Probiotic Strain Lactobacillus casei BL23 Prevents Colitis-Associated Colorectal Cancer

The gut microbiota plays a major role in intestinal health, and an imbalance in its composition can lead to chronic gut inflammation and a predisposition to developing colorectal cancer (CRC). Currently, the use of probiotic bacteria represents an emerging alternative to treat and prevent cancer. Moreover, consumption of these beneficial bacteria may also favorably modulate the composition of the gut microbiota, which has been described in several studies to play an important role in CRC carcinogenesis. In this context, the aim of this study was to assess the protective effect of oral treatment with Lactobacillus casei BL23, a probiotic strain well known for its anti-inflammatory and anticancer properties. First, CRC was induced in C57BL6 mice by a single intraperitoneal injection with azoxymethane (8 mg/kg), followed by four courses of dextran sodium sulfate (2.5%) in drinking water that were separated by an adjustable recovery period. At the time of sacrifice (day 46), tumor incidence, histological scores, and epithelial proliferation were determined in colon samples. Our results show that L. casei BL23 significantly protected mice against CRC development; specifically, L. casei BL23 treatment reduced histological scores and proliferative index values. In addition, our analysis revealed that L. casei BL23 had an immunomodulatory effect, mediated through the downregulation of the IL-22 cytokine, and an antiproliferative effect, mediated through the upregulation of caspase-7, caspase-9, and Bik. Finally, L. casei BL23 treatment tended to counterbalance CRC-induced dysbiosis in mice, as demonstrated by an analysis of fecal microbiota. Altogether our results demonstrate the high potential of L. casei BL23 for the development of new, probiotic-based strategies to fight CRC.

