Room: Karl Dean Ballroom B1
Purpose: The checkpoint blockade immunotherapies like cytotoxic-lymphocyte antigen-4 (CTLA-4), programmed-death-1 (PD-1) and its ligand (PD-L1) have shown long term survival benefits across the several tumor types including melanoma. It has also been reported that a sub-set of patients group is refractory to this therapy. We have investigated the dysregulated metabolism of immunotherapy resistant and sensitive murine melanoma tumors using in-vivo Â¹Â³C-MRS of hyperpolarized pyruvate and ex-vivo Â¹H-NMR spectroscopy.
Methods: 1-Â¹Â³C-labeled pyruvic acid containing 15 mM trityl radical (OX63) was hyperpolarized using a commercial DNP polarizer. 250 Î¼L hyperpolarized pyruvate was administered via a tail-vein catheter to tumor bearing mouse in 8-10 seconds. Anatomical MRI and Â¹Â³C-MRS were obtained using a Bruker 7T scanner (Figure 1). The high-resolution Â¹H-NMR experiments was performed on a 500 MHz spectrometer (Bruker) coupled with cryogenic temperature probe.
Results: B16 melanoma murine tumors sensitive and resistance to CTLA-4, PD-1, and PD-L1 blockade immunotherapy were employed in our metabolic imaging study. Pyruvate metabolism in tumor xenograft was well captured in real-time as shown in Figure 1B. Pyruvate was readily metabolized to lactate. The quantitative flux ratio lactate-to-pyruvate (Lac/Pyr) was determined and used as a treatment response marker of sensitive and resistant tumors. The Lac/Pyr ratios were significantly higher in resistant tumors compared to sensitive tumors (p<0.01, (Figure 1C). The differences in Lac/Pyr ratio among the tumors were observed much sooner than the tumor volume shrinkage. In NMR metabolomics study on ex-vivo tissue samples, we have observed high level of lactate concentration in resistant tumors (Figure 1D) that validates our in-vivo results.
Conclusion: Metabolic imaging with hyperpolarized pyruvate and NMR metabolomics enable distinguishing immunotherapy resistance and sensitive tumors at early time point. Our experimental data suggest that checkpoint blockade immunotherapy resistant tumors acquired hypermetabolic state with upregulated glycolysis to evade the immune response.
MRS, High-resolution Imaging, High-field NMR