T lymphocytes expressing chimeric antigen receptors (CAR) have been shown to be efficient for tretment of hematologic, but less efficient for solid tumors, due to the great complexity of the tumor microenvironment involving: heterogeneity of tumor antigens, difficulty in maintaining and persisting of immune responses, the immunosuppressive millieu, and the dense extracellular matrix. Thereby, macrophages emerge as an exceptional option for the insertion of CAR molecules, given their functional capacity to infiltrate the tumor matrix, to phagocyte, present antigens and secrete cytokines.

To generate human macrophages expressing CAR from umbilical cord blood CD34+ progenitor cells to treat solid tumors.

CD34+ umbilical cord cells were isolated, expanded in vitro for 07 days and differentiated into macrophages using in either StemSpan™ SFEM II (SP) or RPMI 1640 (RP) media with different combinations of the following cytokines: GM-CSF, M-CSF and IL-3, for additional 10 days. After differentiation, the cells were phenotypically characterized by multi-color flow cytometry and morphology (cytospin) and evaluated for their phagocytic capacity after co-culture with Nalm6 leukemic cell line (ratio 1:3). Subsequently, aiming to set-up a cell transformation protocol we used the piggyBac transposon system to insert the green fluorescent protein (GFP) via electroporation on CD34+ cells.

Our data revealed that 93.3% of the CD34+ differentiated cells were CD64+CD14+CD1clowCD163+CD16+CD86lowCD80lowHLA-DR+ and their morphological evaluation indicate a typical macrophage phenotype when compared to classical monocyte-derived macrophages. The phagocytosis assay showed a percentage of 20.1% and 16.2% when cells were differentiated in SP and RP, respectively. Furthermore, 26.8% of CD34+ cells expressed GFP within 24 hours after electroporation with a viability of 60.7%.

Our data indicate that cells differentiated from human CD34+ umbilical cord stem cells showed phenotypic and functional features consistent with human macrophages. In addition, the piggyBac transposon system is an interesting protocol for the insertion of CAR molecules in CD34+ cells. Our next steps involve improving the electroporation efficiency of CD34+ cells and the transformation of CAR-expressing CD34+ cells into CAR-macrophages for their functional characterization against solid tumor cell lines.