The gut microbiota plays a major role in intestinal health, and an imbalance in its composition can lead to chronic gut inflammation and a predisposition to developing colorectal cancer (CRC). Currently, the use of probiotic bacteria represents an emerging alternative to treat and prevent cancer. Moreover, consumption of these beneficial bacteria may also favorably modulate the composition of the gut microbiota, which has been described in several studies to play an important role in CRC carcinogenesis. In this context, the aim of this study was to assess the protective effect of oral treatment with Lactobacillus casei BL23, a probiotic strain well known for its anti-inflammatory and anticancer properties. First, CRC was induced in C57BL6 mice by a single intraperitoneal injection with azoxymethane (8 mg/kg), followed by four courses of dextran sodium sulfate (2.5%) in drinking water that were separated by an adjustable recovery period. At the time of sacrifice (day 46), tumor incidence, histological scores, and epithelial proliferation were determined in colon samples. Our results show that L. casei BL23 significantly protected mice against CRC development; specifically, L. casei BL23 treatment reduced histological scores and proliferative index values. In addition, our analysis revealed that L. casei BL23 had an immunomodulatory effect, mediated through the downregulation of the IL-22 cytokine, and an antiproliferative effect, mediated through the upregulation of caspase-7, caspase-9, and Bik. Finally, L. casei BL23 treatment tended to counterbalance CRC-induced dysbiosis in mice, as demonstrated by an analysis of fecal microbiota. Altogether our results demonstrate the high potential of L. casei BL23 for the development of new, probiotic-based strategies to fight CRC.
Keywords: Lactobacillus casei Bl23, lactic acid bacteria, probiotic, azoxymethane-dextran sodium sulfate, colorectal cancer, immunomodulation inTrODUcTiOn Colorectal cancer (CRC) is a major public health problem and is considered the third most common cancer around the world, with nearly 1.2 million new cases every year and a mortality rate of ~40% (1). The incidence of CRC can be associated with a large number of both genetic (2) and environmental factors (3). In particular, one major risk factor for the development of CRC is chronic intestinal inflammation (4); indeed, patients suffering from inflammatory bowel diseases (IBDs) are six times more likely to develop CRC than healthy individuals (5) Today, the use of probiotics represents a promising strategy for the treatment and prevention of cancer. Probiotics are "live microorganisms, which when administered in adequate amounts confer a health benefit on the host" (6). The most common probiotic strains belong to the genera Bifidobacterium and Lactobacillus. Interestingly, epidemiological studies have shown a lower incidence of CRC in healthy volunteers who regularly consumed fermented dairy products (containing probiotics), especially yogurt (7-10). However, despite encouraging observations of the anticancer effects of probiotics (which have been accumulating for over 30 years), these clues have thus far been poorly investigated, and even today, the mechanisms underlying these beneficial effects are largely unknown. The beneficial role of probiotic bacteria against CRC onset may be explained by three different mechanisms: (i) modulation of the immune response, (ii) induction of cell apoptosis, or (iii) antioxidant activity [reviewed in Ref. (11)]. As a specific example, the food supplement VSL#3 (a mixture of eight probiotic bacteria) has been shown to modulate the immune response and reduce adenoma development in a model of CRC induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) (12). Furthermore, Lactobacillus gasseri and Bifidobacterium longum have been shown to inhibit cellular proliferation and increase phagocytic activity in a model of 1,2-dimethylhydrazine (DMH)-associated CRC and thus reduce the multiplicity of aberrant crypt foci as well as tumor size (13). In addition, the probiotic strain Lactobacillus casei Shirota is able to suppress chemically induced intradermal tumor onset through both an enhancement of the cytotoxicity of natural killer (NK) cells (14) and IL-12 release by dendritic cells (15).
Beyond having cancer fighting effects, the consumption of probiotics may also favorably modulate the composition of the gut microbiota (16). In this context, several studies have confirmed the important role that the gut microbiota plays in CRC carcinogenesis by generating both biochemical and physiological conditions that may increase the number of colonic preneoplastic lesions (17,18). However, despite the well-known role of microbiota dysbiosis in CRC pathogenesis, there are conflicting reports about a specific correlation between the bacterial community composition in the gut and susceptibility to CRC (18)(19)(20). To date, animal studies have revealed that germ-free mice are more predisposed to developing inflammatory-induced CRC than conventional mice (21). Therefore, manipulation of the gut microbiota could be a promising approach to prevent and/or treat CRC.
We have previously reported anti-inflammatory effects of the probiotic strain L. casei BL23 in two different murine models of chemically induced colitis (22)(23)(24). Furthermore, we have recently observed that this strain also displays antitumoral properties in a mouse allograft model of human papilloma virus (HPV)-induced cancer and in a DMH-induced CRC model (25). We attributed this antitumoral effect to a modulation of the immune response and, in particular, to the modulation of regulatory T-cells toward a Th17-biased immune response associated with the expression of regulatory cytokines (IL-6, IL-17, IL-10, and TGF-β) (25). However, the beneficial role of L. casei BL23 in colitis-associated CRC (such as that induced by AOM-DSS) remains to be assessed. Thus, keeping in mind the role of the gut microbiota and chronic intestinal inflammation in CRC carcinogenesis, we decided in this study to further investigate the impact of L. casei BL23 in a mouse model of CRC induced by AOM and DSS. Our results showed that oral administration of L. casei BL23 significantly reduced tumor onset. Histological analyses revealed reduced proliferation in tumor sections, as demonstrated by the upregulation of three genes involved in apoptosis (caspase 7, 9, and Bik) and cytokine IL-22. Illumina sequencing revealed that the gut microbiota of mice treated with L. casei BL23 tended to differ in both community composition and community richness from those of PBS-treated mice. Finally, we provide some clues about the host molecular mechanisms involved in the anticancer effects of this beneficial probiotic bacterium.

Bacterial strains and growth conditions
Lactobacillus casei BL23 (26) was grown in Man, Rogosa, and Sharpe medium at 37°C without agitation overnight. To prepare live bacterial inoculum, strains were washed twice with PBS at 3,000 g and suspended in PBS at a concentration of 5 × 10 9 colony-forming units/ml plus 15% glycerol.

animals and experimental Design
All experiments were handled in accordance with institutional ethical guidelines, and the study was approved by the COMETHEA ethics committee ("Comité d'Ethique en Expérimentation Animale") of the Centre INRA of Jouy-en-Josas and AgroParisTech. Female C57BL/6 mice (6-8 weeks old; Janvier SAS, St. Berthevin, France) were maintained in sterile isolators at the INRA animal facility (n = 5 per cage) with 12 h light cycles and fed irradiated normal chow (R 03-40, SAFE) and water ad libitum.
Temperature and moisture were carefully controlled. Mice were separated into three groups. The first two groups were administered PBS, while the last group was orally administered L. casei BL23. As shown in Figure 1, administration of PBS or L. casei BL23 started a week before tumor induction and was performed every day until sacrifice.

