Sulforaphane Attenuates AOM/DSS-Induced Colorectal Tumorigenesis in Mice via Inhibition of Intestinal Inflammation

Abstract Sulforaphane (SFN) is a compound derived from cruciferous plants. It has received considerable attention in recent years due to its effectiveness in cancer prevention and anti-inflammatory properties. The purpose of this study was to evaluate the antitumor potential of sulforaphane on colitis-associated carcinogenesis (CAC) through the establishment of a mouse model with AOM/DSS. First, AOM/DSS and DSS-induced model were established and administered SFN for 10 wk, and then the severity of colitis-associated colon cancer was examined macroscopically and histologically. Subsequently, immune cells and cytokines in the tumor microenvironment (TME) were quantified. Finally, the influence of sulforaphane was also investigated using different colon cell lines. We found that sulforaphane treatment decreased tumor volume, myeloid-derived suppressor cells (MDSC) expansion, the expression of the proinflammatory cytokine IL-1β, and the level of IL-10 in serum. Also, it enhanced the antitumor activities of CD8+ T cells and significantly reduced tumorigenesis as induced by AOM/DSS. SFN also attenuated intestinal inflammation in DSS-induced chronic colitis by reshaping the inflammatory microenvironment. This work demonstrates that sulforaphane suppresses carcinogenesis-associated intestinal inflammation and prevents AOM/DSS-induced intestinal tumorigenesis and progression.


Introduction
Colorectal cancer (CRC) is the fourth most common cancer in the world (1).Important stages in the development of CRC are closely related to chronic intestinal inflammation, with the most common being inflammatory bowel disease (IBD), and mainly include ulcerative colitis (UC) and Crohn's disease (CD).It is well established that colitis predisposes individuals to colorectal tumorigenesis (2,3).Colitis-associated colorectal cancer (CAC) is a distinct form of colorectal cancer that serves as a valuable model for understanding the role of chronic inflammation in tumor development.The exact cause of chronic inflammation in individuals with inflammatory bowel disease (IBD) and the primary driver of the transition from IBD to CAC remain unclear.Nevertheless, there is growing evidence indicating that immune cells and cytokines within the tumor microenvironment play a crucial role in this process.Myeloid-derived suppressor cells (MDSCs) are a major population of immune cells with immunosuppressive capabilities that infiltrate the inflamed colorectal tissue (4).Although MDSCs are often discussed as a defined population, phenotypic definition of these cells is actively evolving.These cells were initially identified as CD11b + Gr-1 + cells with immune regulatory functions in a mouse model of CD8+ T cell-mediated colitis.According to the different expressions of Ly6G and Ly6C, MDSCs can be further divided into two subgroups of monocyte-derived MDSC(M-MDSCs) and granulocytic MDSC (G-MDSC or PMN-MDSC).Their mouse counterparts have been defined as CD11b + Ly6C + and CD11b + Ly6G + Ly6C low , respectively (5).MDSCs were found to exert direct immunoregulatory effects by competing for cysteine within the tissue environment, leading to the upregulation of iNOS and Arg-1 activities.Consequently, this consumption of L-arginine hampers T cell production (6,7).Moreover, the expansion of MDSCs induced by norepinephrine inhibits T cell proliferation through the production of reactive oxygen species (ROS), which can be toxic to most cell types (8).
There is compelling evidence to suggest that commonly associated dietary and lifestyle changes have a significant impact on the development of colorectal cancer.Epidemiological studies have revealed an inverse correlation between the intake of cruciferous vegetables and colorectal cancer risk (9).Sulforaphane, a natural effective inducer of Nrf2 product isolated from cruciferous vegetables was reported to play a defensive role in plants via its cytotoxic effects on microorganisms and small parasitic animals.It has also been suggested to be beneficial to human health via broad anti-inflammatory and antioxidant effects (10) and thus helps inhibit the development of cancers (11,12).In addition to its preventive effect on cancer, sulforaphane has recently been shown to inhibit the growth of various types of cancer cells by modulating multiple pathways related to cancer progression.It appears to be responsible for the larger part of the plant's health-promoting and cancer-preventive properties beyond its nutrient content (13,14).Furthermore, the extract of broccoli seeds had been approved by the National Health and Family Planning Commission as a new food raw material in 2017, and its domestic application has been affirmed in China.
Emphasizing the importance of understanding the therapeutic effects of SFN, our previous studies have found that SFN accumulates intracellular ROS and induces cell cycle arrest at the G2/M phase, thereby impeding the proliferation of colon cancer cells.However, the effects and mechanisms of SFN on colitis-associated colorectal cancer (CAC) have not been fully investigated.In this study, we used a well-established AOM/DSS-induced CRC model to further evaluate the tumor-suppressive effects and mechanisms of SFN on CRC carcinogenesis.

