Dados do Trabalho

Título

GENERATION OF HUMAN MACROPHAGE EXPRESSING CHIMERIC ANTIGEN RECEPTORS (CAR) DERIVED OF CD34+ CELLS FROM UMBILICAL CORD BLOOD FOR THE TREATMENT OF SOLID TUMORS

Introdução

T lymphocytes expressing CAR have been shown to be less efficient for solid tumors, due to the tumor microenvironment involving: heterogeneity of tumor antigens, difficulty in maintaining of immune responses, the immunosuppressive milieu, and the dense extracellular matrix. Thereby, macrophages emerge as an exceptional option for the insertion of CAR (CAR-Mac), given their capacity to infiltrate the tumor matrix, to phagocyte, present antigens and secrete cytokines.

Objetivo

We aim to generate CAR-Mac from umbilical cord blood CD34+ progenitor cells to treat solid tumors.

Métodos

CD34+ cells were isolated, expanded in vitro for 07 days and differentiated into macrophages using StemSpan™SFEMII (SP) or RPMI1640 (RP) media with different cytokines (GM-CSF, M-CSF and IL-3) for 10 days. Then, the cells were phenotypically characterized by flow cytometry and morphology and evaluated for their phagocytic capacity after co-culture with Nalm6 leukemic cell line. Subsequently, to set-up a cell transformation protocol we used the piggyBac transposon system (PB) to insert the green fluorescent protein (GFP) via electroporation on CD34+ cells.

Resultados

Data revealed that 93.3% of the CD34+ differentiated cells were CD64+CD14+CD1clowCD163+CD16+CD86lowCD80lowHLA-DR+ and their morphology indicate a typical macrophage phenotype, compared to 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%.

Conclusões

Cells differentiated showed features consistent with human macrophages and the PB is an interesting protocol for the insertion of CAR in CD34+ cells. Our next steps are the differentiation of CAR-expressing CD34+ cells into CAR-Mac for their functional characterization.

Palavras-chave

CAR; Macrophage; Cell Therapy.

Financiador do resumo

FAPESP#2022/11481-9, CNPQ#442676/2020-4 and PRONON#25000.027785/2021-21.