A.S.는 AIRC(IG #28807) 및 PRIN(#P2022YE2MX)에서 지원됩니다. M.M.은 AIRC-FIRC 펠로우십(#25558)의 지원을 받습니다. ADN은 혁신 생태계 로마 테크노폴(Innovation Ecosystem Rome Technopole)의 지원을 받ECS00000024 EU-Next Generation EU, PNRR Mission 4 Component 2 Investment 1.5의 자금 지원을 받습니다.

유세포분석 기반 암 면역 주기 모니터링 

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저자는 공개할 이해 상충이 없습니다.

항암 면역 반응을 모니터링하는 것은 TME에서 작용하고 패권을 위한 끊임없는 싸움을 지원하는 복잡한 분자 및 세포 상호 작용을 규명하고 이해하는 데 가장 중요합니다23. 여기에서는 암-면역 주기를 구성하는 단계의 모니터링 및 특성화를 위한 간단한 mFC 및 ToC 지원 프로토콜에 대해 자세히 설명합니다. 쥐 세포주와 마우스 유래 면역 세포를 기반으로 하는 이 프로토콜은 약간?...

지난 수십 년 동안 면역 요법은 암 치료를 위한 최첨단 옵션의 최전선에 있었습니다. 항종양 목적으로 면역 체계를 활용하면 역사적으로 예후가 좋지 않은 다양한 혈액 및 고형 악성 종양에 걸쳐 환자에게 혜택을 줄 수 있는 강력한 개념 증명을 제공했습니다. 면역 요법이 다른 방법으로는 치료하기 어려운 암에 대한 기회를 제공함에 따라 놀라울 정도로 빠른 진행 속도를 경험하고 있습니다. 이러한 발전은 적어도 부분적으로는 암세포와 면역세포 사이의 상호 작용에 대한 정교한 이해에 기인할 수 있습니다. 이러한 상호 작용은 면역 체계가 암세포를 파괴하기 위해 점화하는 피드포워드 "엔진", 이른바 암-면역 주기와 유사합니다. 이 항암 면역 반응은 인식, 처리 및 반응의 세 가지 주요 수준에 걸쳐 진행됩니다. 첫째, 인식 단계에서 종양 형성 중에 생성된 종양 항원(Ag)은 종양 미세환경(TME, 1단계)에서 죽어가는 암세포에 의해 방출되고 종양 침투 수지상 세포(DC, 2단계)에 의해 삼켜집니다. 다음으로, 처리 단계에서 DC는 주요 조직적합성 복합체(MHC) 분자를 통해 포획된 종양 Ag의 에피토프를 제시하고, 표면에 더 높은 수준의 늑골 분자를 발현하고(3단계), 종양 배출 림프절(dLN)로 이동하여 화물을 미성숙 CD8 T 세포에 교차 제시합니다(CD8N, 4단계). 이러한 모든 단계는 종양 Ag 특이적 교차 프라임 CD8 T 세포(CD8C-P)가 활성화되고 effector CD8 T(CD8E) 세포로 성숙한 후 클론 확장(단계 5)을 거치는 최종 반응 과정에서 수렴됩니다. 그런 다음 CD8E 세포는 dLN과 혈액을 통해 TME로 이동하며(6단계) T 세포 수용체(TCR)와 동족 종양 Ags 사이의 상호 작용을 통해 암세포를 특이적으로 인식하고 결합하고, 세포독성 분자[즉, 인터페론(IFN)-γ, 퍼포린 및 그랜자임(Grzs)]을 방출하고, 암세포를 죽이고(단계 7)1, 2. 암세포 사멸은 암-면역 주기에 연료를 공급하기 위해 추가 종양 Ags의 방출로 이어집니다. 사실, 이 모든 단계를 통해 면역 체계는 예상보다 훨씬 더 자주 암세포를 파괴하고 거부합니다. 그러나 암 환자의 경우 이러한 단계 중 적어도 하나가 제대로 작동하지 않습니다. 우리와 다른 연구자들은 암세포가 더 공격적이고 면역 특권을 가진 변이체로 진화하거나 3,4,5 또는 T-세포 효과를 방해함으로써 면역 반응을 지연시키려고 한다는 것을 보여주었다 6,7.
암 연구와 항암제 개발은 모두 암과 면역 세포 간의 관계를 연구할 수 있는 실험 모델, 이른바 종양 면역학(onco-immunology)에 의존합니다. 여기에는 종양 면역학 주기의 각 단계를 종합적으로 재현하는 빠르고 신뢰할 수 있으며 재현 가능하고 저렴한 in vitro 모델에 대해 설명하고 있으며, 면역 감시 및 궁극적으로 면역 편집의 표현형 및 기능적 특징 세트에 대한 빠르고 명확한 관점을 제공합니다.
다매개변수 유세포 분석(mFC)은 암 임상 시험의 기초 암 연구, 진단 및 중개 연구에서 가장 성공적인 단일 세포 분석 도구 중 하나입니다. 각 세포에서 더 많은 특징을 동시에 캡처할 수 있기 때문에 mFC는 종양 면역학에서 최고의 표준 분석 플랫폼으로 자리매김했습니다. TME8,9,10과 같은 이종 및 이형 세포 현탁액의 단일 세포 수준에서 여러 단백질 발현 패턴 및 기능적 특성을 빠르고 재현성 있게 측정할 수 있는 가능성과 높은 감도 및 특이성을 결합합니다. 표현형 및 기능적 발현 패턴은 모두 시간에 민감하기 때문에 실험 설계, 적합한 패널, 대조군 및 적정 항체의 선택, 적절한 시료 처리 및 기기 사용에 세심한 주의를 기울이는 것이 결과의 신뢰성, 비교 가능성 및 재현성을 위해 그리고 실험 결과를 자신 있게 해석하는 데 매우 중요하다11.
Tumor-on-a-chip 미세유체 장치(ToC)는 암 및 면역 세포 역학의 시험관 내 마이크로 스케일 생체 모방을 허용하여 TME를 모델링하고 12,13,14,15를 상호 작용합니다. 구체적으로, ToC는 2차원(2D) 또는 3차원(3D) 배양 설정으로 구성된 다양한 세포 유형을 호스팅할 수 있는 다채널 미세유체 세포 배양 장치로서, 높은 충실도로 모델링할 수 있으며, TME(12)에서 생리학적으로 발생하는 이형 세포 상호 작용 및 화학적 구배의 흐름과 같은 주요 구조 및 기능 단위를 높은 정밀도로 제어할 수 있습니다. 13,14,15. 특히, 면역 화학인력 및 궤적뿐만 아니라 면역 세포와 암세포의 상호 작용을 실시간으로 모니터링하고 타임 랩스 현미경 및 자동 추적 분석을 통해 정량화할 수 있습니다 5,12,13,14,15,16. 또한, ToC는 암의 발병과 진행 및 치료에 대한 반응을 조절하는 중요한 과정을 분석하고 조작할 수 있는 가능성을 제공한다17.
이 기사에서는 mFC와 ToC를 결합하여 암 Ags의 DC 매개 식세포작용(단계 1-3), T 세포 교차 프라이밍(단계 4), 활성화 및 클론 확장(마지막은 5-ethynyl-2'-deoxyuridine(EdU) 및 Cu(I) 촉매 사이클로첨가 [클릭] 기술을 통해 항암 면역 반응의 모든 수준을 연구합니다. 5단계), CD8E 세포가 TME로 귀환(6단계), 마지막으로 CD8E-세포 매개 암세포 사멸(단계 7, 그림 1).
이 연구는 암-면역 주기를 연구하기 위한 간단하고 빠르며 신뢰할 수 있는 표준 프로토콜을 수립하기 위한 노력에 기여합니다. 암 연구, TME 역학 및 치료에 대한 mFC 및 ToC 모델의 개선 및 통합은 이러한 모델이 실험 제어와 함께 생물학적 충실도를 제공하기 때문에 큰 잠재력을 가지고 있습니다. 따라서 이 프로토콜은 자연 및 후천적 면역 감시에 대한 개별 세포 플레이어의 역할과 상호 상호 작용을 특성화, 모니터링 및 적시에 조작할 수 있도록 함으로써 암-면역 주기를 단계적으로 재현하는 데 도움이 됩니다. 이는 궁극적으로 동물 연구를 개선, 축소 및 대체하는 데 도움이 되는 동시에 종양을 능가하고 임상 치료를 안내하는 데 중요한 통찰력을 제공할 것입니다. 마지막으로, mFC 및 ToC의 장점과 한계에 대해 비판적으로 논의하고 최첨단 기술(예: 단일 세포 및 세포 내 분해능에서의 고플렉스 공간 분석)과 비교하여 종양 면역학 연구 및 치료를 발전시킵니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microtubes&#160;</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>100 kV e-beam litography</td>
			<td>Vistec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dishes&#160;</td>
			<td>Greiner Bio One</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Euroclone</td>