Animals
C57BL/6 mice (aged 6-8 wk) were purchased from Cavens Laboratory Animal (Changzhou, China).The mice were kept in standard housing cages under specific pathogen-free conditions.All experimental procedures were reviewed and carried out in accordance with relevant laws and institutional guidelines and approved by the Animal Care and Use Committee of Nanjing Medical University (ID: SYXK(Su) 2018-0020).

AOM/DSS-Induced CAC and DSS-Induced Chronic Colitis Model and Treatment
To establish the CAC model, we injected each mouse intraperitoneally with 12.5 mg/kg of AOM dissolved in PBS.After 7 days, the animals were provided with drinking water containing 2% DSS for 7 days, followed by drinking water for 14 days, and exposed to two more 2% DSS treatment cycles.During every DSS cycle, the mice were gavaged with SFN (75 mg/kg) daily for 10 days.The mice were weighed weekly and sacrificed on the tenth week.DSS colitis was induced by adding 2% DSS to the mice's drinking water for 1 week, followed by normal drinking water for 2 wk.For a total of three cycles, during each DSS cycle, the mice were gavaged with SFN (75 mg/kg) daily for 10 days.Body weights were monitored daily over the course of the experiment.At the end of the experimental period, the mice were sacrificed, and their colon lengths were measured.We used the DAI to quantify colitis severity as previously described (15).After sectioning into 3 µm sections, colon segments were stained with H&E to assess tissue integrity.

Flow Cytometry
Surface and intracellular molecule staining was performed as previously described (16).Briefly, the colon and draining spleen were collected from mice and minced into pieces smaller than 1 mm 3 followed by digestion with collagenase type IV, hyaluronidase, and deoxyribonuclease for 30 min at 37 °C.Then, the cells were filtered through a 70 µm cell strainer and washed twice with staining buffer (PBS containing 2% FBS).Cells were resuspended in a buffer and stained with fluorescently labeled antibodies against CD45, F4/80, CD11b, CD11c, MHC-II, CD3, CD8 or CD4 on ice for 30 min.After a washing step, flow cytometry was performed, and the flow cytometry data were analyzed using FlowJo software (BD, USA).In addition, we quantified a panel of multiple cytokines in the colonic homogenates using the LEGENDplex TM Multi-Analyte Flow Assay Kit according to the manufacturer's protocol.The assay FCS files were analyzed using BioLegend's LEGENDplex™ data analysis software.

RNA Isolation and Real-Time qRT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol and the RNA concentration and integrity were determined by NanoDrop ND2000 spectrophotometry and formaldehyde-agarose gel electrophoresis respectively.The mRNA expression was quantified by real-time qRT-PCR using the PrimeScript RT kit and the SYBR Green PCR master mix (Takara, Dalian, China).The Ct values for mRNA were normalized to GAPDH mRNA.The sequences of the primers for this study are listed in Supplementary Table 1.

Histopathological Examination
Colons were removed from cecum to anus, flushed with cold PBS, their length and weight measured, then fixed as "Swiss-rolls" in 4% paraformaldehyde overnight.The specimens were processed as per standard Hematoxylin and eosin (H&E) staining protocol, formalin-fixed, embedded in paraffin, and sliced into 4 µm sections.The degree of colitis was assessed as previously described (17).

Statistical Analysis
All the data were presented as the mean ± standard error of the mean (SEM).The log-rank test (Mantel-Cox) was used to evaluate survival differences.Statistical significance of the differences was evaluated using a Student's t test or one-way ANOVA.p < 0.01 was considered highly statistically significant, and p < 0.05 was considered statistically significant.

