Genetic engineering of Hoxb8-immortalized hematopoietic progenitors - a potent tool to study macrophage tissue migration

J Cell Sci. 2020 Mar 5;133(5):jcs236703. doi: 10.1242/jcs.236703.


Tumor-associated macrophages (TAMs) are detrimental in most cancers. Controlling their recruitment is thus potentially therapeutic. We previously found that TAMs perform protease-dependent mesenchymal migration in cancer, while macrophages perform amoeboid migration in other tissues. Inhibition of mesenchymal migration correlates with decreased TAM infiltration and tumor growth, providing rationale for a new cancer immunotherapy specifically targeting TAM motility. To identify new effectors of mesenchymal migration, we produced ER-Hoxb8-immortalized hematopoietic progenitors (cells with estrogen receptor-regulated Hoxb8 expression), which show unlimited proliferative ability in the presence of estrogen. The functionality of macrophages differentiated from ER-Hoxb8 progenitors was compared to bone marrow-derived macrophages (BMDMs). They polarized into M1- and M2-orientated macrophages, generated reactive oxygen species (ROS), ingested particles, formed podosomes, degraded the extracellular matrix, adopted amoeboid and mesenchymal migration in 3D, and infiltrated tumor explants ex vivo using mesenchymal migration. We also used the CRISPR/Cas9 system to disrupt gene expression of a known effector of mesenchymal migration, WASP (also known as WAS), to provide a proof of concept. We observed impaired podosome formation and mesenchymal migration capacity, thus recapitulating the phenotype of BMDM isolated from Wasp-knockout mice. Thus, we validate the use of ER-Hoxb8-immortalized macrophages as a potent tool to investigate macrophage functionalities.

Keywords: CRISPR/Cas9; Conditionally immortalized macrophages; ER-Hoxb8; Macrophage migration.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation / genetics
  • Cell Movement / genetics
  • Genetic Engineering
  • Hematopoietic Stem Cell Transplantation*
  • Homeodomain Proteins / genetics
  • Macrophages*
  • Mice


  • Homeodomain Proteins
  • Hoxb8 protein, mouse