			<td>&#160;ET3012</td>
			<td></td>
		</tr>
		<tr>
			<td>15 and 50 mL tubes</td>
			<td>Corning&#160;</td>
			<td>352096; 352070</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer&#160;</td>
			<td>Corning&#160;</td>
			<td>CLS431750</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene tubes&#160;</td>
			<td>Greiner Bio-One</td>
			<td>120180</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer&#160;</td>
			<td>Corning&#160;</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flask</td>
			<td>Euroclone</td>
			<td>&#160;ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Adsorbent wipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allumin foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD107a (LAMP-1) Antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-111-319</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD25 (7D4) Antibody&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-118-678</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD3 (17A2) Antibody</td>
			<td>BioLegend</td>
			<td>100206</td>
			<td></td>
		</tr>
		<tr>
			<td>Aptes</td>
			<td>Sigma Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit with BD GolgiPlug</td>
			<td>BD Biosciences</td>
			<td>555028</td>
			<td></td>
		</tr>
		<tr>
			<td>BD GolgiPlug Protein Transport Inhibitor (Containing Brefeldin A)</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>BD GolgiStop Protein Transport Inhibitor (Containing Monensin)</td>
			<td>BD Biosciences</td>
			<td>554724</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological, Salem</td>
			<td>A1312</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c Monoclonal Antibody (N418)</td>
			<td>eBioscience</td>
			<td>12-0114-81</td>
			<td></td>
		</tr>
		<tr>
			<td>CD137 (4-1BB) Monoclonal Antibody (17B5)</td>
			<td>eBioscience</td>
			<td>17-1371-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 Monoclonal Antibody (17A2)</td>
			<td>eBioscience</td>
			<td>25-0032-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44 Monoclonal Antibody (IM7)</td>
			<td>eBioscience</td>
			<td>11-0441-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (30-F11)</td>
			<td>Invitrogen</td>
			<td>MCD4528</td>
			<td></td>
		</tr>
		<tr>
			<td>CD69 Monoclonal Antibody (H1.2F3)</td>
			<td>eBioscience</td>
			<td>48-0691-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a Monoclonal Antibody (53-6.7)</td>
			<td>eBioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a Monoclonal Antibody (53-6.7)</td>
			<td>eBioscience</td>
			<td>17-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD95 (APO-1/Fas) Monoclonal Antibody (15A7)</td>
			<td>eBioscience</td>
			<td>53-0951-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting slides</td>
			<td>Kova International</td>
			<td>87144E</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium quartz masks</td>
			<td>MB W&#38;A, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit</td>
			<td>Invitrogen</td>
			<td>C10635</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS)</td>
			<td>EuroClone</td>
			<td>ECB4053L</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>EuroClone</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo v10.0.7</td>
			<td>Flowjo, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Granzyme B Monoclonal Antibody (NGZB)</td>
			<td>eBioscience</td>
			<td>12-8898-82</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hotplate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humified cell culture incubator (37&#176;, 5% CO2)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice machine</td>
			<td>Brema Ice Makers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IFN gamma Monoclonal Antibody (XMG1.2)</td>
			<td>eBioscience</td>
			<td>11-7311-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Illustrator CC 2015</td>
