Our investigation in identifying differences in microbiota in human pancreatic model mice cohort feces in comparison to control mice cohort showed several unique bacterial strains that were present in abundance or absent in the fecal microbiome of pancreatic cancer bearing mice. Literature review informed the importance of varying abundance of microbiota in cancer and select bacteria are discussed below.
Lachnoclostridium bolteae was previously named Clostridium bolteae. This microbe is a gram-positive rod and an obligate anaerobe. It is a spore forming organism that resides within the human gut. C. bolteae has flagella which helps allow it to be an opportunistic pathogen. This, as well as several genes providing resistance to ampicillin, erythromycin, lincomycin, ciprofloxacin, and doxycycline make it a problematic constituent of the human microbiome.28 It has been linked to autism as well as pancreatic cancer in human beings.29,30
R. peoriensis is a gram-positive, spore forming, anaerobic bacillus that was characterized in 2003 from a swine manure sample and is an emerging human pathogen.31 It caused bacteremia in a patient with pancreatic cancer. Upon treatment with intravenous metronidazole the patient died of multiple organ failure within a month of being admitted to the hospital.32 In 2022, eight months after a prematurely terminated surgery, a Canadian woman with jejunal adenocarcinoma and endometrial cancer was admitted to hospital and analysis of blood and uterine samples revealed a Clostridium perfringens infection.33 A course of piperacillin-tazobactam, tobramycin and doxycycline was initiated for four days with resolution. Next, she was put on oral amoxicillin-clavulanate for five weeks. Five months later she came to the ER with several severe symptoms and blood samples were positive for R. peoriensis and Clostridium difficile.
Alistipes putredinis is a gram-negative, anaerobic bacterium that is commonly found in the human gastrointestinal tract. Colorectal cancer patients have been demonstrated to be enriched with A. putredinis.34 In IL-10 knockout mice (that also did not make the antimicrobial Lcn2 that prevents bacterial iron siderophores from functioning), Alistipes spp. proliferates in the right side of the murine colon (proximal, cecum) and promote polyp formation. A. putredinis has also been detected in nipple aspirate fluid in women with a history of breast cancer compared to healthy controls.35 Surprisingly, this organism is considered a favorable bacterium for the host when it comes to immunotherapy. Its presence in the gut is associated with successful Anti-PD-L1 therapy.36
Akkermansia muciniphila is a gram-negative, anaerobic organism. A muciniphila is a normal constituent of the mammalian gut microbiome. It is not motile, nor does it form spores. It enjoys the attention of microbiome researchers because it contributes to the turnover of intestinal mucous, tightens gut epithelial cell junctions, and stimulates the immune system. These capabilities make it an attractive probiotic candidate and viable alternatives to Lactobacillus spp. and Bifidobacteria spp.
There are dozens of studies providing evidence that this organism and its products contribute to positive health outcomes, including cancer. Luo et al demonstrated that extracellular vesicles (EV) derived from A. muciniphila could limit tumor size in the murine prostate cancer model.37 This effect may be due to the increase in GZMB+ and IFN- γ+ lymphocytes in tumors. Furthermore, the researchers observed that EV treated mice had more M1 macrophage in the tumor microenvironment. In addition, when THP-1 macrophages were exposed to EVs in vitro they assumed shape alterations and transcription profiles of the M-1 phenotype which potentiates neutralization of tumor cells.
Shi et al obtained tumor samples from patients with colorectal cancer.38 These ex vivo samples were processed and then challenged with A. muciniphila and IL-2. Apoptosis of tumor cells, the ratio of CD8+/CD4+ in CD3+ cells, CD80+CD86 in DD11c+ cells, and IFN-γ+ in CD3+ all were significantly higher than challenge with IL-2 or the microorganism alone. The melanoma and CRC murine models were consequently employed to test the effect of A. muciniphila and IL-2 on tumor volume and survival rate. A. muciniphila and IL-2 treated mice demonstrated statistically significant tumor volume and survival rate.
A. muciniphila has also shown synergy with cisplatin against lung cancer. Investigating the impact of A. muciniphila on Lewis Lung Cancer in mice, Chen et al found that mice treated with cisplatin and A. muciniphila had lower expression of ki67/GAPDH and p53/GAPDH at statistically significant levels compared to mice administered cisplatin only.39 Serum concentration of TNF-α, IL-6, and IFN-γ were also found to be significantly lower in A. muciniphila /cisplatin mice versus mice administered only cisplatin.