cytokine analysis
Mononuclear cells were isolated from spleens by gentle extrusion of the tissue through a 50-μm-mesh Nylon cell strainer (BD). Cells were resuspended in DMEM medium that was supplemented with 10% fetal calf serum, 2 mM l-glutamine, 50 U/mg penicillin, and 50 U/mg streptomycin (Lonza, Levallois-Perret, France). Erythrocytes were lysed with red blood cell lysing buffer (Sigma-Aldrich).

Tumor histology
Colon sections were formalin fixed and embedded in paraffin (4%, VWR, France). Epithelial proliferation was assessed by Ki67 staining according to the manufacturer's instructions, using mouse monoclonal anti-Ki67 antibody (MM1, Leica Biosystems; 1:50). The proliferation index was determined by counting the number of Ki67-positive cells per crypt in three well-aligned crypts.
Two-micron colon sections were used for H&E staining. Histological score was determined using a BX43 Olympus microscope in a blinded manner, via the observation of three parameters: inflammation/cellular infiltration, epithelial lesions, and regeneration.

gene and Protein expression analysis
Colon sections were stored in RNA later (Ambion) at −80°C. RNAs were extracted using the RNeasy mini-kit (Qiagen, Courtaboeuf, France) following the manufacturer's recommendations. RNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA synthesis was carried out from 1 µg of RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA), according to the manufacturer's instructions. RT-qPCR was carried out in a reaction volume of 25 µl with Taqman probes (β-actin: Mm01963702_S1, caspase-9: Mm00516563_m1, caspase-7: Mm01195085_m1, Bik: Mm00476123_m1) (Life Technologies, France) according to the manufacturer's instructions, using an ABI Prism 7700 (Applied Biosystems, USA) thermal cycler. To quantify and normalize the expression data, we used the ΔCt method, using the geometric mean Ct value from β-actin as the endogenous reference gene.

Microbial Dna extraction
DNA was extracted using the Godon technique from stool (T0 = day of AOM treatment) and cecal content (Tf = time final; day of sacrifice) as described by Lamas et al. (27). The DNA pellet was washed with 70% ethanol, dried, and resuspended in 50 µl of Tris-EDTA buffer. DNA suspensions were stored at −20°C until amplification. Reactions were performed using an annealing temperature of 65°C for 30 cycles in a T100 thermocycler (Biorad, France). Sequencing was performed using 460-bp paired-end reads and an Illumina Miseq protocol on the GeT-PLaGe platform (Toulouse, France). Illumina reads were joined using the fastq-join method. The sequences were demultiplexed and quality filtered using the QIIME (version 1.8.0) software package. The sequences were assigned to OTUs using UCLUST algorithm 41 with a 97% threshold of pairwise identity and classified taxonomically using the Greengenes reference database.

statistical analysis
Data were analyzed with Prism software (version 5). All normally distributed data were displayed as mean ± SEM. Comparisons between two groups were performed with a Student's t-test.

L. casei Bl23 Protects against Tumor Development in a colitis-associated crc Model
To determine the potential beneficial effects of the dairy strain BL23 of L. casei on CRC onset, live bacteria were orally administered to mice treated with AOM and DSS. As shown in Figure 2A, mice fed with L. casei BL23 were protected against tumor development: no mouse in this group developed macroscopic tumors, compared to 67% (6/9) of mice that received only PBS (Figures 2A,B). Mice treated with PBS developed a per-mouse average of 2.3 tumors at least 2 mm in diameter ( Figure 2C). Since this CRC model is related to chronic intestinal inflammation, we then assessed DAI and histological scores and intestinal epithelial damage. As shown in Figure 3A, DAI scores increased after each DSS cycle; however, there were no significant differences between any of the treated groups. For the histological scores, mice fed L. casei BL23 showed less damage than control mice did (p = 0.052, Student's t-test; Figures 3B,C). In addition, Ki67 levels (which are expressed in proliferating cells) were significantly lower (p = 0.044, Student's t-test) in mice treated with L. casei BL23 than in control mice (Figures 3D,E).

