Gut microbiota Original research Ginseng polysaccharides alter the gut microbiota and Gut: first published as 10.1136/gutjnl-2020-321031 on 18 May 2021. Downloaded from kynurenine/tryptophan ratio, potentiating the antitumour effect of antiprogrammed cell death 1/ programmed cell death ligand 1 (anti- PD-1/PD- L1) immunotherapy Jumin Huang ,1 Di Liu,2 Yuwei Wang,1 Liang Liu,1 Jian Li,3 Jing Yuan,4 Zhihong Jiang,1 Zebo Jiang,1 WL Wendy Hsiao,1 Haizhou Liu ,2 Imran Khan,1 Ying Xie,1 Jianlin Wu,1 Yajia Xie,1 Yizhong Zhang,1 Yu Fu,1 Junyi Liao,1 Wenjun Wang,1 Huanling Lai,1 Axi Shi,1 Jun Cai,1 Lianxiang Luo,5 Runze Li,1 Xiaojun Yao,1 Xingxing Fan,1 Qibiao Wu,1 Zhongqiu Liu,6 Peiyu Yan,1 Jingguang Lu,1 Mingrong Yang,1 Lin Wang,1 Yabing Cao,7 Hong Wei,3 Elaine Lai- Han Leung1 ► Additional online ABSTRACT supplemental material is Objective Programmed death 1 and its ligand 1 (PD-1/ Significance of this study published online only. To view, PD-L1) immunotherapy is promising for late-stage lung please visit the journal online What is already known on this subject? (http:// dx. doi. org/ 10. 1136/ cancer treatment, however, the response rate needs gutjnl- 2020- 321031). to be improved. Gut microbiota plays a crucial role in ► The gut microbiota plays a crucial role in immunotherapy sensitisation and Panax ginseng has shaping the systemic immune system and For numbered affiliations see has a major influence on the effectiveness end of article. been shown to possess immunomodulatory potential. In this study, we aimed to investigate whether the of anticancer immunotherapy targeting the CTLA-4 and programmed death 1 and its ligand Correspondence to combination treatment of ginseng polysaccharides (GPs) 1 (PD-1/PD- L1) pathways in both preclinical Dr Elaine Lai- Han Leung, Macau and αPD-1 monoclonal antibody (mAb) could sensitise University of Science and tumour models and patients with cancer. http://gut.bmj.com/ the response by modulating gut microbiota. Technology State Key Laboratory The response rate of non- small cell lung cancer Design Syngeneic mouse models were administered ► of Quality Research in Chinese (NSCLC) patients to anti- PD-1 immunotherapy Medicines, Taipa, Macau 123, GPs and PD-1 mAb, the sensitising antitumour effects α is less than 25%, dietary supplements may China; lhleung@ must. edu. mo of the combination therapy on gut microbiota were Professor Hong Wei; influence the gut microbiome and the response assessed by faecal microbiota transplantation (FMT) and weihong63528@ 163. com to anti- PD-1 immunotherapy. Dr Yabing Cao; 16S PacBio single- molecule real- time (SMRT) sequencing. ► Ginseng polysaccharides (GPs), one of the most sumscaoyabing@ hotmail. com on October 1, 2021 by guest. Protected copyright. To assess the immune-related metabolites, metabolomics abundant components of Panax ginseng, have analysis of the plasma samples was performed. JH, DL and YW contributed an important influence on immunomodulation equally. Results We found GPs increased the antitumour and antitumour effects. response to αPD-1 mAb by increasing the microbial Received 5 March 2020 metabolites valeric acid and decreasing L-kynurenine , Revised 5 April 2021 as well as the ratio of Kyn/Trp, which contributed to the Accepted 4 May 2021 INTRODUCTION suppression of regulatory T cells and induction of Teff Lung cancer has the highest morbidity and cells after combination treatment. Besides, the microbial mortality rate worldwide.1 Approximately analysis indicated that the abundance of Parabacteroides 80%–85% of all lung cancers are non- small cell distasonis and Bacteroides vulgatus was higher in lung cancers (NSCLCs). Inhibitors of programmed responders to anti- PD-1 blockade than non- responders death 1 (PD-1) and its ligand PD-L1 are effective in the clinic. Furthermore, the combination therapy therapies for metastatic NSCLC lacking sensi- © Author(s) (or their sensitised the response to PD-1 inhibitor in the mice tising EGFR or ALK mutations.2–6 However, even employer(s)) 2021. Re- use receiving microbes by FMT from six non- responders though these biomarkers were used as the gold permitted under CC BY- NC. No by reshaping the gut microbiota from non- responders standard, the response rate (<25%) is still unsat- commercial re- use. See rights and permissions. Published towards that of responders. isfactory, even leading to hyperprogressive disease 7 8 by BMJ. Conclusion Our results demonstrate that GPs (HPD). Hence, there remains a need for more combined with αPD-1 mAb may be a new strategy to effective first- line treatments for the majority of To cite: Huang J, Liu D, patients with advanced NSCLC and for predictive Wang Y, et al. Gut Epub sensitise non-small cell lung cancer patients to anti-PD-1 ahead of print: [please immunotherapy. The gut microbiota can be used as a biomarkers to identify patients who may benefit 9 include Day Month Year]. novel biomarker to predict the response to anti-PD-1 from new therapies. Extensive