Assessment of myeloid-derived suppressor cell differentiation ex vivo
Ester Blanco1, David Escors2, Grazyna Kochan2
1Oncoimmunology Research Unit, Navarrabiomed-Fundación Miguel Servet, Hospital Universitario de Navarra (HUN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain; Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, Spain.
Abstract:
Myeloid-derived suppressor cells (MDSCs) are major promoters of progression and metastasis in cancer. MDSCs inhibit the anti-tumor immune response through multiple mechanisms. The main MDSC functions in cancer are related to the inactivation of T cells and the establishment of an immunosuppressive tumor microenvironment (TME) through the production of pro-inflammatory cytokines, among other mechanisms. MDSCs are phenotypically similar to conventional myeloid cells, so their identification is challenging. Moreover, they infiltrate the tumors in limited numbers, and their purification from within the tumors is technically difficult and makes their study a challenge. Therefore, several ex vivo differentiation methods have been established. Our differentiation method leads to MDSCs that closely model tumor-infiltrating counterparts. In this protocol, MDSCs are differentiated from bone marrow precursors by incubation in differentiation medium produced by murine tumor cell lines engineered to constitutively express granulocyte-monocyte colony stimulating factor (GM-CSF). These ex vivo-generated MDSC subsets show high fidelity compared to their natural tumor-infiltrated counterparts. Moreover, the high yields of purification from these ex vivo differentiated MDSC enable their use for validation of new treatments in high-throughput assays. In this chapter we describe the engineering of a stable cell line overexpressing GM-CSF, followed by production and collection of conditioned media supporting MDSC differentiation. Finally, we detail the isolation procedure of bone marrow cells and the specific MDSC differentiation protocol.
Insights
Myeloid-derived suppressor cells (MDSCs) are key drivers of cancer progression. This study presents a novel ex vivo differentiation method to generate reliable MDSCs for cancer research and drug development.
Area of Science:
- Immunology
- Cancer Biology
- Cell Biology
Background:
- Myeloid-derived suppressor cells (MDSCs) promote cancer progression and metastasis by suppressing anti-tumor immunity.
- MDSCs create an immunosuppressive tumor microenvironment (TME) via T cell inactivation and cytokine production.
- Identifying and purifying tumor-infiltrating MDSCs is challenging due to their similarity to conventional myeloid cells and low numbers.
Purpose of the Study:
- To establish a reliable method for ex vivo differentiation of myeloid-derived suppressor cells (MDSCs).
- To generate MDSCs that accurately model tumor-infiltrating counterparts for cancer research.
- To facilitate high-throughput drug validation assays by enabling high-yield MDSC purification.
Main Methods:
- Engineering a murine tumor cell line to constitutively express granulocyte-monocyte colony-stimulating factor (GM-CSF).
- Collecting conditioned medium from engineered cell lines to induce MDSC differentiation from bone marrow precursors.
- Isolating bone marrow cells and applying a specific protocol for ex vivo MDSC differentiation.
Main Results:
- The ex vivo differentiation method yields MDSC subsets with high fidelity to natural tumor-infiltrating MDSCs.
- The protocol allows for high-yield purification of differentiated MDSCs.
- The generated MDSCs closely model their in vivo counterparts.
Conclusions:
- This protocol provides a robust method for generating functional MDSCs ex vivo.
- The high-yield and fidelity of these cells support their use in validating novel cancer therapies.
- This approach overcomes challenges in studying MDSCs, advancing cancer immunology research.