			<td>Adobe Systems Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institute of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte 2022A Software</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Cytotox Dye for Counting Dead Cells</td>
			<td>Sartorius</td>
			<td>4632</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte SX5 Live-Cell Analysis System&#160;</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>JuLi Smart Fluorescent Live Cell Imaging Microscope</td>
			<td>Bulldog Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory bench</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory refrigerator (4&#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Safety Cabinet (Class II)</td>
			<td>Angelantoni</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM</td>
			<td>EuroClone</td>
			<td>ECB3004D</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201; 130-042-401&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separators</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-10; 130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MCA205 mouse fibrosarcoma cell line</td>
			<td>Sigma-Aldrich</td>
			<td>SCC173</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiologically controlled animal facility equipped with Class II safety cabine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroCL 21R Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002552</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel&#160;</td>
			<td>Microsoft, Redmond&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Naive CD8a+ T Cell Isolation Kit, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-543</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon ECLIPSE Ts2</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements BR 5.30.0064-BIT</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical litography</td>
			<td>EVG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt and streptomycin sulfate</td>
			<td>EuroClone</td>
			<td>ECB3001D/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet aid</td>
			<td>Drummond Scientific Co., Broomall, PA</td>
			<td>&#160;4-000-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PKH67 Fluorescent Cell Linker Kits</td>
			<td>Sigma-Aldrich</td>
			<td>PKH67GL-1KT</td>
			<td>fluorescent cell linker&#160; kit</td>
		</tr>
		<tr>
			<td>plastic coverslip</td>
			<td>IBIDI</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Thermo Scientific</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive Ion Etching system</td>
			<td>Oxford plasmalab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute 1640 (RPMI 1640)</td>
			<td>EuroClone</td>
			<td>ECB9006L</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)</td>
			<td>Corning- Millipore-Sigma; St. Louis, 
			MO</td>
			<td>CLS4486; CLS4487; CLS4488; CLS4489</td>
			<td></td>
		</tr>
		<tr>
			<td>SL 16 Centrifuge Series</td>
			<td>Thermo Scientific</td>
			<td>75004031</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpels, surgical forceps, scissors and pliers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tips (1&#8211;10 &#956;L, 20&#8211;200 &#956;L, 1000 &#956;L)</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECTD00010; ECTD00020; ECTD00200; ECTD01005</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3000 series</td>
			<td>MicroChem corp, Newton, (MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suite of dermal biopsy punches</td>
			<td>Kai Medical, Tedpella</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dowsil, Dow Corning</td>
			<td>101697</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR beta Monoclonal Antibody (H57-597)</td>
			<td>eBioscience</td>
			<td>12-5961-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMCS&#160;</td>
			<td>Sigma Aldrich</td>
			<td>92360</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Thermo Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA w/ Phenol Red</td>
			<td>EuroClone</td>
			<td>ECM0920</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

