Objective: Programmed death 1 and its ligand 1 (PD-1/PD-L1) immunotherapy is promising for late-stage lung cancer treatment, however, the response rate needs to be improved. Gut microbiota plays a crucial role in immunotherapy sensitisation and Panax ginseng has been shown to possess immunomodulatory potential. In this study, we aimed to investigate whether the combination treatment of ginseng polysaccharides (GPs) and αPD-1 monoclonal antibody (mAb) could sensitise the response by modulating gut microbiota. Design: Syngeneic mouse models were administered GPs and αPD-1 mAb, the sensitising antitumour effects of the combination therapy on gut microbiota were assessed by faecal microbiota transplantation (FMT) and 16S PacBio single-molecule real-time (SMRT) sequencing. To assess the immune-related metabolites, metabolomics analysis of the plasma samples was performed. Results: We found GPs increased the antitumour response to αPD-1 mAb by increasing the microbial metabolites valeric acid and decreasing L-kynurenine, as well as the ratio of Kyn/Trp, which contributed to the suppression of regulatory T cells and induction of T _eff cells after combination treatment. Besides, the microbial analysis indicated that the abundance of Parabacteroides distasonis and Bacteroides vulgatus was higher in responders to anti-PD-1 blockade than non-responders in the clinic. Furthermore, the combination therapy sensitised the response to PD-1 inhibitor in the mice receiving microbes by FMT from six non-responders by reshaping the gut microbiota from non-responders towards that of responders. Conclusion: Our results demonstrate that GPs combined with αPD-1 mAb may be a new strategy to sensitise non-small cell lung cancer patients to anti-PD-1 immunotherapy. The gut microbiota can be used as a novel biomarker to predict the response to anti-PD-1 immunotherapy.

Background & Aims Mutant KRAS promotes glutaminolysis, a process that uses steps from the tricarboxylic cycle to convert glutamine to α-ketoglutarate and other molecules via glutaminase and SLC25A22. This results in inhibition of demethylases and epigenetic alterations in cells that increase proliferation and stem cell features. We investigated whether mutant KRAS-mediated glutaminolysis affects the epigenomes and activities of colorectal cancer (CRC) cells. Methods We created Apc^minKras^G12D mice with intestine-specific knockout of SLC25A22 (Apc^minKras^G12DSLC25A22^fl/fl mice). Intestine tissues were collected and analyzed by histology, immunohistochemistry, and DNA methylation assays; organoids were derived and studied for stem cell features, along with organoids derived from 2 human colorectal tumor specimens. Colon epithelial cells (1CT) and CRC cells (DLD1, DKS8, HKE3, and HCT116) that expressed mutant KRAS, with or without knockdown of SLC25A22 or other proteins, were deprived of glutamine or glucose and assayed for proliferation, colony formation, glucose or glutamine consumption, and apoptosis; gene expression patterns were analyzed by RNA sequencing, proteins by immunoblots, and metabolites by liquid chromatography–mass spectrometry, with [U-¹³C₅]-glutamine as a tracer. Cells and organoids with knocked down, knocked out, or overexpressed proteins were analyzed for DNA methylation at CpG sites using arrays. We performed immunohistochemical analyses of colorectal tumor samples from 130 patients in Hong Kong (57 with KRAS mutations) and Kaplan-Meier analyses of survival. We analyzed gene expression levels of colorectal tumor samples in The Cancer Genome Atlas. Results CRC cells that express activated KRAS required glutamine for survival, and rapidly incorporated it into the tricarboxylic cycle (glutaminolysis); this process required SLC25A22. Cells incubated with succinate and non-essential amino acids could proliferate under glutamine-free conditions. Mutant KRAS cells maintained a low ratio of α-ketoglutarate to succinate, resulting in reduced 5-hydroxymethylcytosine—a marker of DNA demethylation, and hypermethylation at CpG sites. Many of the hypermethylated genes were in the WNT signaling pathway and at the protocadherin gene cluster on chromosome 5q31. CRC cells without mutant KRAS, or with mutant KRAS and knockout of SLC25A22, expressed protocadherin genes (PCDHAC2, PCDHB7, PCDHB15, PCDHGA1, and PCDHGA6)—DNA was not methylated at these loci. Expression of the protocadherin genes reduced WNT signaling to β-catenin and expression of the stem cell marker LGR5. Apc^minKras^G12DSLC25A22^fl/fl mice developed fewer colon tumors than Apc^minKras^G12D mice (P < .01). Organoids from Apc^minKras^G12DSLC25A22^fl/fl mice had reduced expression of LGR5 and other markers of stemness compared with organoids derived from Apc^minKras^G12D mice. Knockdown of SLC25A22 in human colorectal tumor organoids reduced clonogenicity. Knockdown of lysine demethylases, or succinate supplementation, restored expression of LGR5 to SLC25A22-knockout CRC cells. Knockout of SLC25A22 in CRC cells that express mutant KRAS increased their sensitivity to 5-fluorouacil. Level of SLC25A22 correlated with levels of LGR5, nuclear β-catenin, and a stem cell-associated gene expression pattern in human colorectal tumors with mutations in KRAS and reduced survival times of patients. Conclusions In CRC cells that express activated KRAS, SLC25A22 promotes accumulation of succinate, resulting in increased DNA methylation, activation of WNT signaling to β-catenin, increased expression of LGR5, proliferation, stem cell features, and resistance to 5-fluorouacil. Strategies to disrupt this pathway might be developed for treatment of CRC.

Spheroid-based three-dimensional (3D) liver culture models, offering a desirable biomimetic microenvironment, are useful for recapitulating liver functions in vitro. However, a user-friendly, robust and specially optimized method has not been well developed for a convenient, highly efficient, and safe in situ perfusion culture of spheroid-based 3D liver models. Here, we have developed a biomimetic and reversibly assembled liver-on-a-chip (3D-LOC) platform and presented a proof of concept for long-term perfusion culture of 3D human HepG2/C3A spheroids for building a 3D liver spheroid model. On the basis of a fast and reversible seal of concave microwell-based PDMS–membrane–PDMS sandwich multilayer chips, it enables a high-throughput and parallel perfusion culture of 1080 cell spheroids in a high mass transfer and low fluid shear stress biomimetic microenvironment as well as allowing the convenient collection and analysis of the cell spheroids. In terms of reducing spheroid loss and maintaining cell morphology and viability in long-term perfusion culture, the cell spheroids in the 3D-LOC were more safe and efficient. Notably, the polarisation, liver-specific functions, and metabolic activity of the cell spheroids in 3D-LOC were also remarkably improved and exhibited better long-term maintenance over conventional perfusion methods. Additionally, a robust micromilling method that incorporates secondary PDMS coating techniques (SPCs) for fabricating V-shaped concave microwells was also developed. The V-shaped concave microwell arrays exhibited a higher distribution density and aperture ratio, making it easy to form large-scale and uniform-sized cell spheroids with minimum cell loss. In summary, the proposed 3D-LOC could provide a convenient and robust solution for the long-term safe perfusion culture of hepatic spheroids and be beneficial for a variety of potential applications including development of bio-artificial livers, disease modeling, and drug toxicity screening.