Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a “hypermetabolic” phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

In recent years, magnetic core–shell nanoparticles have received widespread attention due to their unique properties that can be used for various applications. We introduce here a magnetic core–shell nanoparticle system for potential application as a contrast agent in magnetic resonance imaging (MRI). MnFe₂O₄–Fe₃O₄ core–shell nanoparticles were synthesized by the wet-chemical synthesis method. Detailed structural and compositional charaterization confirmed the formation of a core–shell microstructure for the nanoparticles. Magnetic charaterization revealed the superparamagnetic nature of the as-synthesized core–shell nanoparticles. Average size and saturation magnetization values obtained for the as-synthesized core–shell nanoparticle were 12.5 nm and 69.34 emu g⁻¹ respectively. The transverse relaxivity value of the water protons obtained in the presence of the core–shell nanoparticles was 184.1 mM⁻¹ s⁻¹. To investigate the effect of the core–shell geometry towards enhancing the relaxivity value, transverse relaxivity values were also obtained in the presence of separately synthesized single phase Fe₃O₄ and MnFe₂O₄ nanoparticles. Average size and saturation magnetization values for the as-synthesized Fe₃O₄ nanoparticles were 12 nm and 65.8 emu g⁻¹ respectively. Average size and saturation magnetization values for the MnFe₂O₄ nanoparticles were 9 nm and 61.5 emu g⁻¹ respectively. The transverse relaxivity value obtained in the presence of single phase Fe₃O₄ and MnFe₂O₄ nanoparticles was 96.6 and 83.2 mM⁻¹ s⁻¹ respectively. All the nanoparticles (core–shell and single phase) were coated with chitosan by a surfactant exchange reaction before determining the relaxivity values. For similar nanoparticle sizes and saturation magnetization values, the highest value of the transverse relaxivity in the case of core–shell nanoparticles clearly illustrated that the difference in the magnetic nature of the core and shell phases in the core–shell nanoparticles creates greater magnetic inhomogeneity in the surrounding medium yielding a high value for proton relaxivity. The MnFe₂O₄–Fe₃O₄ core–shell nanoparticles exhibited extremely low toxicity towards the MCF-7 cell line. Taken together, this opens up new avenues for the use of core–shell nanoparticles in MRI.