Insights into the role of the intestinal microbiota in colon cancer

Abstract

The intestinal microbiota consists of a dynamic organization of bacteria, viruses, archaea, and fungal species essential for maintaining gut homeostasis and protecting the host against pathogenic invasion. When dysregulated, the intestinal microbiota can contribute to colorectal cancer development. Though the microbiota is multifaceted in its ability to induce colorectal cancer, this review will focus on the capability of the microbiota to induce colorectal cancer through the modulation of immune function and the production of microbial-derived metabolites. We will also explore an experimental technique that is revolutionizing intestinal research. By elucidating the interactions of microbial species with epithelial tissue, and allowing for drug screening of patients with colorectal cancers, organoid development is a novel culturing technique that is innovating intestinal research. As a cancer that remains one of the leading causes of cancer-related deaths worldwide, it is imperative that scientific findings are translated into the creation of effective therapeutics to treat colorectal cancer.

Introduction

Over the past decade, major insights have been gained into the role of the intestinal microbiota in colorectal cancer (CRC) development. As the fourth leading cause of cancer related deaths worldwide, the importance of understanding which factors lead to the development of CRC is becoming increasingly evident [Maynard et al. 2014; Brody, 2015]. Causative relationships have been drawn between the presence of certain microbes and the development of cancer. Though each relationship proposed remains complex and highly dependent on the site of cancer development, today, approximately 20% of human malignancies are thought to be influenced by microorganisms [de Martel et al. 2012]. Carcinogenic microbes colonize a large portion of the human population and yet only a small subset of such individuals will develop cancer. For this reason, host genetics, dietary composition, and inflammatory dysregulation are crucial factors in understanding the relationship between microbes and CRC development.

The transformation of a normal intestinal epithelium to one harboring intestinal adenomas or ultimately, intestinal carcinomas, occurs through the acquisition of a multitude of mutations in tumor suppressors and oncogenes [Kraus and Arber, 2009; Stegeman et al. 2013]. These mutations typically begin in intestinal stem cells and can be developed over the course of one’s life [Barker et al. 2009]. It is known that CRC is commonly initiated by mutations in the tumor suppressor gene Adenomatous Polyposis Coli (APC) [Senda et al. 2007]. Mutations in APC cause the formation of benign adenomas and are present in over 80% of CRCs [Davies et al. 2014; Senda et al. 2007; Ghazvini et al. 2013; Seton-Rogers, 2015]. The multidomain APC protein, encoded by the APC gene, plays a vital role in maintaining homeostasis of the gut epithelium and thus, when mutated, can impart functional consequences capable of driving cancerous cell proliferation [Senda et al. 2007; Fatehullah et al. 2013; Bakker et al. 2012]. One of APC’s prominent cellular functions is as a negative regulator of the Wnt/β-catenin signaling pathway and thus inactivation of APC leads to stimulation of the Wnt/β-catenin signaling pathway [Seton-Rogers, 2015]. After activation of this pathway, β-catenin is able to translocate to the nucleus and enable the unregulated proliferation responsible for initiating polyp formation [Bardhan and Liu, 2013]. Microbes are hypothesized to play a role in dysregulating the Wnt/β-catenin signaling pathway as a means of tumor initiation [Garrett, 2015]. After the establishment of an APC mutation, the mutational activation of the EGF receptor (EGFR) signaling pathway is thought to play an important role in CRC progression [Drost et al. 2015]. Thereafter, is a sequential cascade of accumulated mutations in genes such as KRAS, PTEN and TP53 [Davies et al. 2014]. Overall, it is clear that CRC has an underlying genetic basis, however, given the unique composition of the intestinal microenvironment it is likely that other environmental factors can also play a role in CRC initiation and progression.

Microbes can play a key role in dysregulating the immune system and altering host metabolism to allow for CRC development. In this review, we will explore the recent literature published on the role of microbes in inflammation-associated CRC as well as CRC caused by microbial-derived metabolites. We will also introduce an experimental technique capable of modeling the interaction of microbes with epithelial tissue and how this technique is being used to revolutionize drug screening for human CRCs.

