The ability to control the activation of prodrugs by transition metals has been shown to have great potential for controlled drug release in cancer cells. However, the strategies developed so far promote the cleavage of C-O or C-N bonds, which limits the scope of drugs to only those that present amino or hydroxyl groups. Here, we report the decaging of an ortho-quinone prodrug, a propargylated β-lapachone derivative, through a palladium-mediated C-C bond cleavage. The reaction's kinetic and mechanistic behavior was studied under biological conditions along with computer modeling. The results indicate that palladium (II) is the active species for the depropargylation reaction, activating the triple bond for nucleophilic attack by a water molecule before the C-C bond cleavage takes place. Palladium iodide nanoparticles were found to efficiently trigger the C-C bond cleavage reaction under biocompatible conditions. In drug activation assays in cells, the protected analogue of β-lapachone was activated by nontoxic amounts of nanoparticles, which restored drug toxicity. The palladium-mediated ortho-quinone prodrug activation was further demonstrated in zebrafish tumor xenografts, which resulted in a significant anti-tumoral effect. This work expands the transition-metal-mediated bioorthogonal decaging toolbox to include cleavage of C-C bonds and payloads that were previously not accessible by conventional strategies.

Purpose: To identify a molecular signature of macrophages exposed to clinically relevant ionizing radiation (IR) doses, mirroring radiotherapy sessions. Methods: Human monocyte-derived macrophages were exposed to 2 Gy/ fraction/ day for 5 days, mimicking one week of cancer patient’s radiotherapy. Protein expression profile by proteomics was performed. Results: A gene ontology analysis revealed that radiation-induced protein changes are associated with metabolic alterations, which were further supported by a reduction of both cellular ATP levels and glucose uptake. Most of the radiation-induced deregulated targets exhibited a decreased expression, as was the case of cathepsin D, a lysosomal protease associated with cell death, which was validated by Western blot. We also found that irradiated macrophages exhibited an increased expression of the transferrin receptor 1 (TfR1), which is responsible for the uptake of transferrin-bound iron. TfR1 upregulation was also found in tumor-associated mouse macrophages upon tumor irradiation. In vitro irradiated macrophages also presented a trend for increased divalent metal transporter 1 (DMT1), which transports iron from the endosome to the cytosol, and a significant increase in iron release. Conclusions: Irradiated macrophages present lower ATP levels and glucose uptake, and exhibit decreased cathepsin D expression, while increasing TfR1 expression and altering iron metabolism.

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Checkpoint immunotherapy has transformed cancer treatment, yet current approaches targeting adaptive immunity benefit only a subset of patients, leaving innate immunity as a largely untapped therapeutic frontier. Here, we identify CD24 as an innate immune checkpoint that protects colorectal tumors from macrophage-mediated clearance through an evolutionarily conserved recognition mechanism. Using zebrafish xenografts of isogenic colorectal cancer (CRC) cell lines, SW480 and SW620, we show that high CD24 expression in SW620 correlates with an immune-evasive, macrophage-resistant phenotype. Loss of human CD24 dramatically sensitizes tumors to clearance in zebrafish, while pharmacological macrophage depletion abolishes this effect. Mechanistically, CD24 suppresses innate immunity in a multilayered fashion, by limiting myeloid recruitment, dampening TNFα-driven macrophage inflammatory polarization, and blocking phagocytosis. Live imaging further revealed that CD24 constrains macrophages to a restrained, patrol-like state, and that its loss enables them to adopt a highly motile, tumor‑directed, and functionally engaged state, characterized by increased fusion activity and myeloid intercellular interactions. We show that zebrafish macrophages respond to human CD24 despite extensive evolutionary divergence, and glycocalyx profiling revealed broad remodeling of the tumor cell surface upon CD24 loss, suggesting evolutionary conservation of sialic acid-dependent receptor recognition. Transcriptomic analyses identified the Siglec-like gene si:dkey-24p1.7 as a candidate zebrafish macrophage-expressed receptor mediating this response. Finally, analysis of TCGA CRC cohorts revealed that CD24 expression is a stage-dependent prognostic marker, underscoring the clinical relevance of this axis. Together, these findings establish CD24 as a critical orchestrator of innate immune evasion in CRC, while further validating zebrafish xenografts as a powerful platform for dissecting innate immuno-oncobiology in vivo .