Chemotherapy is the commonly used treatment for advanced lung cancer. However, it produces side effects such as the development of chemoresistance. A possible responsible mechanism may be therapy-induced senescence (TIS). TIS cells display increased senescence-associated β-galactosidase (SA-β-gal) activity and irreversible growth arrest. However, recent data suggest that TIS cells can reactivate their proliferative potential and lead to cancer recurrence. Our previous study indicated that reactivation of proliferation by TIS cells might be related with autophagy modulation. However, exact relationship between both processes required further studies. Therefore, the aim of our study was to investigate the role of autophagy in the senescence-related chemoresistance of lung cancer cells. For this purpose, human and murine lung cancer cells were treated with two commonly used chemotherapeutics: cisplatin (CIS), which forms DNA adducts or docetaxel (DOC), a microtubule poison. Hypoxia, often overlooked in experimental settings, has been implicated as a mechanism responsible for a significant change in the response to treatment. Thus, cells were cultured under normoxic (~19% O₂) or hypoxic (1% O₂) conditions. Herein, we show that hypoxia increases resistance to CIS. Lung cancer cells cultured under hypoxic conditions escaped from CIS-induced senescence, displayed reduced SA-β-gal activity and a decreased percentage of cells in the G2/M phase of the cell cycle. In turn, hypoxia increased the proliferation of lung cancer cells and the proportion of cells proceeding to the G0/G1 phase. Further molecular analyses demonstrated that hypoxia inhibited the prosenescent p53/p21 signaling pathway and induced epithelial to mesenchymal transition in CIS-treated cancer cells. In cells treated with DOC, such effects were not observed. Of importance, pharmacological autophagy inhibitor, hydroxychloroquine (HCQ) was capable of overcoming short-term CIS-induced resistance of lung cancer cells in hypoxic conditions. Altogether, our data demonstrated that hypoxia favors cancer cell escape from CIS-induced senescence, what could be overcome by inhibition of autophagy with HCQ. Therefore, we propose that HCQ might be used to interfere with the ability of senescent cancer cells to repopulate following exposure to DNA-damaging agents. This effect, however, needs to be tested in a long-term perspective for preclinical and clinical applications.

Immunotherapy revolutionized cancer treatment in the last decade. Both NK cells and T cells are key components of host immunity against malignant cells that are being extensively investigated in the field of cancer immunotherapy. While approaches have been developed to improve the antitumor activity of NK and T cells, the tumor microenvironment remains an obstacle to effective NK and T-cell–based therapies. Here, we demonstrated that cancer-conditioned medium suppresses the antitumor activity of NK cells. Ammonia, a by-product of cancer cell metabolism, accumulated in cancer-conditioned medium and in the tumor microenvironment, and impaired the cytotoxicity of NK cells as well as the efficacy of antibody-based and chimeric antigen receptor (CAR) NK and CAR T-cell–based therapies in vitro. Ammonia induced NK and T-cell dysfunction by decreasing the amount of mature perforin in secretory lysosomes, which was dependent on its lysosomotropic features and ability to increase pH in acidic compartments. These findings demonstrate that, in addition to its previously described role in promoting tumor growth as a source of nitrogen for tumor biomass, ammonia promotes tumor immune escape by inhibiting both NK and CAR T-cell cytotoxicity. Significance: Ammonia is elevated in the tumor microenvironment and functions as an immunoinhibitory metabolite in cancer by reducing perforin levels, inhibiting NK and T-cell–mediated immunity and limiting the efficacy of immunotherapies.

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

Th17 lymphocytes are a distinct subpopulation of T cells that are characterized by the production of interleukins IL-17, IL-21, IL-22, and IL-26, and high expression of RORγt. These cells play an important role in inflammation and autoimmune diseases. Recent studies using rodent and human models have also highlighted their promising properties as agents in cellular immunotherapy for cancer. However, much less is known about the properties of canine Th17 lymphocytes, despite the domestic dog being an important model used in comparative medicine. In this study, we developed methods of activation and differentiation of canine CD4⁺ T lymphocytes towards the Th17 phenotype. Additionally, we targeted the Wnt/β-catenin signaling pathway to modulate the efficiency of Th17 cells differentiation. CD4⁺ T cells were successfully activated with magnetic EpoxyBeads, and in combination with the appropriate programming medium, they acquired the Th17 phenotype. Furthermore, indomethacin, an inhibitor of the Wnt/β-catenin pathway, significantly increased the efficiency of differentiation, causing elevated production of IL-17 and changed T cell metabolism by promoting oxidative phosphorylation. The protocol elaborated in our study provides an efficient method of canine Th17 lymphocyte differentiation. Our findings also suggested that the modification of the Wnt/β-catenin signaling pathway could be a valuable strategy for optimizing canine Th17 cell differentiation and advancing cell-based immunotherapy.

Supplementary Fig. 8 shows effects of ammonia on perforin gene expression at the transcriptional level

Supplementary Fig. 4 shows that ammonia accumulates in NK cells, affects their cytotoxic potential but not their viability.