The intestinal microbiota in times of health and disease

The intestinal microenvironment consists of an intricate organization of trillions of bacteria responsible for modulating host metabolism and immunity [Bevins and Salzman, 2011; Sekirov and Finlay, 2009; Ahn et al. 2013]. The amount of bacterial cells within the human body has proven challenging to quantify. While it was once estimated that microbial cells outnumber the total number of human cells by 10-fold, more recent estimates hypothesize the number of bacteria within the human body to be on the same order of magnitude as human cells [Bevins and Salzman, 2011; Sender et al. 2016]. The intestinal microbiota offers nutrition, host defense, and immune development benefits to the host [Bevins and Salzman, 2011]. One’s microbial composition is also sensitive to alterations in factors specific to the intestinal microenvironment such as pH, presence of oxygen, mucosal integrity, and nutrient availability [Flint et al. 2012].

During pregnancy and in the early months of life, maternal microbes are essential for the establishment of a child’s microbiota. The birthing process constitutes the first major environmental exposure newborns have to microbes. It is known that newborns delivered vaginally are highly enriched with Lactobacillus, while a Cesarean delivery enriches newborns with Staphylococcus and Propionibacterium [Tamburini et al. 2016]. The transition from newborn to infant to adult provides a lot of opportunities for an individual’s environment to shape the composition of their microbiota (Figure 1). Though quite variable in the early months of human life, an adult microbiota composition develops at the end of the first year of life [Bevins and Salzman, 2011; Blaser and Falkow, 2009; Blaser, 2014]. In healthy adults, Gram-negative Bacteroides and Gram-positive Firmicutes are the prominent resident bacterial populations, while Proteobacteria, Verrucomicrobia, Tenericutes, Deffibacteres and Fusobacteria are lower abundance phyla [Bevins and Salzman, 2011; Irrazabal et al. 2014]. When there is a disturbance that shifts the composition of the normal microbial community, the once prominent obligate anaerobic bacteria are replaced by an increased presence of facultative anaerobic bacteria. This dysbiotic shift can cause an increase of potentially harmful microorganisms capable of inducing inflammatory disorders and CRC [Irrazabal et al. 2014; Ahn et al. 2013]. Zackular and colleagues observed a relationship between bacterial community structure and colon tumorigenesis [Zackular et al. 2015]. Using an azoxymethane (AOM)/dextran sodium sulfate (DSS) model of intestinal tumorigenesis in C57BL/6 mice, the authors treated experimental mice with antibiotics before and after the initial DSS treatment. This initial DSS treatment corresponds to the period during AOM/DSS treatment when inflammatory responses are at their highest. When vancomycin, metronidazole and streptomycin were administered before the first DSS treatment, no improvement in tumor burden was observed. If vancomycin, metronidazole, and streptomycin were administered to the mice after the initial DSS treatment, a significant reduction in tumor burden was observed [Zackular et al. 2015]. They hypothesized that the reduction in tumor burden observed in this study was due to the initial relative abundance of both the genus Lactobacillus and the family Enterobacteriaceae. By studying the bacterial populations within the gut in times of health and disease, we gain important insight as to the possible role of the microbiota in disease development.

Figure 1.

Factors shaping the adult microbiome. Maternal transfer of microbiota to infant can take place while in the placenta, during the birthing process, and through breastfeeding. Whether a mother has a natural birth or a Cesarean delivery, the newborn receives a different bacterial inoculum. Over the course of a child’s life, antibiotic use, dietary composition, and environmental exposures can influence the composition of one’s microbiota.

