The abundance and diversity of GMB has been reported to be associated with not only the occurrence but also the prognosis, especially the treatment outcome of many diseases. Our previous study indicated the GMB was significantly changed in patients with DLBCL, which suggested the GMB might play a role in the pathogenesis of DLBCL. However, whether the GMB is associated with the treatment outcome remains to be defined. In this current study, we aimed to investigate the association of GMB with treatment outcome in patients with DLBCL.
We first analyzed the GMB in pretreatment DLBCL patients (PRG) compared with healthy controls (CG), which repeated our previous research with different cohort of patients[4]. In consistent with our previous research, β-diversity analysis showed that there were significant differences in the GMB between UEG and CG. At 6 levels, a continuous evolutionary relationship we observed. More specifically, the abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, Escherichia-Shigella, Escherichia coli was significantly higher in PRG than in CG, which was consistent with our previous research results to further validate that Proteobacteria phylum is the dominant gut microbiota in untreated DLBCL patients[4]。
The prognosis of the 17 DLBCL patients after 4 courses chemotherapy intervention were assessed by PET/CT efficacy evaluation, which classified these patients into the CRG (n = 10) and NCRG (n = 7). β-diversity analysis show that the abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, Escherichia-Shigella, Escherichia coli was lower in posttreatment (POG) than that in pretreatment (PRG) DLBCL patients and Proteobacteria phylum is no longer the dominant gut microbiota in posttreatment DLBCL patients.These results indicated the GMB has been changed considerably following chemotherapy.
β-diversity analysis found that the abundance of Lactobacillaceae (family), Lactobacillus (genus), Lactobacillus fermentum (species) were significantly higher in CRG than NCRG, while there were no significant differences between CR_PRE and NCR_PRE. These results revealed that the abundance of Lactobacillaceae, Lactobacillus, Lactobacillus fermentum in CRG_PRE and NCRG_PRE were at the same baseline before the 4-courses chemotherapy intervention started, and there existed significant differences in CRG and NCRG after chemotherapy in DLBCL patients. The higher abundance of Lactobacillaceae, Lactobacillus, Lactobacillus fermentum in the posttreatment DLBCL patients suggested that these gut microbiota might be associated with the extinction of lymphomas and as dominant gut microbiota in CRG DLBCL patients, Lactobacillus fermentum was likely a succession of gut microbiota caused by chemotherapy intervention and tumor extinction.
DLBCL patients were treated with 4-courses chemotherapy interventions, such as anti-tumor chemotherapeutic drugs and other therapeutic drugs, which may have a direct impact on gut microbiota. Fusobacteria (phylum) was reported to be sensitive to antibiotics[20]. β-diversity analysis found the abundance of Fusobacteria (phylum), found Fusobacteriia (class), Fusobacteriales (order), Fusobacteriaceae (family), Fusobacterium (genus) were significantly lower in POG than in PRG, and were significantly lower than in CG; there were significant differences in α-diversity analysis between POG and CG, which was significantly lower in POG than in CG. Therefore, we may conclude that these changes of gut microbiota could be caused by the direct effect of chemotherapeutic intervention and were not associated with extinction in lymphomas in DLBCL patients. However, the abundance of Proteobacteria in POG decreased significantly and no longer the dominant gut microbiota, which showed no significant difference between POG and CG at the same baseline, suggesting that the changes of Proteobacteria phylum were related to the extinction of lymphomas in DLBCL patients. The abundance of Lactobacillaceae, Lactobacillus, Lactobacillus fermentum CRG_PRE and NCRG_PRE at the same baseline before chemotherapy and were significantly higher in CRG than NCRG and CG after the the same chemotherapy intervention, indicating that they were obviously dominant GMB in CRG. Therefore, the abundance and diversity of Proteobacteria phylum and Lactobacillaceae, Lactobacillus, Lactobacillus fermentum could be completely excluded from the direct effects of drugs, but may directly related to the outcome of chemotherapy in DLBCL patients.
Studies have shown that Escherichia coli can produce colibactn and cytolethal distending toxin that can cause double-strand breaks in intestinal epithelial cells, leading to gene mutations that eventually lead to tumor formation[21]. Therefore, Proteobacteria phylum are likely to play an important role in the occurrence of DLBCL, which is the focus of the in-depth study of the microenvironment and pathogenesis of DLBCL. Therefore, regulating the abundance of gut Proteobacteria phyla may be a novel strategy for the prevention of DLBCL.