co-culture in vitro systems to reproduce the cancer-immunity cycle

근본적인 암 연구와 효과적인 반격 요법의 개발은 모두 암과 면역 세포 간의 상호 작용, 이른바 암-면역 주기를 자세히 설명하는 실험적 연구에 의존합니다. mFC(Multiparametric Flow Cytometry) 및 ToC(tumor-on-a-chip microfluidic devices)와 결합된 체외 공동 배양 시스템은 암 면역 주기의 각 단계에 대한 간단하고 빠르며 신뢰할 수 있는 모니터링 및 특성 분석을 가능하게 하며 암 면역 감시와 면역 회피 간의 균형을 유지하는 메커니즘을 식별할 수 있도록 합니다. 암과 면역 세포 간의 역동적인 상호 작용에 대한 철저한 이해는 종양을 능가하는 데 중요한 통찰력을 제공하고 환자의 치료 개인화 및 최적화 속도를 가속화할 것입니다. 특히, 여기에서는 쥐 암 세포주와 마우스 유래 면역 세포에서 암 면역 주기의 각 단계의 동적 복잡성을 밝히고 면역 감시에 중점을 두기 위한 간단한 mFC 및 ToC 지원 프로토콜에 대해 자세히 설명합니다. 이 프로토콜의 시간 및 비용 관련 기능을 고려할 때 대규모로 확실히 실현 가능합니다. 더욱이, 약간의 변형을 통해 이 프로토콜은 인간 암 세포주와 인간 말초 혈액 유래 면역 세포에 모두 적용할 수 있으며, 면역 반응의 바이오마커를 식별하기 위해 특정 경로의 유전적 및/또는 약리학적 억제와 결합될 수 있습니다.

