◥ Accumulating evidence indicates that CD8 þ T cells in the tumor microenvironment and systemic CD4 þ T-cell immunity play an important role in mediating durable antitumor responses. We longitudinally examined T-cell immunity in the peripheral blood of patients with non-small lung cancer and found that responders had significantly (P < 0.0001) higher percentages of effector, CD62L low CD4 þ T cells prior to PD-1 blockade. Conversely, the percentage of CD25 þ FOXP3 þ CD4 þ T cells was significantly (P ¼ 0.034) higher in nonresponders. We developed a formula, which demonstrated 85.7% sensitivity and 100% specificity, based on the percentages of CD62L low CD4 þ T cells and CD25 þ FOXP3 þ cells to predict nonresponders. Mass cytometry analysis revealed that the CD62L low CD4 þ T-cell subset expressed T-bet þ , CD27 À , FOXP3 À , and CXCR3 þ , indicative of a Th1 subpopulation. CD62L low CD4 þ T cells significantly correlated with effector CD8 þ T cells (P ¼ 0.0091) and with PD-1 expression on effector CD8 þ T cells (P ¼ 0.0015). Gene expression analysis revealed that CCL19, CLEC-2A, IFNA, IL7, TGFBR3, CXCR3, and HDAC9 were preferentially expressed in CD62L low CD4 þ T cells derived from responders. Notably, longterm responders, who had >500-day progression-free survival, showed significantly higher numbers of CD62L low CD4 þ T cells prior to PD-1 blockade therapy. Decreased CD62L low CD4 þ T-cell percentages after therapy resulted in acquired resistance, with longterm survivors maintaining high CD62L low CD4 þ T-cell percentages. These results pave the way for new treatment strategies for patients by monitoring CD4 þ T-cell immune statuses in their peripheral blood.
Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.
The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2), uroporphyrinogen decarboxylase (UROD), and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 μM for 5 min) did not cause global oxidations but instead had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.
EGFR-mutated lung cancer accounts for a significant proportion of lung cancer cases worldwide. For these cases, osimertinib, a thirdgeneration EGFR tyrosine kinase inhibitor, is extensively used as a first-line or second-line treatment. However, lung cancer cells acquire resistance to osimertinib in 1 to 2 years. Thus, a thorough clarification of resistance mechanisms to osimertinib is highly anticipated. Recent next-generation sequencing (NGS) of lung cancer samples identified several genetically defined resistance mechanisms to osimertinib, such as EGFR C797S or MET amplification. However, nongenetically defined mechanisms are not well evaluated. For a thorough clarification of osimertinib resistance, both genetic and nongenetic mechanisms are essential. By using our comprehensive protein phosphorylation array, we detected IGF1R bypass pathway activation after EGFR abolishment. Both of our established lung cancer cells and patient-derived lung cancer cells demonstrated IGF2 autocrine-mediated IGF1R pathway activation as a mechanism of osimertinib resistance. Notably, this resistance mechanism was not detected by a previously performed NGS, highlighting the essential roles of living cancer cells for a thorough clarification of resistance mechanisms. Interestingly, the immunohistochemical analysis confirmed the increased IGF2 expression in lung cancer patients who were treated with osimertinib and met the established clinical definition of acquired resistance. The findings highlight the crucial roles of cell-autonomous ligand expression in osimertinib resistance. Here, we report for the first time the IGF2 autocrine-mediated IGF1R activation as a nongenetic mechanism of osimertinib resistance in lung cancer at a clinically relevant level. Implications: Using comprehensive protein phosphorylation array and patient-derived lung cancer cells, we found that IGF2 autocrinemediated IGF1R pathway activation is a clinically relevant and common mechanism of acquired resistance to osimertinib.
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