CRISPR-MEDIATED CAR-NK CELL THERAPY TARGETING TUMOR-ASSOCIATED MACROPHAGES IN PEDIATRIC OSTEOSARCOMA

Abstract

Objective: To assess, through a cross-sectional immunophenotypic analysis, the potential responsiveness of pediatric osteosarcoma tumor environments to CRISPR-edited CAR-NK therapy targeting tumor-associated macrophages (TAMs). This study investigates the prevalence of M2 macrophages (CD163+/CD206+) in osteosarcoma specimens and evaluates the ex vivo cytotoxic efficacy of CAR-NK cells against primary TAM-enriched tumor cultures.

Design: A cross-sectional study analyzing 28 pediatric osteosarcoma tumor specimens collected during biopsy or resection at a tertiary care hospital in Pakistan. Immune cell populations, cytokine expression, and checkpoint markers were profiled. Parallel ex vivo co-culture assays were conducted using CRISPR-edited CAR-NK cells targeting CD206.

Results: M2-TAMs were highly enriched in 71% of tumor samples, correlating with increased PD-L1 and CCL2 expression. CRISPR-edited CAR-NK cells demonstrated significantly higher cytotoxic activity and IFN-γ secretion in tumors with high CD206 expression. Multivariate analysis indicated a strong inverse correlation between M1/M2 ratio and CAR-NK cytotoxic effectiveness (r = -0.69, p < 0.01).

Conclusion: This cross-sectional study provides evidence that pediatric osteosarcomas with high TAM burden and M2 dominance could benefit from CAR-NK immunotherapy strategies. These data offer a foundational map of immune heterogeneity in pediatric bone sarcomas and support future in vivo studies and trials.