Major insights have also been gained in recognizing the role of diet in shaping microbial composition. In 2013, Cox and Blaser shed light on the importance of several pathways involved in microbe-induced obesity. Highlighted in this review were the capabilities of microbes to contribute to obesity by increasing energy harvest, altering metabolic signaling, and altering immune inflammation. Interestingly, Cox and Blaser were also able to infer the consequences of a high-fat diet to be associated with the selection for obesogenic microbes: a decrease in Bacteroides and increase in Firmicutes [Cox and Blaser, 2013]. Xu and colleagues examined the role of three dietary interventions (control, high-fat diet, and energy-restricted diet) on the colonic microbiota composition and aberrant crypt foci formation in female C57BL/6N mice [Xu et al. 2016]. All mice were following the AOM-induced colon-tumorigenesis model and were given dietary intervention during the initiation phase of tumorigenesis and during the progression phase. Xu and colleagues observed that mice on the energy-restriction diet during the progression phase had increased Firmicutes and reduced Bacteroides compared with high-fat-diet-consuming mice, irrespective of their diet during the initiation phase. The gut microbiota changes observed in mice receiving dietary intervention only during the initiation phase were not consistently maintained after a change in diet during the progression phase. High-fat diet led to alterations in the microbial abundance and persisted into adulthood. High-fat diet in early life was associated with significantly reduced Bacteroidetes regardless of dietary changes during adulthood. When it came to aberrant crypt foci development, energy restriction during the initiation phase of AOM-induced carcinogenesis significantly increased the amount of aberrant crypt foci in mice on the high-fat diet during the progression phase. These findings shed light on the possible joint role of early life and adult dietary changes on microbiota composition and colon carcinogenesis. Chronic consumption of alcohol has also been suggested as a dietary contribution with the capability of altering the human gut microbiota and increasing one’s chance of developing colorectal cancer [Tsuruya et al. 2016]. Tsuruya and colleagues observed chronic alcoholics to have a reduction in the relative abundance of the obligate anaerobic phylum Bacteroidetes in their gut microbiota and an increased population of the facultative anaerobic genus Streptococcus [Tsuruya et al. 2016]. Overall, many types of dietary changes may play a pivotal role in creating a gut microbiota conducive to colorectal cancer development.

Passenger microbes can be pathogenic in colorectal cancer development

It is clear that individuals with CRC have an altered population of gut microbes compared with healthy individuals. However, whether microbes play a direct or indirect role in CRC development has been a topic of great discussion. In 2012, Tjalsma and colleagues came up with a model for CRC development that highlighted the role of some bacteria as drivers and others as passengers in CRC development [Tjalsma et al. 2012]. Under this classification, bacteria with procarcinogenic capabilities were hypothesized to either cause chronic low-grade inflammation or produce DNA-damaging compounds capable of initiating early CRC events. Shigella, Citrobacter, and Salmonella have all been found to be present in higher proportions at early stages of CRC than in a healthy host, and they disappear as CRC progresses [Dupont, 2009]. While pathogenic-driver bacteria are easily able to colonize the healthy intestinal epithelium, opportunistic passenger bacteria replace driver bacteria in the tumor microenvironment [Tjalsma et al. 2012]. Passenger bacteria, such as Fusobacterium ssp., and members of the Streptococcaceae family, are not present at the time of CRC initiation but rather can take advantage of colonic barrier permeability and tumor microenvironment changes to thrive [Lazarovitch et al. 2013; Marchesi et al. 2011]. Since passenger bacteria enrich colon adenoma and carcinomas, it is particularly relevant to understand whether the presence of these microbes is a consequence of tumorigenesis or rather an active contributor to tumor progression. In 2013, Zackular and colleagues addressed this question by inoculating germ-free mice with the microbiota from tumor-bearing mice and observed that disease could be transferred into germ-free mice [Zackular et al. 2013]. An AOM/DSS model of colitis-associated carcinogenesis was used in this study. In agreement with this work, Kostic and colleagues explored the relationship of an opportunistic passenger Fusobacterium, with colorectal cancer development, and found that when Fusobacterium was administered into Apc^Min/+ mice, accelerated colonic tumorigenesis was observed [Kostic et al. 2013]. Taken together, the work of both Zackular and colleagues and Kostic and colleagues shed light on the possible role of passenger bacteria in colonic pathogenesis. Such research suggests that altering one’s microbial composition may prove to be an effective strategy for combating the development of certain colorectal cancers.

Microbes and inflammation-associated colorectal cancers

Intestinal epithelial cells (IECs) play a crucial role in the maintenance of mucosal homeostasis. Once there is a breach in the intestinal epithelial barrier, innate immune cells are the first responders to the site of injury (Figure 2). The initial inflammatory response is characterized by the activation of the nuclear factor kappa β (NF-kβ), MAPK/ERK, and STAT3 pathways [Grivennikov, 2013; Saleh and Trinchieri, 2011]. The genes within the NF-kβ family of transcription factors are responsible for the regulation of inflammation and apoptosis while the MAP/ERK pathway induces proliferation [Kraus and Arber, 2009; Irrazabal et al. 2014]. Chronic inflammation of the mucosal tissue has the capacity to induce genetic mutations, promote angiogenesis, stimulate proliferation, and inhibit apoptosis [Hyun et al. 2015]. A correlation has been made between persons suffering from chronic inflammation, in the form of inflammatory bowel disease (IBD), and the promotion of CRC [Kraus and Arber, 2009]. Though the role of immune dysregulation in CRC development is complex, an upregulation of inflammatory cells has been found at the site of many CRCs. These inflammatory cells can harbor adverse adaptive and innate responses and result in the overexpression of cytokines and chemokines capable of inducing cancer.

