JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Materials
References
자동 번역
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면역학 및 감염병학

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

Published: April 30, 2021
doi:
10.3791/61895
, , , , , , , ,
1CNR Institute for Photonics and Nanotechnology, 2Dept. of Oncology and Molecular Medicine,Istituto Superiore di Sanità, 3Istituto di Patologia Generale,Università Cattolica del Sacro Cuore, 4Tumor Immunology and Immunotherapy Unit,IRCCS Regina Elena National Cancer Institute

Summary

在免疫疗法和单细胞基因组分析时代，癌症生物学需要新颖的体外和计算工具，以便在适当的时空背景下研究肿瘤 – 免疫界面。我们描述了在2D和3D环境中利用肿瘤免疫微流体共培养的方案，与细胞功能的动态，多参数监测兼容。

Abstract

复杂的疾病模型需要尖端工具，能够提供生理和病理相关、可操作的见解，并揭示原本看不见的过程。密切模拟体内场景的高级细胞测定正在成为可视化和测量影响癌症进展的双向肿瘤 – 宿主相互作用的新方法。在这里，我们描述了两种通用方案，在自然和治疗诱导的免疫监视下，在微设备中重现高度可控的2D和3D共培养，模拟肿瘤微环境（TME）的复杂性。在第1节中，提供了一个实验设置，通过明场延时显微镜监测贴壁肿瘤细胞和浮动免疫群体之间的串扰。作为一种应用场景，我们分析了抗癌治疗的效果，例如所谓的免疫原性癌细胞死亡诱导剂对免疫细胞募集和激活的影响。在第2部分中，3D肿瘤免疫微环境以竞争布局组装。通过长达72小时的荧光快照监测差异免疫浸润，以评估联合治疗策略。在这两种设置中，图像处理步骤都说明了提取过多的免疫细胞参数（例如，免疫细胞迁移和相互作用，对治疗剂的反应）。这些简单而强大的方法可以进一步定制，以模拟TME的复杂性，包括癌症，基质和免疫细胞亚型的异质性和可塑性，以及它们作为癌症进化驱动因素的相互作用。这些快速发展的技术与活细胞高内涵成像的兼容性可能导致大型信息数据集的生成，从而带来新的挑战。事实上，三角形“共培养/显微镜/高级数据分析”为精确的问题参数化铺平了道路，这可能有助于量身定制的治疗方案。我们预计，未来癌症免疫芯片与用于高通量处理的人工智能的整合将在利用预测和临床前工具的能力进行精确和个性化的肿瘤学方面向前迈出一大步。

Introduction

作为实验学科的不同医学分支的演变取决于在受控条件下操纵细胞群和器官功能的能力1。这种能力源于可测量模型的可用性，这些模型能够概括我们体内发生的过程。

在免疫治疗和单细胞基因组分析2的时代，癌症生物学需要利用新兴的体外和计算模型在适当的时空背景下研究肿瘤 – 免疫界面2，3。

肿瘤微环境4（TME）是一种复杂的组织，癌细胞与其他细胞（免疫细胞、基质细胞和内皮细胞）和非细胞（细胞外基质，ECM）成分不断相互作用并动态共同进化。这种复杂景观的动态性质决定了免疫细胞是作为恶性细胞的朋友还是敌人，从而强烈影响疾病进展和对治疗的反应。如今，肿瘤免疫学家、生物信息学家和系统生物学专家的巨大努力正在汇聚在一起，以解决癌症异质性5，6的临床意义，无论是在空间（即，在不同的肿瘤区域）还是在时间（即，在不同的肿瘤进展阶段）5，6，并在单细胞水平上表征癌症和免疫细胞表型和功能。作为这种协同作用的一个例子，先进的计算机视觉技术现在通常用于组织学样本中免疫浸润的空间映射7，8。

在实验模型的前端，桥接动物研究和传统的体外方法，微流体和共培养技术的进步提供了不同类别的微工程细胞模型，如类器官，微生理系统9，10，11（MPS）和器官芯片12，13，14 （OOC）。它们具有共同的特征，即放大细胞生态系统的“大局”视图，并扩大体外潜力以控制微环境因素，同时利用高内涵显微镜15和图像处理方法。