Sulforaphane Alleviated AOM/DSS-Induced Colorectal Tumorigenesis in Mice
In this study, we used a well-established AOM/DSS murine model that mimics chronic intestinal inflammation to investigate whether sulforaphane has a therapeutic role in colitis-associated colorectal tumorigenesis (18).Compared with the control group, there was no statistically significant change in weight and no obvious clinical symptoms were observed over a period of two weeks, indicating that SFN in the current dosing was not toxic (Supplementary Figure 1).For these experiments, 6-8-week-old mice were injected with AOM (12.5 mg/kg) and treated with three cycles of DSS as defined (Figure 1A).During the modeling process, the survival rate of mice was calculated and the mortality rate of mice in the model group was 30%.The SFN group survived, and it was observed that SFN significantly improved the survival rate of mice (p = 0.05) (Figure 1B).We found that SFN treatment significantly attenuated the weight loss caused by DSS (p < 0.05) (Figure 1C).Diarrhea and blood in feces are important clinical parameters to score the severity of AOM/DSS induced intestinal inflammation and tumorigenesis.It revealed that SFN treatment significantly decreased the disease activity index (DAI), a composite score used to evaluate the clinical manifestations of colitis(p < 0.05) (Figure 1D).It is preliminarily speculated that SFN may delay and have a certain inhibitory effect on induced colon cancer.Thus, SFN delayed the death of the mice and maintained the healthy state of the mice.
Reduced colon length often serves as a surrogate macroscopic indication of colonic injury.The results of the colon length showed SFN treatment attenuated AOM/DSS induced colonic injury (p = 0.0009) (Figure 1E).Similarly, the spleen weight of mice in each group was measured and a significantly smaller spleen was observed in the SFN-treated mice than in the model group (p = 0.04) (Figure 1F).An analysis of the tumor diameter and size showed that sulforaphane alleviated AOM/DSS-induced colorectal tumorigenesis(p = 0.03) (Figure 1G).To further assess the colitis-associated tumor, H&E staining was performed.The results revealed normal colon structure and no inflammatory cell infiltration in the control group.However, in the AOM/DSS model group, goblet cells disappeared and inflammatory cell infiltration was severe.SFN hyperplasia glands were disordered, the degree of inflammatory cell infiltration was mild, and the structural change of the colon was not obvious in most cases.This indicated that the SFN could protect the integrity of the colonic mucosa from damage (Figure 1H).Collectively, these findings suggest that treatment with SFN reduces the onset and clinical signs of CRC induced by AOM/DSS.

Sulforaphane Attenuates Inflammation in the Tumor Microenvironment in Mice with Colitis-Related CRC
Innate immunity plays a vital role in controlling intestinal inflammation during the early stage of inflammatory bowel disease (IBD).Mice treated with SFN had less aggressive tumors, and the submucosa surrounding the tumor tissue was less infiltrated with inflammatory cells.Thus, we investigated whether and how SFN treatment impacts colonic immune cell infiltrates.Dendritic cells (DCs), Natural Killer (NK) and tumor-associated macrophage (TAM) cells did not change after the SFN treatment, whether in spleen, intestinal lymph nodes, or colon tumors.However, the SFN treatment significantly decreased colonic CD11b + LY-6G + subpopulation (granulocytic MDSCs (G-MDSCs)) (19,20) in colon tumors who can block the generation of T cells (21) (p = 0.02) (Figure 2A).The colonic MDSC expression of arginase-1, a known critical mediator of MDSC function, was significantly decreased upon the SFN treatment (p = 0.05) (Figure 2D).The proportions of CD3+ T cells in CD45+ cells (p = 0.01) and CD8+ T cells in CD3+ cells were significantly increased (p = 0.03), the CD4/CD8 ratio decreased due to a marked decrease in CD4+ T cells after the SFN treatment (p = 0.02) (Figure 2B&C).These results suggest that SFN may alleviate CAC by reshaping immune cell infiltration in the tumor microenvironment, especially MDSCs.
Furthermore, we monitored the expression of inflammation-associated genes by real-time PCR.The mRNA levels of transcripts for proinflammatory mediators such as IL1β (p = 0.04) and IL10 (p = 0.006) were significantly reduced in the SFN group compared to those in the AOM/DSS group, while TNFα and IFNγ mRNA expression was not significantly altered (Figure 2D).At the same time that the expression of cytokines in serum was detected, it was found that the level of IL-10 in serum decreased significantly after the SFN treatment (p = 0.03) (Figure 2E).Taken together, these results indicate that SFN treatment appears to suppresses the recruitment of MDSCs, while enhancing the recruitment of T lymphocytes through regulating the cytokines expression in the TME.