Our results from this study showed that the composition of gut microbiota in DLBCL patients had a succession after 4 courses chemotherapy intervention and Proteobacteria phylum was no longer the dominant gut micorbiota which was replaced by Lactobacillus fermentum. The change and succession of these gut microbiota composition can be excluded from the direct effect of therapeutic drugs on microbiota. Lactobacillus fermentum became the dominant microbiota in complete remission patient, which was related to the changes in lymphoid tumor and resulted from the gut microbiota succession caused by chemotherapy intervention and tumor extinction. Recent studies have indicated that signifificant microbiome alterations represented an increase in Lactobacillus during nCRT (neoadjuvant chemoradiotherapy)[22]. Therefore, the product of Lactobacillus fermentum may directly involve in anti-tumor,synergistically with chemoradiotherapy, and enhance the therapeutic effect of chemotherapeutic drugs.
Recent studies have found that some microbial bacteria have shown potential anti-tumor activity, such as Lactobacilli in vitro experiments. Lactobacillus rhamnosus GG can not only suppress the growth of tumor cells but also has anti-metastasis function[23–26]. Bladder cancer patients have been shown to have reduced recurrence of superficial bladder cancer by oral Lactobacillus casei[27] and the underlying mechanism might be related to the direct effect of anti-tumor immune responses induced by the activated host NK cells and macrophages [28]. Lactobacillus casei inhibited colon cancer in two ways: direct stimulation of immune cells and secreting metabolites with anti-tumor effect. Lactobacillus fermentum has the same effect, but its inhibition of colon cancer cells was less intense than Lactobacillus casei[29]。
Studies have shown that the anti-tumor effect of Lactobacilli is achieved by mTOR (molecular target of rapamycin) and Wnt/β-cantenin signaling pathway, which can significantly up-regulate secretory crimp-related protein 2 (SFRP2) and reduce CCND1、mTOR、S6K1、EIF4E expression[30]. Wnt/β-catenin signaling pathway consists of Wnt proteins, transmembrane receptors, cytoplasmic proteins, nuclear transcription factors, and downstream target genes[31]. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of mTOR signaling pathway is an important pathway leading to cell growth and tumor proliferation[32]. By regulating the expression of relevant signal transduction genes in mTOR and Wnt/β-cantenin signaling pathways in tumor cells, Lactobacilli plays an important role in tumor cell growth. And studies have shown that, activation of mTOR complexes can lead to the phosphorylation of various signaling molecules, and thus over-expression of cyclinD1、Bcl2 proteins leads to tumor formation. Therefore, the results from our study showed that the abundance of Lactobacillus fermentum was significantly higher in CRG than that of the NCRG, suggesting that Lactobacillus fermentum may inhibit DLBCL [29]。
PICRUSt was used to predict the microbial function based on the 16S rRNA gene sequencing data, which showed that the Thiamine metabolism function of gut microbiota in PRG and POG was significantly lower than that of CG. High dose Thiamine can reduce pyruvate dehydrogenase kinase activity and play anti-tumor role in xenograft mice[33]. Therefore, it is speculated that the gut microbiota causes the DLBCL micro-environment to remain in the state of low thiamine, which leads to the over-expression of pyruvate dehydrogenase kinase, increasing the glycolytic process of cancer cells leading to tumor growth. Pentose phosphate pathway function was significantly higher in PRG than POG, but there was no significant difference in PRG and CG. Studies have shown that over-expression of enzymes with different oxidation and non-oxidation branches in the pentose phosphate pathway increases cisplatin resistance, and inhibition of pentose phosphate pathway restores cisplatin sensitivity[34]. Therefore, gut microbiota may increase the function of pentose pathway to promote the resistance of tumor cells to chemotherapeutic drugs. Glycine,serine and threonine metabolism function was significantly higher in NCRG than in CRG, and the two groups were at the same baseline before chemotherapy intervention. Studies have shown that glycine and serine are involved in mitochondrial carbon synthesis during cancer development, and serine can also act as a bridge between mTOR signaling pathways and DNA methylation[35]. In melanoma cells with low serine, p53 protein-mediated modulation of glycerate phosphate dehydrogenase (PHGDH) enhances apoptosis[36].