Figure 2.

The role of dysbiosis and immune dysfunction in colon carcinogenesis. (1) The intact intestinal epithelium. (2) Microbial dysbiosis, breach of epithelial barrier, and bacterial translocation. (3) Activation of inflammatory pathways associated with immune dysregulation and inflammation-associated colorectal cancer development.

There have been many seminal studies published in mouse models over the past decade linking the innate immune system with microbial dysregulation and the development of CRC. In both Apc^min/+ spontaneous and carcinogen-induced models of intestinal tumorigenesis, Rakoff-Nahoum and Medzhitov showed that activation of the myeloid differentiation factor 88 (MyD88), an adaptor protein for the microbial sensing Toll-like receptor (TLR) and IL-1/IL-18 signaling pathways, can promote intestinal tumor development in the Apc^min/+ mouse model [Rakoff-Nahoum and Medzhitov, 2007]. Using another model of colon carcinogenesis, Salcedo and colleagues noticed that the Myd88^-/- genotype is protective for polyp formation in the AOM/DSS model of colitis-associated carcinogenesis [Salcedo et al. 2010]. These results suggest that Myd88 may be promoting tumorigenesis through its role in the IL-1/IL-18 signaling pathway. Repressed IL-1/IL-18 signaling, in the absence of Myd88 may be unable to control microbial composition. Given that mice lacking caspase-1 signaling, an essential signal in the production of IL-1 and IL-18, have an increased susceptibility for polyp development [Salcedo et al. 2010], one can speculate that inflammasome signaling through caspase-1 and the production of IL-1/IL-18 are essential for host defense against inflammation-driven CRC. Salcedo and colleagues further explored this hypothesis by treating IL18^-/- and IL18R^-/- mice with AOM/DSS and found these mice to be more susceptible to polyp formation than wild-type (WT) mice [Salcedo et al. 2010]. This research suggests that in inflammation-driven CRC, activation of the Myd88 pathway contributes to carcinogenesis by signaling through the IL-18 receptor. Chen and colleagues attributed a protective effect to Nod1, a NLR family member, in AOM/DSS-induced chemical carcinogenesis [Chen et al. 2008]. Nod1 is an innate immune and epithelial cell receptor known to activate through the NFKβ signaling pathway [Athman and Philpott, 2004]. Chen and colleagues found that Nod1^-/- mice develop significantly more tumors than WT mice and that treatment of Nod1^-/- mice with antibiotics before AOM/DSS treatment suppressed intestinal tumor formation [Chen et al. 2008]. Couturier-Maillard and colleagues similarly observed that a loss of Nod2 combined with treatment in the AOM/DSS model of colitis-associated carcinogenesis resulted in an upregulation of inflammatory cytokines and an increase in intestinal epithelial dysplasia [Couturier-Maillard et al. 2013]. This observation was hypothesized as caused by the microbial dysbiosis seen after loss of Nod2.