L. casei Bl23 Displays antiproliferative activities
As immunomodulation and the induction of cell apoptosis are among the main probiotic-related protective mechanisms against CRC, we examined changes in both the immune response and apoptosis pathways due to L. casei BL23 treatment. We first determined the levels of both local (i.e., colon and mesenteric lymphoid node (MLN) samples) and systemic (i.e., spleen samples) cytokines that are involved in inflammation and carcinogenesis, including IL-22, IFN-γ, IL-10, IL-21, TNF-α, IL-6, and IL-17A (Figures S1-S3 in Supplementary Material). As shown in Figure 4A, colonic IL-22 (a cytokine that promotes proliferation of cancer cells) (28) levels were lower (p = 0.057, Student's t-test) in L. casei BL23-treated mice compared to controls. Other cytokines that have been linked to CRC carcinogenesis, such as IFN-γ, TNF-α, IL-6, IL-10, IL-21, and IL-17A, were assessed in MLN ( Figure S1 in Supplementary Material), in spleen ( Figure  S2 in Supplementary Material), and in colon samples ( Figure S3 in Supplementary Material), but no significant difference was observed between treated and control mice.
In addition, we performed colic gene expression analysis of genes involved in apoptosis, specifically, caspase-7, caspase-9, and Bik. Our results reveal that L. casei BL23 induced a significant increase compared to controls in the expression of the executioner caspase-7 ( Figure 4B, p = 0.017, Student's t-test) and the initiator caspase-9 ( Figure 4C, p = 0.028, Student's t-test), together with  an increase of the apoptotic gene Bik (Figure 4D, p = 0.082, Student's t-test). Finally, we determined the level of truncated caspase-7 (which corresponds to the active form of caspase-7 in apoptosis). For this, we selected two mice treated with L. casei BL23 and protected against tumor development, which presented high caspase-7 RNA expression levels, and a mouse treated with PBS (not protected against tumors), which presented low levels of caspase-7 RNA expression. As shown in Figure 4E, western blot results confirmed that the mice protected against tumor onset produced higher levels of the active form of caspase-7 compared to the non-protected mouse. Altogether, these data suggest that L. casei BL23 has an antiproliferative and apoptotic effect in this CRC model; a detailed proposal of the mechanisms of action of L. casei BL23 against CRC is shown in Figure 5.

impact of L. casei Bl23 Treatment on Microbiota richness and Diversity
To determine the impact of L. casei BL23 on the gut microbiota, we analyzed microbiota richness and diversity after BL23 oral treatment. This analysis provided a total of 1,709,762 high-quality and classifiable reads, with an average of 5,000 (n = 51) reads per sample. First, beta diversity (Bray-Curtis distance) was analyzed in microbial samples using principal components analysis, which reduced the dimensionality of the data set. While no grouping was observed before AOM challenge (T0; Figure 6A), the microbiota of mice treated with L. casei BL23 tended to diverge from those of PBS-treated animals at Tf (sacrifice; Figure 6B, p = 0.1, Anosim). Then, we estimated community richness (alpha diversity; Shannon and Simpson indexes) at T0 and Tf. As shown in Figure 6C, AOM injection resulted in a reduction in alpha diversity with respect to that found in the group treated with PBS but not AOM (Shannon index, 6.4 ± 0.22 in PBS/ AOM-treated group versus 6.8 ± 0.46 in PBS/no AOM group, p = 0.028, Student's t-test). Despite the fact that the effects of L. casei strain BL23 were not statistically significant (Simpson index, 483.3 ± 111.8 in BL23/AOM-treated group versus 436.8 ± 54.7 in PBS/AOM-treated group, ns, Student's t-test; Figure 6D), these results provide intriguing clues about this strain's interactions with other members of the gut microbiota. Finally, bacterial communities were characterized at the phylum level (Figure 6E). Firmicutes was the dominant phylum in the L. casei BL23/AOM treated group (51 ± 10%), with Bacteroidetes ranked second (45 ± 10%). In contrast, Bacteroidetes was the most abundant phylum in both PBS/AOM and PBS/no AOM treated groups (52 ± 12 and 53 ± 13%, respectively), followed by Firmicutes (44 ± 12 and 43 ± 13%, respectively). The third most abundant phylum in all groups was Proteobacteria, with approximately 3-4% of reads. By using linear discriminant analysis (LDA) coupled with effect size measurements (linear discriminant analysis effect size), we found that Prevotella, Ruminococcaceae, and Lactobacillus were the key groups that were overrepresented in the L. casei BL23-treated mice ( Table 1). At the species level, only Lactobacillus zeae was significantly more abundant (p = 0.004, Student's t-test) in these mice, with a LDA score of 2.58. This was not surprising, since this species could in fact correspond to strain BL23 of L. casei. Indeed, 16S RNA analysis has revealed a close relationship (99% similarity) between our focal strain and L. zeae (26).