如今，最先进的MPS和OOC系统已经开始包括免疫学方面，在现有的组织和共培养物中结合不同亚型的免疫细胞，从而探索和测量各种过程，如炎症性疾病，伤口愈合，粘膜免疫以及对毒素或日常食品的反应16。TME芯片模型10，11，12，13，14，15，16，17也与可灌注微血管18，19，20，21集成在一起，已被开发用于研究细胞类型依赖性相互作用，物理和化学扰动以及细胞毒性活性浸润淋巴细胞22，以及临床相关的免疫调节剂23。

在这里，我们提供多功能协议，从芯片中的细胞加载到图像处理工具，以利用2D（第1节）和3D（第2节）设置16中的高级肿瘤免疫微流体共培养，与动态，多参数24 监测和细胞功能的可视化兼容。这是利用斐济免费软件及其工具箱25，26在样品管理和数据分析中保持易用性和灵活性的实现。

第1节中描述的微流体装置旨在进行贴壁癌症和浮动免疫细胞的2D共培养。该平台经过验证，可用于在存在基因突变27 和/或免疫缺陷28的情况下体外测量免疫细胞行为。在这里，我们通过利用基于Trackmate（斐济软件中实现的插件）的半自动方法，说明了在延时明场图像中跟踪免疫细胞的步骤。该程序能够提取免疫迁移 29 的运动学描述符和对靶向癌细胞的反应（即相互作用时间），无论是否用免疫原性细胞死亡诱导剂27处理。

重要的是，这些从时间序列图像中提取的参数可以用先进的数学机器进行处理。作为这种方法潜力的一个例子，我们的小组最近发表了一项基于随机过程和统计力学的数学方法的分析，以模拟细胞网络属性并提供免疫细胞行为的参数化描述（即，有偏差或不相关的随机游走，高度或不协调的运动30，31）。

第二部分中提供的3D设置基于共培养方案，以竞争方式重建嵌入两个凝胶区域中的更复杂的免疫功能TME，具有不同的细胞类型和药物组合。这里描述了图像处理步骤，以测量在不同时间点在基质胶内培养的人A375M黑色素瘤细胞中染色免疫细胞的浸润，以评估抗肿瘤剂组合32。选择A375M系，一种以高度转移表型为特征的A375P衍生细胞系来评估其在免疫细胞存在下的转移能力32。

所描述的模型可以完全符合不同的细胞源（小鼠和人类永生化或原代细胞系、类器官、异种移植物等）。在我们实验室最近的研究中，通过将高内涵视频显微镜与图像分析相结合，将竞争性 3D 布局应用于研究：i） 抗肿瘤（抗体依赖性细胞介导的细胞毒性，ADCC）免疫反应，并剖析成纤维细胞在 HER2+ 乳腺癌片上模型中对曲妥珠单抗治疗耐药中的作用33;ii）骨髓细胞（即癌症相关巨噬细胞）在肿瘤逃避和T细胞募集机制中的作用34;iii）免疫治疗方案的功效，特别是基于干扰素α条件树突状细胞（IFN-DC），在胶原基质中用药物治疗的结肠癌细胞培养，并评估有效运动和随后的吞噬事件35;iv） 骨髓来源的嗜酸性粒细胞向 IL-33 处理或未处理的黑色素瘤细胞的趋化迁移36.

这些先进的模型可以作为观察窗口，了解免疫质地在癌症转移和耐药机制中的作用，但需要努力将研究结果转化为临床，缩小与基础研究的差距37。

作为一种新兴场景，利用自动化高内涵显微镜的强大功能与使用更具生理相关性的微系统相结合，为处理、处理和解释数百甚至数千千兆字节的多参数数据带来了新的潜在挑战，这些数据可以从单个实验活动中生成。这意味着OOC实验与基于人工智能38，39，40，41，42（AI）的算法直接联系，既可用于高级自动化分析，又可以生成特征，这些特征可以依次输入癌症免疫相互作用的计算机模型43，以及令人兴奋的新应用，例如预测药物筛选测定的开发44。

不断扩大的努力集中在疾病模型的设计以及优化策略上，以实施具有单细胞多组学读数的大规模扰动筛选。这无疑将有助于开发并有望在适当程度的方法标准化的同时，临床实施系统肿瘤免疫学芯片方法，以获得对免疫疾病和癌症传播机制的新见解。

