PU.1: C/EBPα ratio-directed reprogramming converts B-ALL cells into stable M1 macrophages for scalable cancer immunotherapy Free

Macrophages exhibit remarkable functional plasticity, making them a promising therapeutic tool against infections, cancers, and tissue regeneration challenges. However, their ability to adapt can also pose issues: within the tumor microenvironment, macrophages may adopt a pro-cancer M2 phenotype, hindering cancer treatment efficacy. Additionally, producing sufficient macrophages for therapy remains challenging. Our study identified transcription factors influencing macrophages polarization and presents an optimized reprogramming strategy using B-ALL cells to generate stable M1-polarized macrophages. These macrophages retain normal functions and offer therapeutic scalability.

By analyzing transcriptome data from various macrophage types, we identified a strong correlation between the SPI1/CEBPA transcription factor ratio and M1 polarization, with higher ratios correlating with M1-type polarization. Utilizing this insight, we reprogrammed RCH-ACV B-ALL cells using two approaches: C/EBPα overexpression or combined high PU.1 and low C/EBPα expression. These interventions generated REP-M2 and REP-M1 macrophages, at 100% efficiency and exhibiting M2 and M1 phenotypes, respectively. Notably, REP-M1 cells maintained their polarization characteristics after withdrawal of the exogenous transcription factor expression, persistently expressing M1-specific markers (e.g., CD80, CD86) even when challenged with potent M2-polarizing cytokines like IL-4 and IL-13.

To evaluate therapeutic functionality, we engineered REP-M1 cells with chimeric antigen receptors (CARs) targeting EGFRvIII and HER2. CAR(EGFRvIII)-REP-M1 cells exhibited robust phagocytosis of EGFRvIII-expressing K562 leukemia cells and A549 lung cancer cells, achieving 45-65% and 25-35% phagocytosis at 2:1 and 1:1 E:T，respectively, in 24 hours. Confocal imaging confirmed internalization rather than surface adhesion. Similarly, CAR(HER2)-REP-M1 cells efficiently engulfed SW480 colorectal cancer cells that naturally express HER2, achieving 25% phagocytosis at a 5:1 E:T in 24 hours. Notably, co-culture with cancer cells significantly increased pro-inflammatory cytokine expression in CAR-REP-M1 cells. Expression of IL1B, IL6, and TNF increased 210-, 66-, and 6-folds in CAR(HER2)-REP-M1 cells after co-culture with SW480 cells, suggesting CAR-REP-M1 cells may impact cancer cells through mechanisms beyond direct phagocytosis. Indeed, co-culture induced apoptosis and modestly reduced proliferation in target cancer cells. Remarkably, at 30:1 E:T, both CAR-REP-M1 cells eliminated targets within 48 hours. In vivo, CAR(EGFRvIII)-REP-M1 cells slowed A549-EGFRvIII tumor growth and prolonged survival in tumor-bearing mice, while CAR(HER2)-REP-M1 cells showed comparable efficacy against SW480 tumors. Collectively, these data demonstrate the therapeutic potential of engineered REP-M1 cells.

The safety of REP-M1 cells for potential therapeutic applications was also evaluated. Multiple lines of evidence support their safety:1) After cell reprogramming, REP-M1 cells lose their ability to proliferate, confirmed by an EdU incorporation assay. 2) Following the withdrawal of the cell reprogramming inducer, REP-M1 cells retained their macrophage characteristics over a 25-day observation period in vitro. Neither reverting to B-ALL cells nor the outgrowth of residual untransdifferentiated B-ALL cells was observed. 3) No resurgence of B-ALL was observed when 10⁷ REP-M1 cells,irradiated at a dose that eliminated RCH-ACV cells,were transplanted into NOD-Prkdc^-/- Il2rg^-/- (NCG)mice over 2.5-months. Importantly, irradiation minimally affected CAR-REP-M1 cell function. Irradiated CAR(EGFRvIII)-REP-M1 cells demonstrated phagocytosis efficiency against K562-EGFRvIII cells comparable to non-irradiated controls.Critically, CAR-REP-M1 cells could be generated in large scale suspension culture, and cryopreservation had minimal functional impact, suggesting their potential as ready-to-use therapeutic product.

In conclusion, with a carefully designed cell reprogramming strategy, we converted B-ALL cells into stably M1-polarized macrophages that demonstrated strong anti-cancer activity as engineered CAR-REP-M1 cells. These findings underscore the potential of CAR-REP-M1 cells as a scalable, effective and safe therapy against various cancers.