DiscUssiOn
There is now mounting evidence pointing to an important link between both chronic colic and rectal damage (present, for example, in IBD patients) and CRC carcinogenesis. Indeed, the risk of developing colitis-associated cancer increases by ~1% in IBD patients (5). To better understand the mechanisms related to tumor onset, different preclinical animal models of CRC have been developed, such as the AOM-DSS model used in this study (29).
L. casei BL23 has been previously studied for its anti-inflammatory activities in different models of chemically induced colitis (22,23). Furthermore, L. casei has been widely studied in different murine models of cancer (14,(30)(31)(32). In particular, L. casei BL23 has antiproliferative effects in the mouse allograft model of HPV-induced cancer and protects against DMH-induced CRC (25). Here, we decided to explore the impact of oral administration of L. casei strain BL23 in a murine model of CRC induced by AOM and DSS. Strikingly, our results revealed that L. casei BL23 significantly reduced tumor development, since all treated mice were tumor free at the end of the experiment. In addition, we also found some clues about the molecular mechanisms involved in the protective effect against cancer, and it appears that, in this model, L. casei BL23 acts mainly via the inhibition of cell proliferation. Indeed, this strain is able to downregulate proliferation, as observed through a decrease in Ki67. In addition, L. casei BL23 was also able to increase apoptosis via upregulation of caspase-9, caspase-7, and Bik.
Given the reported anti-inflammatory properties of L. casei BL23, we also assessed the cytokine profiles of treated mice. With the exception of a reduced histological score, L. casei BL23 had no significant effects on cytokine regulation. However, a weak decrease was observed in IL-22 levels in colons from mice treated with BL23. This cytokine has been recently implicated in CRC development in both humans and APC min/+ murine model (33). Thus, it appears that IL-22 levels are enhanced in tumor tissues and that mice displaying lower levels of this cytokine are protected from tumorigenesis. It was recently reported that the beneficial effects of a strain of Lactobacillus reuteri in a model of CRC induced by AOM-DSS were mediated by a histidine decarboxylase (HDC), which downregulated IL-22 expression (34). We performed in silico analyses to search for the nucleotide sequence of the HDC cluster in the L. casei BL23 genome, but were unable to find a corresponding sequence region (data not shown). However, it is still possible that another protein produced by this strain may act directly on IL-22 regulation/expression. The main sources of IL-22 are NK cells, γδ T cells, and lymphoid tissue inducer cells, as well as TH17 and TH22 cells. To determine which cell types are affected by L. casei BL23 treatment, future experiments will need to examine the correlation between each population of cells in the lamina propria of mice and IL-22 downregulation.
Finally, several reports have described disruption of the composition of the microbiota in CRC (17,18). Therefore, we analyzed the bacterial diversity (16S rDNA) in fecal samples from our mice and found that L. casei BL23 tended to restore the diversity disrupted by AOM injection. In agreement with previous reports (20), Firmicutes was the dominant phylum in the AOM/PBS-treated group. However, the introduction of L. casei BL23 reversed the ratio of Firmicutes to Bacteroidetes. Few individual species were affected by L. casei treatment, with the exception of L. zeae, which is actually considered a synonym of L. casei strain BL23 (26). However, future investigations should also consider analyzing the bacterial communities in the colic mucosa from tumor sections, because the assemblages present in stools are not necessarily an accurate reflection of the intestinal environment.
In conclusion, our work revealed that L. casei strain BL23 protected against CRC in an AOM/DSS model. Although this bacterium has well-known anti-inflammatory properties, we speculate instead that the protection observed here occurs through a reduction in cell proliferation and the induction of apoptosis.