Sulforaphane Attenuated Intestinal Inflammation in DSS-Treated Mice
SFN treatment has a protective effect on CAC.Chronic inflammation predisposes tissue to tumor development.We hypothesized that SFN may affect the development of CAC during the chronic inflammation stage.Similarly, we used the DSS method to induce chronic inflammation in mice to explore the role of SFN in this process (Figure 3A).As shown in our study, mice receiving DSS in their drinking water exhibited a significant loss in body weight as compared to the SFN group (p < 0.05) (Figure 3B).Also, the disease activity index (DAI) was significantly decreased after the SFN treatment (p < 0.05) (Figure 3C).Furthermore, the mean colon length of the mice treated with DSS was significantly shorter than that of the SFN-treated group (p = 0.05) (Figure 3D).Similarly, the spleen weight of the mice in each group was counted and the spleen was significantly smaller in the SFN-treated mice than in the DSS group (p = 0.04) (Figure 3E).Also, histologic examination showed that the SFN treatment significantly alleviated the characteristics of DSS-induced pathological symptoms, including an increased nucleoplasmic ratio, nuclear polarity extinction, and gland hyperplasia (Figure 3F).These results may suggest that SFN could attenuate intestinal inflammation.
When the colonic immune cells of DSS-treated mice were analyzed, the results were similar to those of CAC induced by AOM/DSS.The SFN treatment significantly decreased colonic granulocytic myeloid-derived suppressor cells (G-MDSCs) in colon tissue (p = 0.03) (Figure 4A).The proportions of CD3+ T cells in CD45+ cells (p = 0.05) and CD8+ T cells in CD3+ cells were significantly increased (p = 0.007), CD4 + T cells and the ratio of CD4/CD8 also decreased significantly after the SFN treatment (p = 0.04) (Figure 4B&C).We also measured various cytokines in the colonic tissues of DSS-treated mice to assess the extent of intestinal inflammation.The mRNA levels of IL1β (p = 0.03) and IL10 (p = 0.05) were significantly reduced in the SFN group compared to those in the control group (Figure 4D).These data indicate that SFN affects immune cells infiltration during the development of colitis, allowing decreased MDSCs and increased T-cell infiltration.

