Background Cancer is one of the leading causes of death worldwide and incidence rates have systematically increased over the years.¹ Immunotherapy, a treatment strategy that harnesses the immune system to specifically target tumor cells, has completely changed the outlook for cancer patients by becoming the standard of care for various types of cancer.^2–5 However, immunotherapeutics are not effective in all cancer patients and often do not yield durable clinical responses.^6–8 Therefore, new tactics in promoting anti-tumor immune responses and to specifically delivery drugs to tumors need to be sought. Carbon monoxide (CO) is a gaseous mediator with several pleiotropic and immunomodulatory effects that has been found to have tumor-suppressive roles in numerous cancer models.^9–18 However, the role of CO in cancer immunity is virtually unknown. Our project aims to dissect the molecular mechanisms and the cellular entities underlying CO-mediated effects by coupling the anticancer potential of CO-releasing molecule 3 (CORM-3) with the tumor-targeting capacity of a recombinant form of human serum albumin (rHSA)¹⁹ into a single agent.

Methods We selected two syngeneic murine colorectal cancer models, CT26 and MC38, to induce tumors in BALB/c and C57BL/6 mice, respectively. Tumor-bearing mice were treated with our conjugate of rHSA with CORM-3 (rHSA-CORM) to assess efficacy and survival. Additionally, we performed an immune profiling and in vitro culture of macrophages to dissect the mechanisms subjacent to rHSA-CORM anti-tumor effects.

Results A substantial attenuation of tumor growth and an increase in overall survival was seen in mice following rHSA-CORM treatments. No differences were observed between treatment groups in tumor-bearing immunocompromised mice (NSG). Immune characterization showed a reduction in tumor-infiltrating monocytes and tumor-associated macrophages (TAMs), whereas NK cells presented higher expression of IFN-y and TNFa and CD8 T cells less PD-1 and CTLA-4 double expression with rHSA-CORM treatments. Using an in vitro model of macrophage differentiation we observed that monocytes were more sensitive to CO levels than macrophages.

Conclusions rHSA-CORM treatments demonstrated significant anticancer effects in mice, attenuating tumor growth and prolonging survival. By characterizing the immune composition of tumors we showed that rHSA-CORM treatments reduced TAMs, releasing immune cells from the suppressive environment of CT26 tumors and enabling the establishment of antitumor immune responses. In vitro macrophage differentiation demonstrates that CO mustn’t act by specifically impairing the monocyte-to-macrophage differentiation process, but by affecting the viability of monocytes before differentiation. Further studies are ongoing to clarify how rHSA-CORM influences monocyte homeostasis and macrophage generation.

Acknowledgements Karine Serre and Gonçalo Bernardes have greatly contributed to the design and conceptualization of the project Cong Tang, Inês Albuquerque, Ana Raquel Coelho, Maria Rebelo, Bárbara Sousa, Miguel Chaves-Ferreira performed or assisted in experiments, having contributed to the investigation conducted in the course of this project.

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Ethics Approval Animal studies were conducted in compliance with the EU and national legislation and were approved by the Portuguese veterinary authority (license no. 018085\2021) and the institutional animal ethics committee.