Engineering macrophages with immunomodulatory nanoparticles for photoacoustic image-guided cancer immunotherapy

Introduction: : Enhancing the antitumor activity of macrophages—such as boosting their ability to phagocytose cancer cells and secrete inflammatory cytokines—offers a promising strategy for treating solid tumors. Recent advances such as CAR-macrophages and macrophages engineered to improve phagocytosis through anti-CD47 antibody treatment have demonstrated promising results in preclinical studies. However, these approaches currently lack mechanisms to track the administered macrophages and evaluate their therapeutic impact over time, making it difficult to perform longitudinal and adaptive treatment interventions.
Incorporating imaging techniques and contrast agents enables a potential strategy for real-time, non-invasive monitoring of macrophages within the tumor microenvironment. By labeling macrophages with contrast agents, it becomes possible to track their movement, infiltration, and retention in tumors over time - providing valuable insights into treatment response. However, current macrophage engineering studies rarely report monitoring features. Photoacoustic (PA) imaging, which integrates optical and ultrasound imaging properties, stands out as a powerful tool for monitoring adoptively transferred contrast agent-labeled cells in a non-invasive manner. Contrast agents such as gold nanoparticles convert the absorbed laser light from high-intensity laser pulses into sound waves, allowing light-induced ultrasonic visualization.
In this study, we present a macrophage engineering strategy designed for ultrasound/photoacoustic (US/PA) image-guided cancer immunotherapy. We synthesized a nanoparticle formulation that offers both PA imaging contrast and immunomodulation of macrophages. When macrophages were labeled with these nanoparticles, they exhibited enhanced antitumor phenotype and enabled longitudinal PA imaging over time. Our findings highlight a promising direction for the development of image-guided macrophage-based cellular immunotherapies.

Materials and

Methods: : Macrophages were derived from murine bone marrow cells. Bone marrow cells were extracted from the femur and the tibia from 5 - 8 weeks old Balb/c mice. The cells were cultured with 20 ng/mL M-CSF for differentiation into macrophages. Fresh media was added on day 3 and the cells were used on days 5-6. The hyperbranched gold nanoconstructs (HBGNCs) were synthesized via surface-blocker assisted seeded growth. Briefly, the surface of 35 nm-sized gold nanospheres was partially blocked during the seeded growth. The dimension was approximately 100 nm. The resulting HBGNCs shape and optical properties were analyzed by TEM imaging and UV-vis-NIR spectrometry. For PA imaging, macrophages were labeled with varying concentrations (ODs) of HBGNCs for 16 hours with the HBGNCs added to culture media. Afterwards, cells were washed with PBS and collected. The cell solution was embedded in 8% (w/v) gelatin phantom. The phantom was submerged in water and imaged under Vevo2100/LAZR imaging system at 800 nm with varying cell densities. The effect of particle labeling on key cellular functions, such as viability, migration, and antitumoral functions (CD80, CD86, MHCII, and CD206 expression) were investigated by confocal microscopy and flow cytometry.

Results, Conclusions, and Discussions:: Results and Discussions
The labeling efficiency of macrophages increased with concentrations of HBGNCs. The size and the granularity of macrophages increased as the HBGNCs were internalized (Fig. 1a). The concentration of HBGNCs did not significantly affect cell viability until optical density 1 (OD 1, at 800 nm), and began to decrease from OD 2 (Fig. 1b). Further labeling experiments were performed with OD 1 concentration.
HBGNC-labeled macrophages were embedded in tissue-mimicking gelatin phantoms and imaged under US/PA imaging system (Fig. 1c). The PA signal increased with increasing cell concentrations from 0 to 2000 cells per microliter, suggesting that PA signals reflect the number of labeled macrophages (Fig. 1d).
Next, interferon gamma (IFNg), an immunomodulatory cytokine that reprograms macrophages into antitumor phenotype, was conjugated to HBGNCs (HBGNC-G). Representative antitumor surface markers (MHCII, CD86, and CD 80) and protumor surface marker (CD206) were analyzed following incubation with PBS, free IFNg, HBGNC, and HBGNC-G, by flow cytometry. HBGNC-G-treated macrophages showed the highest expression of all three antitumor markers. Also, HBGNC alone stimulated macrophages to some degree. Similarly, HBGNC-G-treated macrophages had the lowest expression of CD206 (Fig. 1e).
The same four groups of macrophages were subjected to transwell migration assay (Fig. 1f). IFNg, a macrophage migration stimulant, showed the highest migration at 6 hour point. However, HBGNC and HBGNC-G-labeled macrophages displayed the highest migration at 24 hour point (Fig. 1g), suggesting a high accumulation of macrophages at the tumor tissues over time post adoptive transfer.

Conclusions
Our study introduces a new type of macrophage-mediated cancer immunotherapy strategy with US/PA image guidance. HBGNC-labeled macrophages yielded strong PA signals without significantly affecting the viability, and HBGNC-G formulation strongly reprogrammed macrophages into antitumor phenotypes. Moreover, both HBGNC and HBGNC-G-labeled macrophages showed the highest migration ability over IFNg-primed macrophages. These results collectively indicate that adoptive transfer of HBGNC-G-labeled macrophages may lead to high tumor accumulation and therapeutic efficacy.

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