Discussion
Chronic intestinal inflammation is increasingly recognized due to its implication in the pathogenesis of CRC.Three types of inflammation can be distinguished based on when they affect CRC pathogenesis: chronic inflammation preceding tumorigenesis, tumor-elicited inflammation, and therapy-induced inflammation.These types of inflammation promote tumor transformation of colonic epithelial cells, cell proliferation and invasion, immune cell infiltration, and immune responses involved in all stages of colorectal cancer development (22,23).
Despite an increased understanding of molecular biology and the genomics of cancer, drugs cause severe side effects and resistance inevitably develops, reactivating tumor growth and dissemination.It has been reported that some natural plant extracts suppress intestinal inflammation and prevent AOM/   cascade (27).PEITC reduces the properties of cancer stem cells and regulates genes involved in inflammatory, immune, and chemokine-related signaling (28).Similarly, SFN, another isothiocyanate compound found in cruciferous plants, has demonstrated potent anticancer effects.In our study, SFN exhibited significant antitumor effects both in vitro (Figure 1) and in vivo, without causing toxicity.In vitro, SFN decreased cell viability, increased Nrf-2 and HO-1 expression, induced ROS accumulation, and arrested cell cycle progression at the G2/M phase, effectively inhibiting colon cancer cell proliferation (Supplementary Figure 2).In vivo, in an AOM/DSS-induced colitis-associated intestinal tumorigenesis model, SFN treatment alleviated colitis-related symptoms, extended the survival of colitis mice, delayed the development of CAC, and reduced tumor burden (Figure 1).In addition, DSS was used to simulate the chronic inflammation stage before CAC formation.SFN could slow down weight loss, prolong colon length and reduce tissue damage (Figure 3).
The AOM/DSS model-induced CAC has been extensively studied in terms of changes in immune cells and cytokines within the immune microenvironment.Chronic colonic disorders promote the recruitment and activation of MDSCs, which are immature myeloid cells found in inflammatory sites, secondary lymphoid organs, IBD patients, and tumor tissues (29,30).MDSCs play a critical role in CAC progression by promoting chronic inflammation, facilitating blood vessel formation, creating an immunosuppressive tumor microenvironment, and promoting the stemness of colon cancer cells.They infiltrate the inflamed intestinal tissues or tumor microenvironment, contributing to disease progression (31).MDSCs interact with innate and adaptive immune cells and play a crucial role in negatively regulating the immune response to tumors (32).In innate immunity, MDSCs can inhibit the function of NK cells (33), inhibit T-cell activation and function (34) and inhibit DC cell antigen presentation and subsequent proliferation and activation of CD4 + T cells leading to persistent stimulation of inflammation (35).In terms of adaptive immunity, MDSCs can competitively consume cysteine, upregulate the activities of iNOS and Arg-1, mediate ARG catabolism and suppress the T-cell response.Targeting the STAT3 pathway by a selective inhibitor could decrease ARG1 expression in MDSCs and partially reverse the lower expression levels of effector molecules on CD8+ T lymphocytes (36).MDSCs also inhibit the immune response of T cells by producing reactive oxygen species (ROS) (37).In our study, we observed that in CRC tissue induced by AOM/DSS, the proportion of MDSCs was significantly increased.However, treatment with SFN led to a reduction in MDSC production and the expression of Arginase.Additionally, SFN treatment increased the proportions of T-cell markers CD3 + and CD8 + , indicating enhanced T-cell activity (Figure 2).Tumor-infiltrating T cells play a crucial role in preventing the spread of colorectal cancer (38).
We also examined the expression of related cytokines and found that SFN treatment resulted in decreased levels of IL-1β and IL-10, leading to reduced intestinal inflammation (Figure 2).IL-1β is a key pro-inflammatory cytokine that, along with IL-1α and IL-18, coordinates the immune response by regulating IL-6 and TNFα, contributing to autoimmune inflammatory responses (39).In the context of CAC, IL-1β produced by neutrophils can induce intestinal mononuclear phagocytes to produce IL-6, promoting tumor formation (40).IL-10, on the other hand, is widely accepted as an immunosuppressive cytokine and is thought to promote tumor immune escape by dampening the anti-tumor immune response in the tumor micro-environment.Lack of IL-10R in colorectal tissue could lead to severe spontaneous colitis, which increases the risk of CRC initiation (41).However, the role of IL-10 in cancer development is complex.Some studies have shown that IL-10 can act as a tumor-promoting factor in CRC.On the other hand, IL-10 deficiency has been found to enhance the efficacy of immunotherapy, reduce MDSC and Treg levels in the tumor microenvironment (TME), and promote anti-tumor responses (42).Park et al. found that IL-10 produced by MDSCs plays a critical role in mediating the immunoregulatory effects of these cells (43).SFN treatment significantly reduced colonic MDSCs in colon tissue, which could affect the secretion of IL-10.Therefore, SFN may suppress MDSC production, restore T-cell recruitment, and contribute to a reduction in tumor growth and progression.Chronic inflammation is known to predispose tissue to tumor development.Through our research, we found that SFN exhibits antibacterial and anti-inflammatory effects, making it a potential beneficial dietary supplement for improving health (44).In vitro studies with various cell lines (45,46) and a few in vivo studies have shown that SFN has an inverse relationship with inflammatory responses.Additionally, SFN has been reported to reverse DSS-induced gut dysbiosis and increase the production of volatile fatty acids (47,48).
In our study, we used a DSS-induced chronic colitis model to investigate the effect of SFN on inflammation.After three cycles DSS induction, mice in the DSS group showed significant weight loss, shortened colon length, and histopathological symptoms associated with chronic colitis.However, oral SFN administration improved colon length and histopathological score, indicating a reduction in the inflammatory severity of chronic colitis (Figure 3).Inflammation to cancer transformation is a complex process involving immune cells and cytokines (49).Few studies have explored the effects of SFN on immune cells and cytokines during DSS-induced chronic inflammation.Our study revealed that SFN treatment significantly alleviated DSS-induced pathological symptoms, decreased colonic MDSCs, and increased the proportions of CD3 + T cells and CD8 + T cells (Figure 4).Acetylcholine was found to promote the secretion of interleukin-10 by M-MDSCs and suppress inflammation (50).Moreover, we observed that SFN treatment significantly reduced the expression of IL-1β and IL-10 (Figure 4), further supporting its inhibitory effects on chronic colitis.