Over the past few decades, immunotherapy has been at the forefront of cutting-edge options for cancer treatment. Harnessing the immune system with antitumor purposes has provided powerful proof-of-concept for patient benefit across diverse hematologic and solid malignancies with historically poor prognoses. As immunotherapy is offering an opportunity for otherwise hard-to-treat cancers, it is experiencing an astoundingly rapid pace of progress. Such progress can be, at least in part, attributed to the refined understanding of the interplay between cancer cells and immune cells. This interplay resembles a feed-forward &#34;engine&#34; the immune system ignites to destroy cancer cells, the so-called cancer-immunity cycle. This anticancer immune response progresses across three main levels: recognition, processing, and reaction. Firstly, in the phase of recognition, tumor antigens (Ags) produced during tumor formation are released by dying cancer cells in the tumor microenvironment (TME, step 1) and engulfed by tumor-infiltrating dendritic cells (DCs, step 2). Next, in the phase of processing, DCs present the epitopes of the captured tumor Ags through the major histocompatibility complex (MHC) molecules, express on their surface higher levels of costimulatory molecules (step 3), and move to tumor-draining lymph nodes (dLNs) to cross-present their cargo to na&#239;ve CD8 T cells (CD8N, step 4). All these steps converge in the final process of reaction during which tumor Ag-specific cross-primed CD8 T cells (CD8C-P) are activated, mature into effector CD8 T (CD8E) cells, and undergo a clonal expansion (step 5). CD8E cells then leave the dLNs and home through the blood to the TME (step 6) where they specifically recognize and bind to cancer cells through the interaction between their T cell receptor (TCR) and their cognate tumor Ags, release cytotoxic molecules [i.e., interferon (IFN)-&#947;, perforins and granzymes (Grzs)] and kill cancer cells (step 7)1,2. Cancer cell killing leads to the release of further tumor Ags to fuel the cancer-immunity cycle. As a matter of fact, through all these steps, the immune system destroys and rejects cancer cells far more often than supposed. However, in cancer patients, at least one of these steps does not work properly. We and others showed that cancer cells seek to stall the immune response by either evolving into more aggressive and immune-privileged variants3,4,5 or hampering T-cell effectiveness6,7.
Cancer research and cancer drug development both rely on experimental models that allow the study of the relationship between cancer and immune cells, the so-called onco-immunology. Here are described fast, reliable, reproducible, and low-cost in vitro models that comprehensively reproduce each step of the onco-immunology cycle and is offered a rapid and clear view of the phenotypic and functional feature sets of immunosurveillance and eventually immunoediting.
Multiparametric flow cytometry (mFC) is one of the most successful single-cell analytical tools in fundamental cancer research, diagnosis, and translational research in cancer clinical trials. As it allows to simultaneously capture more features in each cell, mFC has earned its place as a gold-standard analysis platform in onco-immunology. It couples high sensitivity and specificity with the possibility to measure multiple protein expression patterns and functional properties quickly and reproducibly at a single cell level from heterogeneous and even heterotypic cell suspensions, as those from the TME8,9,10. As both phenotypic and functional expression patterns are time-sensitive, careful attention to experiment design, the selection of suitable panels, controls, and titred antibodies, and to appropriate sample processing and instrumentation use are critical for the reliability, comparability, and reproducibility of results and to confidently interpret experiment outcome11.
Tumor-on-a-chip microfluidic devices (ToCs) model the TME by allowing in vitro microscale biomimetics of cancer and immune cell dynamics and interplays12,13,14,15. Specifically, ToCs are multichannel microfluidic cell-culture devices able to host diverse cell types organized in either two-dimensional (2D) or three-dimensional (3D) culture settings and able to model with high fidelity and to control with high precision, key structural and functional units such as heterotypic cellular interactions and flows of chemical gradients that physiologically occur in the TME12,13,14,15. In particular, immune chemoattraction and trajectories as well as immune cell interaction with cancer cells, can be monitored in real-time and quantified by time-lapse microscopy and automated tracking analysis5,12,13,14,15,16. Furthermore, ToCs offer the possibility to both analyze and manipulate crucial processes regulating cancer onset and progression and response to therapy17.
In this article, mFC with ToCs are combined to study all the levels of the anticancer immune response going through DC-mediated phagocytosis of cancer Ags (steps 1-3), T cell cross-priming (step 4), activation and clonal expansion (the last by means of 5-ethynyl-2&#39;-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition [click] technology, a highly sensitive and accurate methodology, step 5), CD8E cell homing to the TME (step 6) and, finally CD8E-cell-mediated cancer cell killing (step 7, Figure 1).
This work contributes to the effort toward establishing simple, fast, and reliable standard protocols to study the cancer-immunity cycle. The improvement and integration of mFC and ToC models into cancer research, TME dynamics, and response to therapy hold great potential as these models provide biological fidelity along with experimental control. Hence, this protocol helps recreate, in a stepwise manner, the cancer-immunity cycle by making it possible to characterize, monitor, and timely maneuver the roles of individual cell players and their reciprocal interactions upon natural and acquired immunosurveillance. This ultimately will help refine, reduce, and replace animal studies while providing critical insights to outsmart tumors and guide clinical care. Finally, mFC and ToC advantages and limitations are critically discussed and compared with state-of-the-art technologies (e.g., high plex spatial analyses at single cell and even sub-cellular resolution) to push onco-immunology research and therapy forward.

