An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption.

Although haemoglobin is a known carrier of oxygen in erythrocytes that functions to transport oxygen over a long range, its physiological roles outside erythrocytes are largely elusive1,2. Here we found that chondrocytes produced massive amounts of haemoglobin to form eosin-positive bodies in their cytoplasm. The haemoglobin body (Hedy) is a membraneless condensate characterized by phase separation. Production of haemoglobin in chondrocytes is controlled by hypoxia and is dependent on KLF1 rather than the HIF1/2α pathway. Deletion of haemoglobin in chondrocytes leads to Hedy loss along with severe hypoxia, enhanced glycolysis and extensive cell death in the centre of cartilaginous tissue, which is attributed to the loss of the Hedy-controlled oxygen supply under hypoxic conditions. These results demonstrate an extra-erythrocyte role of haemoglobin in chondrocytes, and uncover a heretofore unrecognized mechanism in which chondrocytes survive a hypoxic environment through Hedy.

Subtype-based analysis of cell-in-cell structures in non-small cell lung cancer.

Lung cancer is ranked as the leading cause of cancer-related death worldwide, and the development of novel biomarkers is helpful to improve the prognosis of non-small cell lung cancer (NSCLC). Cell-in-cell structures (CICs), a novel functional surrogate of complicated cell behaviors, have shown promise in predicting the prognosis of cancer patients. However, the CIC profiling and its prognostic value remain unclear in NSCLC. In this study, we retrospectively explored the CIC profiling in a cohort of NSCLC tissues by using the "Epithelium-Macrophage-Leukocyte" (EML) method. The distribution of CICs was examined by the Chi-square test, and univariate and multivariate analyses were performed for survival analysis. Four types of CICs were identified in lung cancer tissues, namely, tumor-in-tumor (TiT), tumor-in-macrophage (TiM), lymphocyte-in-tumor (LiT), and macrophage-in-tumor (MiT). Among them, the latter three constituted the heterotypic CICs (heCICs). Overall, CICs were more frequently present in adenocarcinoma than in squamous cell carcinoma (P = 0.009), and LiT was more common in the upper lobe of the lung compared with other lobes (P = 0.020). In univariate analysis, the presence of TiM, heCIC density, TNM stage, T stage, and N stage showed association with the overall survival (OS) of NSCLC patients. Multivariate analysis revealed that heCICs (HR = 2.6, 95% CI 1.25-5.6) and lymph node invasion (HR = 2.6, 95% CI 1.33-5.1) were independent factors associated with the OS of NSCLC. Taken together, we profiled the CIC subtypes in NSCLC for the first time and demonstrated the prognostic value of heCICs, which may serve as a type of novel functional markers along with classical pathological factors in improving prognosis prediction for patients with NSCLC.

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment.

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 ± 1.5% (**p < 0.01), 57.49 ± 2.4% (**p < 0.01), and 20.17 ± 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 ± 0.5% (**p < 0.01) and 15.0 ± 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis.

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture.

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.