肿瘤物质摄取的量化

Richard N. Hanna; Catherine C. Hedrick

doi:10.21769/BioProtoc.1974

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Peer-reviewed

Quantification of Tumor Material Uptake

肿瘤物质摄取的量化

Richard N. Hanna email

CH Catherine C. Hedrick

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1974 浏览次数: 10852

评审: Kristopher MarjonMartin V KolevElizabeth V. Clarke

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The authors used this protocol in:

Cover of Science, featuring study using the protocol.

Nov 2015

实验方案合集

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Abstract

Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.

Keywords: Cancer (癌症)

Background

Recent studies have demonstrated that extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris shed from tumors are important mediators of tumor metastasis, growth and evasion of the immune response (Vader et al., 2014; Pucci and Pittet, 2013; Pucci et al., 2016). Immune cells have the ability to remove, respond and transport this circulating tumor material (Hanna et al., 2015; Pucci et al., 2016; Headley et al., 2016). This protocol offers a novel approach to quantify tumor material uptake by specific immune cell populations, and may be adapted to test immune targets that regulate tumor material uptake. This protocol may assist in better understanding the immune response to extracellular tumor material, with the hope of eventually developing novel therapies targeting extracellular tumor material in cancer development.

Materials and Reagents

30 gauge insulin syringe (BD, Ultra-Fine^TM, catalog number: 328431 )
15 ml tubes
70 μm cell strainers (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22363548 )
15 mm Petri dish
5 ml syringe (BD, Luer-Lok^TM, catalog number: 309646 )
96-well v-bottom plate
Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP), B16F10 green fluorescent protein (B16F10-GFP) or other fluorescent-tagged tumor cell line (AntiCancer.com, http://www.anticancer.com/Fluorescent_protein_cell_lines_April_2010.pdf)
Syngeneic recipient mice. C57BL/6J mice (The Jackson Laboratory, catalog number: 000664 ) for LLC and B16F10 tumors.
TrypLE (Thermo Fisher Scientific, Gibco^TM, catalog number: 12604013 )
Dulbecco's phosphate-buffered saline (DPBS) (GE Healthcare, Hyclone^TM, catalog number: SH30013.02 )
UltraPure^TM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15575020 )
Red blood cell (RBC) lysis buffer (10x) (BioLegend, catalog number: 420301 )
Fc Block (CD16/32) (BD, Pharmingen^TM, catalog number: 553141 )
Fluorochrome conjugated antibodies:
CD11b-FITC (BioLegend, catalog number: 101206 )
CD115-APC (BioLegend, catalog number: 135510 )
Ly6C-APC-Cy7 (BioLegend, catalog number: 128026 )
CD45-BV421 (BioLegend, catalog number: 103134 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
Live/Dead Fixable Blue (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: L34962 )
Sodium azide (Sigma-Aldrich, catalog number: S2002 )
Flow buffer (FB) (see Recipes)
RBC lysis buffer (see Recipes)

Equipment

Cell culture flask
Centrifuge
Multiparameter flow cytometer with optimal excitation and detectors for tumor fluorophore (BD, LSRII or similar) or Amnis imaging cytometer (ImageStream X Mark II)

Software