The innate immune system may be the first line of host defense against pathogenic microbes but it is by no means the only immune response. Several research groups have begun to elucidate the activation of adaptive immune cells in tumor development once microbes have breached mucosal barriers. Rag2^-/- mice orally inoculated with Helicobacter hepaticus cannot control microbe-driven inflammation and have increased intestinal tumors when compared with WT controls [Erdman et al. 2003]. This suggests that lymphocytes play an important role in controlling microbially induced CRC. Wu and colleagues found that enterotoxigenic Bacteriodes fragilis (ETBF), which secretes B. fragilis toxin, was capable of inducing tumors in Apc^min/+ mice [Wu et al. 2009]. The hypothesized mechanism of action was through STAT3-mediated Th17 response, since blocking IL-17 and IL-23, cytokines known to amplify the Th17 response, led to reduced colonic dysplasia and tumor formation [Wu et al. 2009]. They later found that ETBF was, in fact, limiting the availability of IL-2 to uncommitted T cells by allowing Th17 polarization and promoting colonic neoplasia [Geis et al. 2015]. Segmented filamentous bacteria (SFB), which are thought to be related to type 1 Clostridia, have been shown to promote inflammation within the gut by inducing T-helper 17 cells to protect murine hosts from enteric infections [Schnupf et al. 2013; Wu et al. 2009]. Though this first line immune response to protect the host from SFB is essential, the prolonged presence of these bacteria is associated with increased risk of autoimmune disease in mice [Schnupf et al. 2013]. The gut microbiota has been shown to not only influence the homeostasis of the immune system but to also alter T-cell populations within the lamina propria. IL-10 is an anti-inflammatory cytokine broadly expressed by cells of the adaptive immune system, whose absence has been associated with the onset of inflammation-induced CRCs [Nagamine et al. 2008; Saraiva and O’Garra, 2010]. Arthur and colleagues found a 100-fold increase in Escherichia coli NC101 in the lumen of IL-10^-/- mice relative to WT controls [Arthur et al. 2012]. E. coli NC101 harbors the genotoxic polyketide synthase (pks) island that encodes Colibactin, and administration of these bacteria to AOM-treated germ-free IL-10 mice led to a significant increase in tumor development. A follow-up study showed that inflammation was controlling specific genes present in the tumour-promoting E. coli pks island [Arthur et al. 2014]. Overall, the recent literature suggests that the adaptive immune system plays an important role in CRC initiation.

Microbial-derived metabolites and colorectal cancer

Short chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are normal products of bacterial fermentation in the colon. Butyrate, a metabolite known to be produced by members of the phylum Firmicutes, has become a metabolite of great significance in the colonic microenvironment. Asides from its function in the epigenetic control of gene expression and its ability to maintain intestinal barrier integrity, butyrate has a controversial role in CRC development [Belcheva et al. 2015]. Using experimental rodent models and case-controlled human studies, diets rich in fiber have been shown to be protective in the development of CRC [Bandera et al. 2007]. Despite the plethora of experimental data describing butyrate in cancer prevention, published literature remains inconclusive on the role of butyrate in colon cancer versus healthy tissue. Butyrate has been shown to stimulate proliferation in normal colonic epithelial cells [Donohoe et al. 2012]. Butyrate’s capability to function in this manner raises concerns of its role in interacting with certain genetic backgrounds to influence CRC development. Mandir and colleagues found that a diet rich in resistant carbohydrates when administered to Apc^min/+ mice significantly increased polyp development in the colon [Mandir et al. 2008]. Building on the previous work of Mandir and colleagues in the Apc^min/+ mouse model, butyrate was also shown to stimulate proliferation in Msh2-deficient colon epithelial cells [Belcheva et al. 2014]. A reduction in the levels of butyrate in colon, either through the administration of a low-carbohydrate diet or the administration of antibiotics, reduced polyp formation in the Apc^min/+Msh2^-/- mouse model. Thus, it is not only important to consider the type of bacterially derived metabolite produced in the colon, but also how this metabolite is interacting with host genetics.

The bacterial-derived metabolite uracil has been linked to the production of reactive oxygen species (ROS) in the gut [Lee et al. 2015]. Reactive oxygen species are known to affect tissues by initiating genetic mutations capable of promoting proliferation or inducing apoptosis. By exploring the intestines in drosophila, Lee and colleagues observed that uracil induced dual oxidase (DUOX)-dependent ROS production acted as an innate immune mechanism to defend against opportunistic pathogenic bacteria. Dual oxidase 2 (DUOX2), a hydrogen peroxide generator located on the intestinal epithelial cell membrane, is the main hydrogen peroxide generator in the intestinal epithelium [Sommer and Bäckhed, 2014; Grasberger et al. 2015; Lee et al. 2015; Roy et al. 2015]. In mice, DUOX2 regulates the interaction between the intestinal microbiota and mucosa, in an effort to maintain immune homeostasis [Grasberger et al. 2015]. Though this research begins to raise questions about the role of bacterial-derived metabolites in reactive oxygen species’ production and immune modulation, further research is needed to explore the implications for CRC development.