Protocol

1. 贴壁细胞和浮动细胞 2D 共培养的芯片设计 注意：2D共培养布局（图1A-C）的特征在于三个腔室（100μm高），由两组微通道阵列（500 x 12 x 10μm3，L×W×H）互连。中间室形成两个封闭的死胡同隔室，其阻止在加载步骤2.5期间溢出到肿瘤部位的浮动免疫细胞。这种设备类型可用于单细胞（贴壁?…

Representative Results

肿瘤免疫浸润是宿主抗肿瘤反应的一个参数。肿瘤在浸润白细胞的组成、密度、位置和功能状态方面存在异质性，与癌细胞的相互作用可以成为预测病程和治疗反应的临床相关信息的基础。从这个意义上说，微流体技术可以用作补充和特权体外工具，以探索肿瘤的免疫质地，以及监测对抗癌疗法的反应。微流控测定、活细胞成像和跟踪软件的耦合可以建立可靠的定量方法，以量化免疫细胞在不同?…

Discussion

所描述的方法试图设计一种通用方法，以可调节的复杂程度概括肿瘤免疫学领域的两个重要方面，这可以从采用更相关的体外模型中受益。第一个涉及肿瘤细胞群方面，其中处理单细胞特征可能会导致更好地描述异质性以及相关的生物学和临床意义，包括对治疗的抵抗力，对转移的治疗，干细胞和分化等级。故事的另一面是TME，包括非癌性成分（免疫和基质细胞，血管）和化学/物理景观（ECM成分…

Disclosures

The authors have nothing to disclose.

