Isolation of Myeloid-derived Suppressor Cells from Mouse Tumor and Determining Their Migration Potential In Vitro

Summary

This article outlines the procedures to isolate myeloid-derived suppressor cells from mouse solid tumors and perform an in vitro assay with the cells to determine their response migration potential to certain soluble factors like cytokines and chemokines.

Abstract

The importance of the immune response in cancer and other diseases (like diabetes mellitus, alzheimers, cystic fibrosis) is now known, and the manipulation of the immune system as a therapy to treat cancer is gaining attention. The immune system regulates tumorigenesis both negatively and positively. The myeloid-derived suppressor cells (MDSCs) are a population of immune cells that are increased during cancer, inflammation, and infection. These cells influence the immune response and effectively suppresses the anti-tumor T cell response. They serve as potential targets for therapeutic intervention to effectively use the immune system to inhibit tumorigenesis. To better understand how such intervention can be applied it is important to study these cell types. Using mouse ovarian tumors, we describe the isolation of MDSCs from solid tissue using gentle dissociation techniques. We further describe how MDSCs are isolated from such dissociated tissue based on the expression of cell surface markers with the help of flow cytometry. Additionally, we describe the procedure to perform an in vitro MDSC migration assay to determine the migration potential of these cells in response to soluble factors like cytokines and chemokines.

Introduction

In recent years a number of studies have focused on understanding the role of immune cells in cancer development and progression. One way by which tumor cells evade the immune system is through the expression of immunosuppressive factors that activate, upregulate, and attract immune suppressive cells like MDSCs in the tumor microenvironment¹.

MDSCs are a population of immature myeloid cells that are generated in the bone marrow. Under normal conditions, these immature cells differentiate into mature myeloid cells like macrophages, monocytes, or dendritic cells². Under pathologic condition, the....

Protocol

All procedures were performed under the guidance of University of Texas at MD Anderson IACUC review board.

1. Reagent Preparation

1. Preparation of medium for migration assay
   1. Prepare 500 mL of complete RPMI media for the migration assay: RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% pen-strep (penicillin 500 U/mL and streptomycin 500 µg/mL).
2. Preparation of buffers for staining MDSCs<.......

Discussion

We have described the methodology to isolate MDSCs from mouse ovarian tumor. The same method can be utilized for isolating MDSCs or other immune cells from any solid normal tissue or solid tumor using cell-specific markers. Additionally, depending on the nature of the tissue the incubation time with the dissociation buffer will need to be optimized.

The isolation of viable immune cells from tumor tissue depends on performing the different isolation steps from dissecting tumors to obtain the so.......

Materials

Name	Company	Catalog Number	Comments
Collagenase IV	Thermo Fisher	17104019	Tumor dissociation
Cell strainer	Falcon	352350	Cell strain
RBC lysis buffer	Biolegend	420301	Cell culture medium
DMEM+Glutamax	Gibco	10569010	Cell culture medium
RPMI	Gibco	11875093	Cell culture medium
FBS	Gibco	10082147	Serum for Cell culture
Phosphate Buffered Saline (PBS)	Sigma	D8537-500ML	PBS
Trypsin	Invitrogen	25200-056	Cell dissociation
Penstrep	HyClone	SV30010	Antibiotic
Sterile water	Invitrogen	10977-015	For dilution of buffers
BV605-CD11b	Biolegend	101237	Antibody for MDSC labeling
FITC-GR1	Biolegend	108405	Antibody for MDSC labeling
APC-Cy7-CD45	Biolegend	103115	Antibody for lymphocyte labeling
PE-Cy7-Ly6C	Biolegend	128017	Antibody for MDSC labeling
APC-Ly6G	Biolegend	127613	Antibody for MDSC labeling
TNFα	Sigma	T7539	Cytokine
TNFα neutralizing antibody	Biolegend	506309	neutralizing antibody
Ghost Dye Violet 450	Tonbo Biosciences	13-0863	Cell viability dye
UltraComp Beads	Invitrogen	01-2222-42	Compensation beads
Cell culture inserts	Corning	353097	Migration chamber
LSR Fortessa X-20	BD Biosciences	LSR Fortessa X-20	Fluorescent Cell analyzer
BD FACSAria Fusion	BD Biosciences	BD FACSAria Fusion	Fluorescent Cell sorter
Cell countess	Cell countess	Cell countess	Cell countess
Flow Jo			Software for Analysis of flow data
Prism			Plotting of graph and statistical analysis
C57BL/6 mice	Taconic	B6-F	mice for tumor generation