Studying the structure and function of the intestine with organoids

Organoid cultures represent a fascinating new tool for studying the influence of genetic and environmental factors on organ development. Unlike immortalized cancer cell lines, organoids are able to maintain a near-physiological environment for epithelial cells [Kohan et al. 2011; Dedhia et al. 2016]. First described by Sato and colleagues in 2009, organoids are 3D tissue-like structures capable of mimicking epithelial development and organization [Sato and Clevers, 2015]. Though initially created using small intestinal crypts and termed ‘mini-guts’, organoids have since been derived from the stomach, colon, brain and prostate [Kohan et al. 2011; Wang et al. 2014]. Within the gastrointestinal epithelium, Lgr5+ cells within the crypt are used to initiate the organoid culture. Initiator cells, such as the Lgr5+ cells, initially form symmetric cyst-like structures [Sato and Clevers, 2015; Sato et al. 2011; Hubert et al. 2016]. During the initial week of culture, these cyst-like structures begin to generate new crypt protrusions. During the first few weeks of organoid culture, once cyst-like organoid rich in stem cells differentiate into flower-like structures comprising all of the differentiating cells of the intestinal epithelium [Sato and Clevers, 2015; Cao et al. 2015]. Though microbes from the colonic tissue where the stem cells have been harvested from are not typically cocultured with organoids, lasting effects from the presence of certain metabolites in the epithelium can still be apparent during organoid culture. If dietary composition has imposed lasting changes to the structure and function of stem cells, organoid development will also be affected by this imprint. Beyaz and colleagues shed light on the impact of dietary composition on stem-cell function by observing that high-fat diet increased Lgr5+ stem-cell number and self-renewal capacity [Beyaz et al. 2016]. Organoids derived from mice on a high-fat diet were found to give rise to more secondary organoids than controls [Beyaz et al. 2016]. The ability of stem cells to self-renew and give rise to functional, differentiated epithelial cell types makes them superbly suited for the study of how fundamental changes in epithelial structure may induce long-lasting structural and functional effects [Barker, 2014]. Highlighting their importance within the epithelium, it comes as no surprise that stem cells can accumulate mutations capable of giving rise to cancerous growth [Barker, 2014]. Engineering organoids has become an important tool for cancer assessment. By interfering with gene expression with either short hairpin RNA knockdown or CRISPR/Cas9-mediated genome editing, the importance of specific genes in the development of the normal epithelium can be assessed [Drost et al. 2015]. Genetic alterations of organoids, through adenovirus-mediated efficient gene transfer, has also been successfully performed and maintained for up to 10 days in organoid culture [Wang et al. 2014]. Organoids derived from Apc-deficient murine cell lines have described the development of a cyst-like organoid structure, as opposed to a conventional ‘flower’-like structure with many new crypt protrusions [Cao et al. 2015]. Organoids genetically engineered to harness other cancer-promoting mutations have similarly been described [Saito et al. 2016; Schwank et al. 2013; Salahudeen and Kuo, 2015; Phillips, 2014; Dedhia et al. 2016; Patman, 2015]. From observing how intestinal stem cells differentiate, to elucidating the role of mutated genes in disease progression, the study of organoid development and growth can reveal novel insight into the function of stem cells within the context of the intestinal epithelium.

Using organoids to model microbiome–host interactions

Organoids cultured in the presence of microbes allow for in-depth assessment of microbe–host relationships. Farin and colleagues treated small intestinal organoids with microbial antigens and bacteria to assess how degranulation of Paneth cell function was affected [Farin et al. 2014]. They introduced microbial constituents to the organoid culture media to allow for baso-lateral exposure or sheered the organoids to allow for luminal exposure. Regardless of the exposure mechanism, Farin and colleagues found that Paneth cell degranulation was not induced by direct stimulation with microbial antigen or bacterial exposure [Farin et al. 2014]. Rather, this paper attributed a key function in the proinflammatory cytokine interferon γ in Paneth cell degranulation. Zhang and colleagues investigated the effects of Salmonella colonization on murine intestinal organoid function and noticed that the introduction of Salmonella to organoid culture disrupted tight junctions, reduced expression of Lgr5, and activated NFkB signaling [Zhang et al. 2014]. Another study explored the role of Salmonella on intestinal epithelial development and used microinjection technology to expose the lumen of human-induced pluripotent stem-cell-derived organoid to microbes, and observed that Salmonella injected into the lumen resulted in epithelial invasion [Forbester et al. 2015]. Despite the abundant ambient exposure of this organoid culturing system to oxygen, Clostridium difficile, an obligate anaerobe, was observed to survive up to 12 hours within the organoid lumen [Leslie et al. 2015]. In addition, when toxins from C. difficile were purified and administered to organoids, disruption of intestinal barrier function was observed. Therefore, organoids can be grown to better characterize relationships between microbes and the intestinal epithelium.