Materials

Cell culture materials 
50 mL tubes Corning-Sigma Aldrich, St. Louis, MO CLS430828 centrifuge tubes
5-aza-2'-deoxycytidine DAC Millipore-Sigma; St. Louis, MO A3656 DNA-hypomethylating agent
6-well plates Corning-Sigma Aldrich, St. Louis, MO CLS3506 culture dishes
75 cm2 cell culture treated flask Corning, New York, NY 430641U culture flasks
A365M American Type Culture Collection (ATCC), Manassas, VA
CVCL_B222		 human melanoma cell line
Doxorubicin hydrochloride Millipore-Sigma; St. Louis, MO D1515 anthracycline antibiotic 
Dulbecco's Modified Eagle Medium DMEM EuroClone Spa, Milan, Italy ECM0728L Culture medium for SK-MEL-28  cells
Dulbecco's Phosphate Buffer Saline w/o Calcium w/o Magnesium EuroClone Spa, Milan, Italy ECB4004L saline buffer solution
Fetal Bovine Serum EuroClone Spa, Milan, Italy ECS0180L ancillary for cell culture
Ficoll GE-Heathcare 17-1440-02 separation of mononuclear cells from human blood. 
hemocytometer Neubauer Cell counter
Heparinized vials Thermo Fisher Scientific Inc., Waltham, MA Vials for venous blood collection
interferon alpha-2b Millipore-Sigma; St. Louis, MO SRP4595 recombinant human cytokine
L-Glutamine 100X EuroClone Spa, Milan, Italy ECB3000D ancillary for cell culture
Liquid nitrogen
Lympholyte cell separation media Cedarlane Labs, Burlington, Canada Separation of lymphocytes by density gradient centrifugation
Lymphoprep Axis-Shield PoC AS, Oslo, Norway
Matrigel Corning, New York, NY 354230 growth factor reduced basement membrane matrix
MDA-MB-231  American Type Culture Collection (ATCC), Manassas, VA  HTB-26 human breast cancer cell line
Penicillin/ Streptomycin 100X   EuroClone Spa, Milan, Italy ECB3001D ancillary for cell culture
Pipet aid Drummond Scientific Co., Broomall, PA 4-000-201 Liquid handling
PKH26 Red Fluorescent cell linker Millipore-Sigma; St. Louis, MO PKH26GL red fluorescent cell dye
PKH67 Green fluorescent cell linker Millipore-Sigma; St. Louis, MO PKH67GL green fluorescent cell dye
RPMI-1640 EuroClone Spa, Milan, Italy ECM2001L Culture medium for MDA-MB-231 cells
serological pipettes (2 mL, 5 mL, 10 mL, 25 mL, 50 mL) Corning- Millipore-Sigma; St. Louis, MO CLS4486; CLS4487; CLS4488; CLS4489; CLS4490 Liquid handling
sterile tips (1-10 μL, 10-20 μL, 20-200 μL, 1000 μL) EuroClone Spa, Milan, Italy ECTD00010; ECTD00020; ECTD00200; ECTD01005 tips for micropipette
Timer
Trypan Blue solution Thermo Fisher Scientific Inc., Waltham, MA 15250061 cell stain to assess cell viability
Trypsin EuroClone Spa, Milan, Italy ECM0920D dissociation reagent for adherent cells
Cell culture equipment
EVOS-FL fluorescence microscope Thermo Fisher Scientific Inc., Waltham, MA Fluorescent microscope for living cells
Humified cell culture incubator  Thermo Fisher Scientific Inc., Waltham, MA 311 Forma Direct Heat COIncubator; TC 230 Incubation of cell cultures at 37 °C, 5% CO2
Juli Microscope Nanoentek
Laboratory refrigerator (4 °C) FDM
Laboratory Safety Cabinet (Class II) Steril VBH 72 MP Laminar flow hood
Optical microscope Zeiss
Refrigerable centrifuge Beckman Coulter
Thermostatic bath
Microfabrication materials 
3-Aminopropyl)triethoxysilane (Aptes) Sigma Aldrich A3648 silanizing agent for bonding PDMS to plastic coverslip
Chromium quartz masks / 4"x4", HRC / No AZ  MB W&A,  Germany optical masks for photolithography
Glass coverslip, D 263 M Schott glass,  (170 ± 5 µm) Ibidi, Germany 10812
Hydrogen Peroxide solution 30% Carlo Erba Reagents 412081 reagents for piranha solution
Methyl isobutyl ketone Carlo Erba Reagents 461945 PMMA e-beam resist developer
Microscope Glass Slides (Pack of 50 slides) 76.2 mm x 25.4 mm  Sail Brand 7101 substrates for bonding chips
Miltex Biopsy Punch with Plunger, ID 1.0mm Tedpella dermal biopsy punches for chip reservoirs
PMMA  950 kDa Allresist,Germany AR-P. 679.04 Positive electronic resists for patterning optical masks
Polymer untreated coverslips Ibidi, Germany 10813 substrates for bonding chips
Prime CZ-Si Wafer,  4”, (100), Boron Doped Gambetti Xenologia Srl, Italy 30255
Propan-2-ol Carlo Erba Reagents 415238
Propylene glycol monomethyl ether acetate (PGMEA) Sigma Aldrich 484431-4L SU-8 resists developer
SU-8 3005 Micro resist technology,Germany C1.02.003-0001 Negative Photoresists
SU-8 3050 Micro resist technology,Germany C1.02.003-0005 Negative Photoresists
Suite of Biopunch, ID 4.0 mm, 6.0 mm, 8.0 mm Tedpella 15111-40, 15111-60, 15111-80 dermal biopsy punches for chip reservoirs
Sulfuric acid 96% Carlo Erba Reagents 410381 reagents for piranha solution
SYLGARD 184 Silicone Elastomer Kit Dowsil, Dow Corning 11-3184-01 Silicone Elastomer (PDMS)
Trimethylchlorosilane (TMCS) Sigma Aldrich 92360-100ML silanizing agent for SU-8 patterned masters
Microfabrication equipment
100 kV e-beam litography Raith-Vistec EBPG 5HR
hotplate
Optical litography system EV-420 double-face contact mask-aligner
Reactive Ion Etching system Oxford plasmalab 80 plus system
Vacuum dessicator
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MicrofluidicCo-cultureTumor MicroenvironmentImmune ResponseCell Interactions2D3DReal-time ImagingMicroscopyTumor CellsImmune CellsAnti-cancer TreatmentsMatrigelFluorescent Dyes
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De Ninno, A., Bertani, F. R., Gerardino, A., Schiavoni, G., Musella, M., Galassi, C., Mattei, F., Sistigu, A., Businaro, L. Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments. J. Vis. Exp. (170), e61895, doi:10.3791/61895 (2021).
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