암-면역 주기 모니터링 및 종양 미세환경 역학 탐구

암-면역 주기를 재현하기 위한 Co-Culture In Vitro 시스템

This protocol allows simple, faster, and reliable monitoring and characterization of each step of the cancer immunity cycle, and thus led to the identification of the mechanisms responsible for keeping the balance between cancer-immune surveillance and immune evasion. The interaction and evolution of cancer and immune cells in the tumor microenvironment have emerged as crucial factors detecting therapeutic outcomes. Currently, several technologies are advancing research in our field, including flow cytometry, single-cell transcriptomics, special transcriptomics, proteomics, tumor-on-a-chip models, and in-vivo models.Our protocol offers significant advantages over other techniques as it provides a simple, fast, reliable, and low cost method for monitoring and characterizing each step of the cancer immunity cycle. In the future, we want to study the dynamics of the tumor microenvironment and immunogenic and immune therapies.

mFC는 cross-presentation21,22에 특화되고 그림 2A와 같이 gated된 널리 알려진 phagocyte subset인 CD11c+ DC가 이전에 PKH67 형광 세포 링커로 표지된 UV 방사선 조사된 MCA205 암세포의 자가사멸체를 집어삼킬 수 있는 능력을 평가했습니다. 예상대로 CD11c+ DC는 4°C가 아닌 37°C에서 시험관 내에서 세포사멸 MCA205 세포를 효율적?...