Conclusions
In summary, the use of SFN for the prevention and treatment of colorectal tumorigenesis mainly involves activating the anti-oxidative stress defense system and inhibiting intestinal inflammation, particularly through the modulation of MDSCs.Our findings highlight the potential of SFN as a dietary supplement that could be beneficial for individuals at risk of colorectal cancer or those undergoing treatment for chronic colitis.

Figure 1 .
Figure 1.inhibitory effects of SFN on aoM/dSS-induced colorectal tumorigenesis in mice.(a) induction procedure for aoM/ dSS-induced CaC in C57BL/6 mice and SFN treatment.(B) Survival curves from the indicated treatment cohorts.(C) Body weight of mice that were gavaged with water (a/d means aoM/dSS group, n = 7) or SFN (a/d + SFN means aoM/dSS + SFN group, n = 7) (d) disease activity index during each treatment cycle.(e) Colon length of each group.(F) Spleen weight of each group.(G) Number and size distribution of colorectal tumors.(H) H&e staining of colon sections and corresponding histological scores.error bars represent the mean ± Sd. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2 .
Figure 2. treatment decreases colonic myeloid-derived suppressor cells (MdSCs) in aoM/dSS-induced colorectal tumorigenesis models.(a) the absolute number of MdSCs (Cd11b + Ly6G+) in Cd45+ cells of the colon tumors were determined by flow cytometry.(B) the absolute number of Cd3+ cells in Cd45+ cells of the colon tumors were determined by flow cytometry.(C) the absolute number of Cd4+ Cd8+ cells in Cd3+ cells of the colon tumors were determined by flow cytometry (n = 7 for both the a/d and a/d + SFN groups).(d) Mouse serum cytokines were detected by using fluorescence-encoded beads suitable for use on various flow cytometers.(e) the mrNa expression levels of antitumor immune response-related molecules in the colon were determined by rt-PCr and calculated by the 2 -ΔCt method.the data are representative of three independent experiments.the statistical significance of differences was determined by unpaired two-tailed Student's t test.*p < 0.05, error bars indicate the means ± SeMs.

Figure 3 .
Figure 3. inhibitory effects of SFN on dSS-induced uC in mice.(a) induction procedure for dSS-induced uC in C57BL/6 mice and SFN treatment.(B) Body weight of mice that were gavaged with water (dSS group, n = 7) or SFN (dSS + SFN group, n = 7) (C) disease activity index during the dSS treatment cycle.(d) Colon length of each group.(e) Spleen weight of each group.(F) H&e staining of colon sections and corresponding histological scores.error bars represent the mean ± SeMs.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4 .
Figure 4. SFN treatment decreases colonic myeloid-derived suppressor cells (MdSCs) in dSS-induced uC models.(a) the absolute number of MdSCs (Cd11b + Ly6G+) in Cd45+ cells of the colon tumors were determined by flow cytometry.(B) the absolute number of Cd3+ cells in Cd45+ cells of the colon tumors were determined by flow cytometry.(C) the absolute number of Cd4+ Cd8+ cells in Cd3+ cells of the colon tumors were determined by flow cytometry (n = 7 for both the dSS and dSS + SFN groups).(d) the mrNa expression levels of antitumor immune response-related molecules in the colon were determined by rt-PCr and calculated by the 2 -ΔCt method.data are representative of three independent experiments.the statistical significance of differences was determined by unpaired two-tailed Student's t test.*p < 0.05, error bars indicate the means ± SeMs.