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Abstract
Background More than half of patients with stage IV breast cancer will eventually develop liver metastasis. Liver metastases are hard to treat, with a very low five-year survival rate of approximately 11%. Recent discoveries highlight the crucial role of macrophages in cancer, making them an appealing target for therapeutic strategies. In the tumor microenvironment, tumor-associated macrophages (TAMs) are predominantly immunosuppressive Arg1+ M2-like macrophages that promote cancer growth, metastasis, and drug resistance. Studies, including our own, suggest that converting TAMs to the anti-tumorigenic M1-like phenotype through macrophage reprogramming shows promising results for cancer eradication. This study utilized CRISPR gene editing delivered via lipid nanoparticles (CRISPR-LNP) to modulate macrophage polarization in the treatment of breast cancer metastasis.
Methods To obtain sustainable change in macrophage polarization, we are utilizing CRISPR gene editing and have successfully loaded them into LNP platform (CRISPR-LNP) (figure 1A). CRISPR screening using LNP revealed that RICTOR gene targeting enabled reprograming of pro-tumorigenic M2-like TAMs to anti-tumorigenic M1 phenotype in vitro and in vivo. In a syngeneic liver metastasis mouse model, we assessed single and multi-dose biodistribution, treatment efficacy, and immune landscape alterations.
Results In the 4T1 breast cancer liver metastatic model, intravenously injected CRISPR-Rictor-LNP nanoparticles concentrated in liver metastases 24 hours post-injection (figure 1B). 75% of the fluorescence detected across the entire body at 24h time point was confined in the metastatic regions. The treatment resulted in a modest increase in macrophage numbers and a significant shift from immunosuppressive CD206+ macrophages to inflammatory CD80+ macrophages within tumor lesions, increasing from approximately 5% to 9% of the total cell population (figure 1C). Imaging Mass Cytometry data further indicated an almost 80% reduction in proliferating cell numbers. Single-cell RNA sequencing analysis showed a depletion of exhausted T-cells and regulatory T-cells (figure 1F&G), with a similar trend observed in macrophage population shift (figure 1E). The treatment also led to significant increase in survival of animals treated with the therapy (figure 1D).
Conclusions These results highlight the potential of reprogramming macrophages from cancer-promoting to cancer-suppressing phenotypes as a promising approach to inhibit cancer growth and enhance the presence of cytotoxic CD8+ T-cells. This strategy could be used effectively as a standalone therapy or in combination with immune checkpoint inhibitors to achieve synergistic effects and effectively eliminate cancer cells. We will conduct further studies will focus on assessing safety, optimizing accumulation in the tumor microenvironment, minimizing exposure to healthy cells, as well as expanding this therapeutic approach to other types of breast cancer metastases.
CRISPR-Rictor-LNP characteization and in vivo evaluation. A) CR-RIC-LNP design. B)Rhodamine tagged CR-Ric-LNP confined to the liver metastatic lesions C) IMC quantification data. D) Kaplan-Meier survival curve. E) Cell quantification from scRNAseq F&G) Remodeling of T-cell dynamic after therapy
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