Harnessing a therapeutic microbiome as an alleviating strategy for dysbiosis and recurrence in colorectal cancer patients undergoing radiotherapy

Wouters Shari

Promoter

Kumar-Singh Samir, (UA), Samir.Kumar-Singh@uantwerpen.be

SCK•CEN Mentor

Ahmed Mohamed Mysara
mohamed.mysara.ahmed@sckcen.be
+32 14 33 28 36

SCK•CEN Co-mentor

Verslegers Mieke
mieke.verslegers@sckcen.be
+32 14 33 28 67

Expert group

Microbiology

PhD started

2018-10-01

Short project description

Recent data suggest a strong association between the gut microbiome and the development of colorectal cancer (CRC), one of the world’s most widely identified malignancies (Jobin, 2013). A significant shift in composition was reported when comparing the gut microbiome of CRC patients with healthy individuals, a phenomenon commonly referred to as dysbiosis. Particularly, the presence of certain types of bacteria such as Bacteroides fragilis and Escherichia coli  have been associated with an increased risk for the development of CRC (Wu et al., 2009; Eaton and Yang, 2015), while Lachnospiraceae species seem to have an antitumor effect in the colon by producing metabolites such as short-chain fatty acid butyrate promoting apoptosis of colonic cancer cells (Hague et al., 1995; Ruemmele et al., 2003). Various observations suggest that microbial dysbiosis leads to loss of protective and the enrichment of cancer-promoting populations (Jobin, 2013; Zackular et al., 2013).


Radiotherapy, plays a vital adjunctive role in the management of CRC recurrence (that range from 20 to 30% after curative surgery). However, the usage of radiotherapy has been known to cause mucositis and radiation induced ulceration which expose the underlying tissues to bacteria, thereby changing the host–microbial interaction and as such driving substantial changes in the gut microbiome leading to intestinal dysbiosis (Meyers, Sasson and Sigurdson, 2003; Fuzun et al., 2004; Bowne et al., 2005).  This dysbiosis is often characterised by symptoms such as diarrhoea, rectal bleeding and malabsorption which is accompanied by epithelial destruction and mucosal ulceration, that could also have prognostic values for tumour recurrence.


Fecal microbial transplantation (FMT) re-establishes the balance of nature within the gut microbiome. This 1950 old remedy,(Eiseman et al., 1958), is nowadays mainly used for the recurrent Clostridium difficile infection (CDI) with an average 91% success rate (Bakken et al., 2011). Additionally, FMT was able to cure the irritable bowel disease and showed remarkable improvement in 52% of patients (Borody et al., 1989). Although antibiotics and probiotics have been available for a long time, the exact agent or combination of agents within FMT that affect the cure is unknown and it is likely that a structured network of microorganisms is responsible for the efficiency (Bakken et al., 2011; Allegretti and Hamilton, 2014). It is for these reasons that FMT was only recently re-evaluated in several trials (www.clinicaltrials.gov) and few companies have recently been established (OpenBiome /USA and NDFB/Netherlands) to prepare FMTs and investigate additional applications.


The application of FMT to cancer patient is a novel niche, where only a few publications are available. In 2015, FMT was applied to resolve the CDI in a single cancer patient (Blackburn et al., 2015) and a more recent publication studied the relationship between gut microbiome and radio-sensitivity –in mice, in which they reported a protective characteristic of FMT when exposed to lethal radiation doses (Cui et al., 2017). Yet the application of FMT in CRC mouse model has never been tested before which highlights the relevance of this proposal.

 


 

Objective

It is well known that radiotherapy (and surgical intervention) will cause disturbance of the gut flora in CRC patients. However, what is lacking and of tremendous importance is whether this dysbiosis will be towards a cancer promoting or protective populations and it association to the CRC recurrence. We hypothesis that these interventions will result in a disrupted microbiome (dysbiosis) harbouring cancer promoting microbial communities that plays a role in the recurrence of CRC. This hypothesis will be investigated in patients as well as animal models. We believe that a longitudinal study combining clinical samples and animal studies would give us insight on these questions. 
In addition, we also intend to investigate the application of the fecal microbiota transplantation (FMT) on the treatment of CRC patients. We hypothesis that FMT is able to resolve radiotherapy induced dysbiosis and protect against CRC recurrence. A Longitudinal study involving mouse models together with the clinical samples will be used. This will give insight on the protective characteristics of fecal microbial transplantation and will provide a comprehensive understanding of a core gut microbiota that will protect colorectal patients undergoing radiotherapy (from recurrence and dysbiosis). The specific aims are enumerated below:


Aim 1.1. To identify temporal shift patterns in the gut microbiome of hospitalized and recurrent CRC patients undergoing radiotherapy and surgical interventions (WP1.1).


This will be evaluated via 16S rRNA metagenomics performed on CRC patients samples obtained from Antwerp University. In a clinical setting, CRC patients (undergoing curative operation combined with radiotherapy) will be included and samples will be collected pre/post operation and pre/post irradiation. Patients will be monitored 12 months post intervention (and samples will be collected in case of recurrence). These samples will be compared to healthy individuals (from the Flemish microbiome project and healthy patients sample collected by UA). 


Aim 1.2. To validate the dysbiosis in the gut microbiome in irradiated AOM/DSS mouse models and investigate its relation to the recurrence of CRC (WP1.2).


The azoxymethane and dextran sulfate sodium (AOM/DSS) mouse model is a well-established model of colitis-associated CRC in wild type mice that recapitulates the progression from chronic inflammation to dysplasia and adenocarcinoma in humans (Fazio et al., 2011; Thaker et al., 2012) and often used to study the microbiome of CRC (Zackular et al., 2013; Baxter et al., 2014). These AOM/DSS mice will be subjected to pelvic irradiation where the microbiome will be assessed (compared to the pre-cancerous/pre-irradiation communities) compared to the non-irradiated controls of AOM/DSS mice. Another setting will be applied to assess the recurrence, where the induction of the cancer will be assessed post-irradiation and sizes and number of the polyps (as well as the microbial communities) will be compared between those mice and non-irradiated controls. Unlike the clinical setting, the pre-cancerous samples will be available and the individual differences (in food, genetics and habits) will be controlled in the mouse model (emphasising the need for the animal model). 


Aim 2. To investigate the fecal microbiota transplantation (FMT) ability to resolve radiation induced dysbiosis and protect against CRC recurrence in AOM/DSS mice (WP2.1)


A Longitudinal study involving mouse models together with the clinical samples will be used. To assess the ability of the FMT to resolve radiation induced dysbiosis, FMT will be inoculated to a group of AOM/DSS mice. Samples will be collected during FMT and pre/post irradiation, and afterwards analysed using 16S rRNA metagenomics and compared to the non-transplanted controls. While for the recurrence, irradiated mice will be inoculated with FMT (compared to a control) and CRC will be chemically induced. Both the number and sizes of the number of the polyps (histologically) as well as the microbial community will be assessed using 16S metagenomics.