Abstract Background cAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations. Findings We have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα) that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments. Conclusions The modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.

Malignant pleural mesothelioma (MPM), a rare malignancy with a poor prognosis, is mainly caused by exposure to asbestos or other organic fibers, but the underlying genetic mechanism is not fully understood. Genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. We found that the MPM had R282W, a key TP53 mutation, and genome-wide allelic loss or loss of heterozygosity, a distinct genomic alteration not previously described in MPM. We identified frequent inactivating SETDB1 mutations in this patient and in 68 additional MPM patients (mutation frequency: 10%, 7/69) by targeted deep sequencing. Our observations suggest the possibility of a new genetic mechanism in the development of either MPM or multiple primary cancers. The frequent SETDB1 inactivating mutations suggest there could be new diagnostic or therapeutic options for MPM.

Background and aims

Targeting costimulatory receptors with agonistic antibodies is a promising cancer immunotherapy option. We aimed to investigate costimulatory receptor expression, particularly 4-1BB (CD137 or tumor necrosis factor receptor superfamily member 9), on tumor-infiltrating CD8⁺ T cells (CD8⁺ tumor-infiltrating lymphocytes [TILs]) and its association with distinct T-cell activation features among exhausted CD8⁺ TILs in hepatocellular carcinoma (HCC).

Approach and results

Tumor tissues, adjacent nontumor tissues, and peripheral blood were collected from HCC patients undergoing surgical resection (n = 79). Lymphocytes were isolated and used for multicolor flow cytometry, RNA-sequencing, and in vitro functional restoration assays. Among the examined costimulatory receptors, 4-1BB was most prominently expressed on CD8⁺ TILs. 4-1BB expression was almost exclusively detected on CD8⁺ T cells in the tumor-especially on programmed death 1 (PD-1)^high cells and not PD-1^int and PD-1^neg cells. Compared to PD-1^int and 4-1BB^neg PD-1^high CD8⁺ TILs, 4-1BB^pos PD-1^high CD8⁺ TILs exhibited higher levels of tumor reactivity and T-cell activation markers and significant enrichment for T-cell activation gene signatures. Per-patient analysis revealed positive correlations between percentages of 4-1BB^pos cells among CD8⁺ TILs and levels of parameters of tumor reactivity and T-cell activation. Among highly exhausted PD-1^high CD8⁺ TILs, 4-1BB^pos cells harbored higher proportions of cells with proliferative and reinvigoration potential. Our 4-1BB-related gene signature predicted survival outcomes of HCC patients in the The Cancer Genome Atlas cohort. 4-1BB agonistic antibodies enhanced the function of CD8⁺ TILs and further enhanced the anti-PD-1-mediated reinvigoration of CD8⁺ TILs, especially in cases showing high levels of T-cell activation.

Conclusion

4-1BB expression on CD8⁺ TILs represents a distinct activation state among highly exhausted CD8⁺ T cells in HCC. 4-1BB costimulation with agonistic antibodies may be a promising strategy for treating HCCs exhibiting prominent T-cell activation.