Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Summary

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

Abstract

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system¹, and the tetracycline regulated gene expression systems.

The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

Introduction

Tumor associated macrophages (TAMs) contribute to tumor development by promoting tumor growth, metastasis, and regulating the immune response². Cancer cells recruit inflammatory monocytes, which infiltrate tumor and differentiate into pro-tumorigenic TAMs³. Infiltration of the tumor with TAMs correlates with poor clinical outcome and has been linked to the immunosuppressive role of macrophages^4,5. However, the mechanisms of the recruitment of macrophages to the tumor are not well explored and a better understanding of the involved pathways is crucial for further ....

Protocol

The protocol section that uses human monocytes obtained from healthy volunteers follows the guidelines of the Children's Hospital Los Angeles Human Research Ethics Committee and has been approved by the Institutional Review Board under the Human Material Protocol number: CCI 08-00208.

1. Preparation of Cancer Cell Lines with Tetracycline-regulated shRNA Expression

1. Cloning of shRNA PAI-1 into Tet-pLKO-puro vector^8,9
   1. Design shRNA oligomers that contain AgeI and EcoRI cleavage site at the 5' end, the sense sequence, a 6....

Results

Three shRNA sequences were tested for the most efficient KD of PAI-1. For this, shRNA sequences against PAI-1 and scrambled (Table 1) were cloned into Tet-pLKO-puro expression vector following the protocol described above. The HT-1080 fibrosarcoma cell line was stably transfected with generated constructs and the cells were treated with doxycycline for 3 days. The expression of PAI-1 was verified by Western blotting (Figure 2A

Discussion

Composed of a variety of cell types, the TME is crucial for the development of cancer. To ascertain optimal growth conditions, cancer cells attract monocytes through secretion of chemotactic factors. Here we present a protocol for studying the mechanism of monocyte recruitment by tumor cells in vitro. For this purpose, a combination of inducible gene expression system, purification of primary human monocytes, and as an example of a migration assay, the Boyden chamber assay, is used.

T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tet-pLKO-puro lentiviral vector	Addgene	21915
FBS (Tetracycline-free)	Omega Scientific	FB-15
Histopaque	Sigma	10771-500ML
EasySep Human Monocyte Enrichment Kit	StemCell	19059
EasySep Magnet	StemCell	18002
EcoRI HF	NEB	R3101S
AgeI HF	NEB	R3552S
Cut Smart buffer	NEB	B7200S
EndoFree Plasmid Maxi Kit	Qiagen	12362
PROMEGA Wizard SV Gel and PCR Cleanup System	PROMEGA	A9281
psPAX vector	Addgene	12260
pMD2.G vector	Addgene	12259
One Shot Stbl3 Chemically Competent E. coli	Thermo Fisher Scientific	C737303
Hema 3 stain set for Wright-Giemsa stain	Protocol	123-869
HEK293 cells	ATCC	CRL-1573
PBS	Corning	21-031-CV
XhoI restriction enzyme	NEB	R0146S
Ligase	NEB	M0202S
Ligase buffer	NEB	M0202S
EDTA 0.5 M solution	Thermo Fisher Scientific	R1021
Lipofectamine 2000	Invitrogen	11668019
The Big Easy" EasySep Magnet	Stem Cell	18001
0.1% Poly-L-Lysine solution	Sigma-Aldrich	P8920
Sodium Acetate	Sigma-Aldrich	S7670
Sodium butyrate	Sigma-Aldrich	B5887
0.45 μM syringe filters	VWR International	28145-479
NaOH	Sigma-Aldrich	S5881-500g
Tris(hydroxymethyl)aminomethane (Tris)	Invitrogen	15504-020
Sodium Chloride(NaCl)	Sigma-Aldrich	S7653-5 kg
Magnesium Chloride (MgCl2)	Sigma-Aldrich	M8266-100g
dithioerythritol (DTE)	Sigma-Aldrich	B8255-5g
ethanol (EtOH)	Sigma-Aldrich	792780
tryptone	Amresco	J859-500g
yeast extract	Fluka	70161-500g
Bacto agar	BD	214010
ampicillin	Sigma-Aldrich	A9393-5g
Dulbecco’s Modified Eagle’s Medium (DMEM)	Corning	10-013-CV
phosphate-buffered saline (PBS)	Corning	21-031-CV
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375
puromycin	Sigma-Aldrich	P9620
Doxycycline hyclate	Sigma-Aldrich	D9891-25g
Roswell Park Memorial Institute (RPMI)	Corning	10-040-CV
0.5 M EDTA pH 8.0	Thermo Fisher Scientific	R1021
Falcon Permeable Support for 24 Well Plate with 8.0μm Transparent PET Membrane	Becton Dickinson	353097
Bovine Serum Albumin	Sigma-Aldrich	A7906
Cotton-Tipped Applicator	Medline	MDS202000
Protocol Hema 3 STAT pack	Fisher Scientific Company	123-869
Resolve Immersion Oil, High Viscosity	Richard Allan Scientific	M4004
Penicillin Streptomycin	Gibco	15140122