여기에서는 암-면역 주기를 구성하는 단계를 모니터링하고 특성화하기 위한 간단하고 빠르며 신뢰할 수 있는 다중 파라메트릭 유세포 분석 및 종양 온 칩 지원 프로토콜에 대해 자세히 설명합니다. 실제로, 암과 면역 세포 간의 상호 작용에 대한 철저한 이해는 종양을 능가하고 임상 치료를 안내하는 데 중요한 통찰력을 제공합니다.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

co-culture-in-vitro-systems-to-reproduce-the-cancer-immunity-cycle

Research

JoVE Journal

Cancer Research

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

457 조회수.  Università Cattolica del Sacro Cuore. 이 프로토콜은 암 면역 주기의 각 단계에 대한 간단하고 빠르고 신뢰할 수 있는 모니터링 및 특성화를 가능하게 하며, 이를 통해 암 면역 감시와 면역 회피 사이의 균형을 유지하는 메커니즘을 식별할 수 있습니다. 종양 미세환경에서 암과 면역 세포의 상호 작용과 진화는 치료 결과를 감지하는 중요한 요인으로 부상했습니다. 현재 유세포 분석, 단?...

비디오: 암-면역 주기 모니터링 및 종양 미세환경 역학 탐구

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

연구

암 연구학

Inducing Cancer Cell Death and Evaluating CD8 T Cells Activation Through Co-Culture and Flow Cytometry Analysis

To begin, plate 3 times 10 to the power of 6MCA205 fibrosarcoma cells in 10 milliliters of complete growth medium in a 100 millimeters Petri dish. Irradiate cells with ultraviolet light and incubate at 37 degrees Celsius with 5%carbon dioxide for at least six hours to let them die. Wash in vitro differentiated dendritic cells, or DCs, twice, at 1100G for four minutes in a complete growth medium.Count the bone marrow-derived empty DCs using the Trypan blue dye exclusion test. Centrifuge the irradiated cancer cells at 1100G for five minutes. Set the co-culture of empty DCs with irradiated apoptotic cells at a two to one ratio in a 12-well plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide.The next day, wash the cells twice at 1100G for five minutes in 10 milliliters of complete growth medium in 15 milliliter tubes. For cell surface staining, re-suspend the bone marrow-derived DCs in 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees Celsius in the dark. After washing the cells with FACS buffer, add 100 microliters of PBS containing vitality fixable near-infrared dye for 30 minutes.Wash the cells once with PBS before re-suspending them in the FACS buffer. Identify the cells of interest based on their FSCA and SSCA properties. Then plot FSCA and FSCH to exclude cell doublets and clumps from the analysis.Plot 780 to 60 nanometer emission band pass filter area and SSCA to remove dead cells. Using 575 to 26 and 530 to 30 nanometer emission band pass filters, detect cell positivity for CD11C marker and PKH67 fluorescent cell linker in two parameter density plots. After counting, culture the CD8 T cells with bone marrow-derived DCs that had taken up apoptotic MCA205 cells at a two to five to one ratio at 37 degrees Celsius and 5%carbon dioxide for 72 hours.Add EdU to the co-culture medium at 10 micromolar final concentration and incubate for 16 to 20 hours. Centrifuge the CD8 cross prime twice at 1100G for five minutes and stain for surface markers in cold FACS buffer for 20 minutes at four degrees Celsius in the dark. After washing the cells twice in FACS buffer, re-suspend them in 100 microliters of PBS containing the vitality fixable aqua dye for 30 minutes.Wash the cell suspensions once with three milliliters of 1%BSA in PBS in flow tubes. Add 100 microliters of 4%paraformaldehyde for 15 minutes for cell fixation. Incubate the cells in 100 microliters of saponin-based permeabilization and wash reagent for 15 minutes.Add 500 microliters of the reaction cocktail and incubate for 30 minutes in the dark. After washing, re-suspend the cells in 100 microliters of saponin-based permeabilization and wash reagent. Identify the cells of interest based on their FSEA and SSEA properties.Then plot FSEA and FSEH to exclude cell doublets and clumps from the analysis. Plot 525 to 50 nanometer emission band pass filter area and SSCA to remove dead cells. Using 530 to 30 and 575 to 26 nanometer emission band pass filter density plots, detect cells'positivity for CD8a and CD3.Analyze cells for Alexa Fluor 647 EdU incorporation in a single parameter histogram using a 660 to 620 nanometer band pass emission filter. CD11c positive dendritic cells effectively engulfed apoptotic MCA205 cancer cells at 37 degrees Celsius, demonstrating temperature-dependent phagocytosis in early cancer immunity cycle stages. After phagocytosis, dendritic cells showed increased levels of CD86 and MHC-II along with reduced levels of PDL1.The clonal expansion of CD8 positive T cells once activated by mature dendritic cells was evident with approximately 20%proliferation rate. Using tumor-on-chip models, the chemotactic response of these T cells towards cancer cell released alarmins was observed.

