_ALL was recognized as non-interventional by the CPP South-East IV dated 04/11/2015 under the reference L15-188. An amended version was recognized as non-interventional on 20/09/2016 under reference L16-160. The CIRCAN_ALL study was declared to the IT and freedom correspondent of the Hospices Civils de Lyon on 01/12/2015, under the reference 15-131. Blood collection was performed when physicians observed the earliest in....

结果

The first pre-requisite was to obtain uncontaminated (infectious agent-free) collections of CTCs for tissue culture and avoid IF background generated. The decontamination protocol enabled cleaning of all the pipes and pumps, and it resulted in the collection of CTCs with a good recovery rate without bacterial contamination. The enriched samples were compared without and with the decontamination protocol workflow of the spiral microfluidic device. To validate the decontamination protocol, the A549 cell line was used in ab.......

讨论

Two major points were raised in the present study, the first with regards to performance of the workflow for its transfer to clinical applications, and the second concerning the decrease in subjectivity for the analysis of fluorescence images obtained.

A performant and optimized workflow for CTC enumeration was initially determined using customizable IF assay after cell enrichment via a CTC label-free microfluidic system (spiral microfluidic device). Using this workflow, a pilot study confirme.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
4'，6-二脒基-2-phé尼龙哚 （DAPI）	Ozyme	BLE 422801	储存条件：+4 °C
BD Facs Clean – 5 L	BD Biosciences	340345	漂白剂基清洁剂。储存条件：室温
漂白剂 1% 清洁液 100 mL	Biolidics	CBB-F016012	漂白剂。储存条件：室温
牛血清白蛋白 （BSA） 7.5%	Sigma	A8412	储存条件：+4 °C
CD45 单克隆抗体（克隆 HI30） Alexa Fluor 647	BioLegend	BLE304020	储存条件：+4 >C
CellProfiler 软件	Broad Institute		图像分析软件
离心装置	Hettich	4706	储存条件： 室温
离心管 50 mL	康宁	430-829	储存条件： 室温
离心管 15 mL	生物利度	CBB-F001004-25	储存条件： 室温
ClearCell FX-1 系统	Biolidics	CBB-F011002	螺旋微流控装置。储存条件：室温
Coulter Clenz 清洁剂 – 5 L	Beckman Coulter	8448222	通用清洁剂。储存条件：室温
CTChip FR1S	Biolidics	CBB-FR001002	微流控芯片。储存条件：室温
Cytospin 4	ThermoFisher	A78300003	储存条件：室温
稀释剂添加剂试剂 – 20 mL	生物利度	CBB-F016009	储存条件：+4 °C
EZ Cytofunnels ThermoFisher		A78710003	带棉花的样品室。储存条件：室温
FcR 封闭剂	Miltenyi Biotec	130-059-901	储存条件：+4 °C
胎牛血清 （FCS）	Gibco	10270-106	储存条件：+4 °C
Fluoromount	Sigma	F4680	安装解决方案。储存条件：室温
Fungizone - 50 mg	Bristol-Myers-Squibb	90129TB29	抗真菌试剂。储存条件：+4 >C
FX1 输入吸管，带锁帽	Biolidics	CBB-F013005	吸管。储存条件：室温
KovaSlide	Dutscher	50126	腔室载玻片。储存条件： 室温
PanCK 单克隆抗体（克隆 AE1/AE3） Alexa Fluor 488	ThermoFisher	53-9003-80	储存条件：+4 °C
多聚甲醛 16%	ThermoFisher	11490570	固定液。储存条件：+4 >C
PD-L1 单克隆抗体（克隆 29E2A3） - 藻红蛋白	BioLegend	BLE329706	储存条件：+4 °C
培养皿	Dutscher	632180	储存条件：室温
磷酸盐缓冲盐水 （PBS）	Ozyme	BE17-512F	储存条件：+4 °C
磷酸盐缓冲盐水超纯 1x 级 – 1 L	1st Base 实验室	BUF-2040-1X1L	储存条件：室温
Pluronic F-68 10%	Gibco	24040-032	抗结合溶液。储存条件：室温
聚赖氨酸载玻片	ThermoFisher	J2800AMNZ	储存条件：室温
聚丙烯锥形管 50 mL	Falcon	352098	储存条件：室温
RBC 裂解缓冲液 – 100 mL	G Biosciences	786-649	储存条件：+4 °C
RBC 裂解缓冲液 – 250 mL	G Biosciences	786-650	储存条件：+4 °C
重悬缓冲液 （RSB）	生物利度	CBB-F016003	储存条件：+4 °C
Shandon Cytopsin4 离心机	ThermoFisher	A78300003	专用离心机。储存条件： 室温
硅隔离器	Grace bio-Labs	664270	储存条件： 室温
无菌 去离子水 – 100 mL	1st Base Laboratory	CUS-4100-100ml	储存条件： 室温
直式荧光显微镜 Axio Imager D1	蔡司		储存条件： 室温
手术 无菌袋装	SPS 实验室	98ULT01240	储存条件： 室温
注射器 BD Discardit II 20 mL 无菌	BD Biosciences	300296	储存条件： 室温
注射器过滤器 0.22 µm 33 mm 无菌	ClearLine	51732	储存条件：室温
Zen lite 2.3 Lite 软件	蔡司		显微镜相关软件