Applying organoid insights into the development to colorectal-cancer therapeutics

Deriving intestinal organoids can assist researchers in understanding how to treat cancers with complex genetic and environmental etiologies [Dedhia et al. 2016]. Stem cells from tumor biopsies can be embedded in a basement membrane-like matrix and allowed to differentiate as organoids. During the first week of culture, organoids from tumor biopsies can inform researchers of any developmental perturbations affecting stem cells, and other lineages of differentiating epithelial cells [Sato et al. 2011; Koo et al. 2012]. A stunted organoid phenotype is typically the first visual clue that there is an abnormality in epithelial development. Organoids cultured from tumor stem cells typically retain a spherical morphology, while phenotypically normal organoids develop an aspherical morphology with lots of new crypt protrusions [Drost et al. 2015; Saito et al. 2016]. The most important means in which organoids can be used to advance cancer therapeutics is as a first-pass screen for drug sensitivity. Unlike cancerous cell lines harboring genetic mutations, organoid development takes tissue directly from the patient and is able to most closely replicate the physiology and environment of the tumor. Before a cancer therapeutic is given to a patient, it can first be given to organoids derived from the patient’s cancerous tissue. Several groups have begun to create organoid biobanks from human intestinal tumor biopsies to test the efficacy of cancer therapeutics on any given tumor before administering the therapeutic to the patient [Van De Wetering et al. 2015; Dedhia et al. 2016; Sato and Clevers, 2013] (Figure 3). This method allows for personalization of cancer treatments so that both host genetics and individual variability are considered together. In brief, one tissue biopsy from normal intestinal tissue and the other from tumorous tissue is harvested from a patient. Organoids are then cultured in appropriate organoid culture media, and then treated in a high-throughput manner with a panel of possible therapeutic drugs that can be used to treat the patient’s cancer [Van De Wetering et al. 2015]. Based on the sensitivity of the organoids to the drug, physicians can determine an appropriate therapeutic regimen for their patients. The time frame is still in the process of being optimized for this process as currently, organoid biobanking takes 2–3 weeks. Such an organoid culturing and drug-screening platform can greatly reduce the need for experimental trial and error on a human patient. Organoids are changing the face of cancer treatment.

Figure 3.

The multiple uses for organoids in modeling microbe–host interactions and providing personalized cancer therapies. (1) Microbes can be administered either to cell culture media or microinjected into the organoid lumen to closely model microbial activity within the gut. Though most published studies derive mouse organoids, organoids can also be derived from human tissue for experimentation (2) Organoid biobanks represent an innovative and effective way to develop personalized cancer therapeutics. Using healthy and tumorous tissue from patients with cancer, response to therapeutic intervention can be closely predicted.

Conclusion

New insights are rapidly being gained into the role of the microbiome in CRC development. Research suggests a key role for microbes in the development of an inflammatory environment in which cancer cells can thrive. Microbes can also influence the development of cancer by producing metabolites that influence host metabolism. As increased resources are invested into the development of therapeutic treatments for CRC, the importance of understanding the relationship between microbes, stem-cell function, and intestinal development is becoming increasingly evident. Phenotypically monitoring organoid development can revolutionize our understanding of intestinal development and the initiation of disease (Figure 3). Though key steps have been taken in combining organoid development with microbes, further research can be performed to even better mimic in vivo conditions. Such innovations will undoubtedly bring us closer to understanding how to use organoids in modeling cancer development. In pursuit of developing effective, personalized cancer therapeutics, organoid biobanks are well on their way to allowing for fast and efficient results about how tumorous tissue will respond to drug treatment [Brody, 2015; Compte et al. 2013]. There are many drugs that confer great efficacy in a small subset of patients; however, these can be ineffective in other patients. Organoids will continue to serve as a great resource in allowing researchers to create individualized treatments to specific cancers [Van De Wetering et al. 2015]. In conclusion, literature published in recent years shows great promise in allowing us to understand the active role of microbes in CRC development as well as shedding light on the CRC diagnostic techniques of tomorrow.