In a multi-institutional study, researchers found that in patients with NSCLC, RCC, or urothelial carcinoma being treated with d PD-1/PD-L1 mAb had significantly different outcomes based on if they had also received antibiotics proximal to the first cancer treatment.40 Progression free survival and overall survival were lower in those patients who took a course of antibiotics compared to those who did not. Next, the team found that metagenomic diversity corresponded with clinical outcomes in RCC and NSCLC patients that were newly diagnosed. Upon commencing PD-1 blockade therapy, the patients could be divided into those that responded to therapy and those that were non respondent. A. muciniphila was one bacterium that was at elevated abundance in the responders. Furthermore, germ free and specific pathogen free MCA-205 sarcoma mice given fecal microbiome transplants from responder NSCLC and RCC patients displayed smaller tumor sizes with PD-1 blockade than mice receiving non responder FMT or responder FMT without PD-1 blockade. To link a specific gut microbe to synergy with PD-1 blockade, gut dysbiosis was promoted in RET-melanoma mice. Then oral gavage with A. muciniphila alone or with E. hirae was performed to allow colonization of the gut. Tumor size was lower in mice receiving bacterial gavage relative to control but lowest in mice receiving both bacteria.
A more recent study by the previously mentioned group investigated the effectiveness of PD-1 blockade based on detection of A. muciniphila (Akk) in feces.41 The Akk+ cohort showed a 28% objective response rate compared to 18% in the Akk- cohort. In Akk+ and Akk- patients receiving solely PD-1 blockade, the ORR was 41% and 19% respectively. Transcriptomic analysis of tumor biopsies demonstrated distinct signatures between responders and non-responders to PD-1 blockade. These genes were relevant to activation of CD4+ T helper cells, exhaustion, and interferons.
This group also discovered that Akk+ patients had significantly different abundances of the bacterium. Fecal microbiome proportion ranged from 0.035% to 66.210%. This allowed the establishment of the Akkhigh group (>4.799%) and an Akklow group. Overall survival percentage was significantly higher in the Akklow group (27.2 months) compared to Akk-(15.5 months), and Akkhigh (7.8 months). In those patients receiving antibiotics, the overall survival percentage of Akklow patients was more pronounced (27.2 months) compared to Akk-(15.5 months) and Akkhigh (7.8 months). Akkhigh also showed elevated levels of Lachnoclostridium bolteae after antibiotic exposure.
Interestingly, there are also other pockets of research that show a negative side to A. muciniphila especially in the murine colorectal cancer model.42-44 This could be due to subtle variations in experimental design such as the use of live/pasteurized forms of the bacteria, antibiotic usage, presence of dysbiosis, dosage, or route of administration.45
As its name implies, A. muciniphila is a chemoorganoheterotroph that utilizes every sugar found in mucin for carbon and energy.46 Some cancers are known to result in high levels of mucous secretion (e.g., adenocarcinomas, colorectal, gastric, lung, ovarian, and pancreatic). Thus, the elevated levels of A. muciniphila in the experimental group of our study could be due to the high amounts of mucous in the gastrointestinal tracts of these mice, which selected for the bacterium. This possibility invokes the concept of “driver” versus “passenger” bacteria in oncology studies.47 Oncobionts such as Fusobacterium nucleatum can become enriched in certain tissue where they interact with host cells to promote cancer. Once sufficient momentum has been attained, other bacteria become selected for and proliferate at these sites. Knowledge of bacteria physiology and metabolism must be leveraged in all future oncology studies to form a more accurate model of cancer. Fortunately, more sensitive biotechnological tools as well as the rise of integrated STEM research, and multidisciplinary teams are facilitating this course of action.
Our investigation in analysis of the pancreatic cancer model mice fecal microbiome by 16S rDNA sequencing and phylogenetic analysis resulted in identification of several bacterial strains that were either present in abundance or absent in the feces of cancer microbiome in comparison to the healthy mice. Further studies will be needed for clarifying the significance of these findings and the role of these bacteria in pancreatic cancer.