research has been doi:10.1136/ immunotherapy. carried out identifying new combinations of PD-1/ gutjnl-2020-321031 PD- L1 pathway inhibitors with other treatments Huang J, et al. Gut 2021;0:1–12. doi:10.1136/gutjnl-2020-321031 1 Gut microbiota was to define the synergistic antitumour effect of GPs and inves- Significance of this study tigate whether the potential effect is related to gut microbiota modulation and its associated treatment mechanisms. What are the new findings? Gut: first published as 10.1136/gutjnl-2020-321031 on 18 May 2021. Downloaded from GPs potentiated the antitumour effect of an PD-1 ► α METHODS monoclonal antibody (mAb) in Lewis lung cancer- bearing Mouse experiments mice. The 8–12 weeks old C57BL/6J mice and humanised PD-1 ► Combination therapy with GPs and an αPD-1 mAb increased knock- in (HuPD-1) mice were reared in independently vented the activated CD8+ T cell population and reduced the Foxp3+ cages at the animal facility of the State Key Laboratory of Quality regulatory T cell population in the periphery, consistent with Research in Chinese Medicine, Macau University of Science and a decrease in the kynurenine/tryptophan ratio. Technology. NSCLC responders and non- responders to pembrolizumab ► Approximately 5×105 Lewis lung cancer (LLC) cells and exhibited distinct gut microbiota diversity as detected by 16S 5×104 B16- F10 cells were subcutaneously inoculated into the PacBio SMRT sequencing, and two differentially abundant right flanks of mice. A total of 24 mice from different litters species, Parabacteroides distasonis and Bacteroides vulgatus were equally divided into four groups: Vehicle (treated with were found. PBS), αPD-1 monoclonal antibody (mAb) (250 µg/mouse, clone: ► Combination therapy with GPs and αPD-1 mAb reshaped RMP1-14, Bio X Cell), GPs (200 mg/kg) and GPs plus αPD-1 the composition of the gut microbiota from non- responders mAb group. Daily oral treatment with GPs was administrated towards that of the responders, which reinstated the after tumour inoculation and injection of αPD-1 mAb 5 times response to the αPD-1 mAb in mice transplanted with PD-1 at 3- day intervals on day 9 when tumour volumes were approx- non- responder faecal samples. imately 50 mm3. Tumour volumes and body weights were How might it impact on clinical practice in the foreseeable measured every 3 days. future? ► Gut microbiota status, such as alpha diversity, can be Antibodies and flow cytometry used as a biomarker to predict the response to anti-PD-1 At the endpoint of the experiment, the blood, spleen and tumours immunotherapy in NSCLC patients. were harvested for flow cytometry analysis. Lamina propria ► GPs represent a novel class of prebiotics to enhance the mononuclear cells (LPMCs) were isolated using a LP dissociation response to anti-PD-1 immunotherapy in NSCLC patients. kit (Miltenyi Biotec, Germany) according to the manufacturer’s ► P. distasonis and B. vulgatus can be used as adjuvants for instructions. Initially, the intraepithelial lymphocytes (IELs) were anti- PD-1 immunotherapy. disrupted from the mucosa by shaking the tissue in a prediges- tion solution. Then, the LP tissue was further treated enzymat- ically and mechanically dissociated into a single- cell suspension containing LPMCs by using a gentle MACS dissociator. or drugs with immunomodulatory effects which may enhance 10 11 For FACS analysis, single-cell suspensions were stained with antitumour response. Recently, gut microbiota has fuelled http://gut.bmj.com/ the following antibodies: PerCP anti-mouse CD45, APC anti- great enthusiasm in cancer immunotherapy. Such as Bacteroides mouse CD3, FITC anti- mouse CD4, and PE/Cy7 anti-mouse fragilis, Bacteroides thetaiotaomicron, Bifidobacterium, Akker- CD8 (Biolegend, clone 30- F11; clone 17A2, clone RM4-5, and mansia muciniphila and Faecalibacterium spp. have been shown clone 53–6.7, respectively, USA). For intracellular staining, cells to have favourable responses to cancer immunotherapy in both were stimulated for 4–6 hour at 37°C with PMA (50 ng/mL), preclinical tumour models and patients with cancer.12–15 Strat- ionomycin (1 µg/mL) and BD Golgi STOPT. After being fixed egies to modulate gut microbiota have thus been proposed to and permeabilised, the cells were stained with APC/Cy7 anti- on October 1, 2021 by guest. Protected copyright. treat patients with cancer and act as novel response prediction mouse interferon (IFN)-γ, PE/Dazzle 594 tumour necrosis factor biomarkers. (TNF)-α, PE antimouse granzyme B (GZMB), PE antimouse Panax ginseng has been widely used in Asia for thousands FoxP3, PerCP/Cy5.5 antimouse ROR-γt and PE anti- mouse inter- of years, not only as a medicine but also as a dietary supple- leukin-17A (Biolegend, clone XMG1.2, clone 506346, clone: ment. The long- term administration of ginseng extracts has 259D, clone MF-14, clone Q31-378, and clone TC11- 18H10.1, been shown to modulate the rat gut microbiota by increasing respectively, USA).