암세포 사멸 유도 및 Co-Culture 및 유세포 분석을 통한 CD8 T 세포 활성화 평가

Enhancing CD8 T Cell Responses Through Re-stimulation with Cancer Cells and Evaluation of Tumor Reactivity

After recovering CD8 cross-primed from coculture, centrifuge them at 1100 g for 10 minutes in 10 milliliters of complete growth medium. Resuspend 1 x 10 to the power of 7 total cells in 100 microliters of dead cell removal microbeads, and incubate for 15 minutes at room temperature. Place separation columns in the magnetic separator, and rinse them with binding buffer.Transfer cell suspensions into separation columns, and collect the flow-through. After washing the column, collect the unlabeled cells that pass through, and combine them with previously collected flow-through. Centrifuge enriched live CD8 cross-primed at 1100 g for five minutes in a complete growth medium.After counting, culture CD8 cross-primed cells with fresh MCA205 cells at a two-to-one ratio in 12 well plates, and incubate at 37 degrees Celsius and 5%carbon dioxide for 72 hours. Before recovering CD8 effector, add monensin and brefeldin to the cancer immune cell cocultures for six hours. Then recover CD8 effector, and centrifuge twice at 1100 g for five minutes.Resuspend cells in three different primary antibody mixes prepared in cold FACS buffer, and incubate for 20 minutes at four degrees Celsius, protecting from light. Add 100 microliters of fixation solution to the cell palate from Mix C and incubate for 20 minutes at four degrees Celsius in the dark. Resuspend the cells in a cold wash buffer, and incubate for 30 minutes at four degrees Celsius.After washing cells twice in FACS buffer, add 100 microliters of PBS containing the Vitality Fixable Aqua Dye to cell pellets for 30 minutes. Identify cells of interest based on their FSCA and SSCA properties. Then plot FSCA and FSCH to exclude cell doublets and clumps from the analysis.Plot 525/50 nanometer emission band-pass filter and SSCA to remove dead cells, Using 660/20 and 780/60 nanometer emission band-pass filters, detect cell positivity for CDAA and CD3 in two parameter density plots. Further, analyze cells for CD44, CD25, and CD69 markers for CD137 marker, and for interferon gamma positive and granzyme B for Mix A, B, and C, respectively. For tumor killing assay, supplement the coculture medium with a real-time cell death quantification dye.After warming the plate to 37 degrees Celsius in the live cell analysis system, perform data scanning every two hours for up to 72 hours. Centrifuge the cells twice at 1000 g for five minutes. Stain cells for surface markers with 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees celsius in the dark.Then add the vitality dye propidium iodide to stain the cells for a gating strategy. Identify the cells of interest based on their FSCA and SSCA properties. Then plot FSCA and FSCH to exclude cell doublets and clumps from the analysis.Use a 450/50 nanometer emission band pass filter to detect cancer cells negative for CD45. In a single parameter histogram, using a 610/620 nanometer band-pass emission filter, analyze cells for propidium iodide incorporation as mean fluorescent intensity. Through multi-parameter flow cytometry, the surface expression levels of the early activation marker CD69, late activation markers CD44 and CD25, membrane expression of CD137, and CD107, tumor reactivity markers were enhanced.The intracellular levels of cytotoxic molecules, granzyme B, and interferon gamma positive were progressively and significantly increased, reaching a peak of expression at 72 hours of coculture. Cognate cocultured MCA205 cells underwent significant levels of CD8 effector mediated death.