Mouse Model Of Colorectal Cancer Research Articles

Effects of Irinotecan on Tumor Vasculature and Oxygenation: An in vivo Study on Colorectal Cancer Model

The effects of chemotherapy on the tumor microenvironment remain poorly investigated, although these are crucial for the drug delivery and anti-tumor activity and can modify physiological behavior of cancer cells. Optical coherence angiography and phosphorescence lifetime spectroscopy are promising optical methods to characterize both tumor vasculature and its oxygenation, respectively. In this study, we used these methods to explore the influence of irinotecan, a widely used chemotherapeutic agent, on tumor angiogenesis and oxygen content in a mouse model of colorectal cancer in vivo. For the first time, it was shown that irinotecan at conventional dosing reduces tumor oxygenation and blood perfusion via antiangiogenic mechanism of action. Immunohistochemistry for hypoxia and for the endothelial cell marker CD31 verified the in vivo findings. These data show the prospects of the optical techniques to monitor tumor microenvironment in the course of chemotherapy, which can help to optimize and individualize the treatment.

Сomparative analysis of acridineacetate-containing compounds’ radio-sensitizing effect during malignant tumor experimental radiotherapy in a metastatic colorectal cancer model in balb/c mice

Radio-sensitizing compounds are used to increase radiotherapy effectivity and reduce negative pathologic systemic effects of radiation. These radio-sensitizers selectively pile-up in tumor tissue and enhance necrobiotic processes caused by irradiation. Unfortunately, broad introduction of radio-sensitizers into clinical practice is hampered by their low effectivity and substantial negative side effects. This what determines radio-sensitizers effectivity studies high actuality in a colorectal cancer mice model. The study involved 156 male BALB/c mice who had been transplanted with colorectal cancer. After the transplantation they were injected with acridineacetate containing compounds Cyclopherone and Procitol. A comparative assessment was undertaken of tumor growth dynamics and metastatic rate. A combination of gamma-irradiation with radio-sensitizing compounds produces a more substantial therapeutic effect according to all parameters of the experimental colorectal cancer growth and development than the isolated effect of radiotherapy or antitumor drugs. A considerable slowing down of tumor growth on the 14th and 28th day of the study was revealed as well as a reliable inhibition of lymphogenous and hematogenous metastatic spread of neoplasma during the entire study and a statistically valid increase of lifespan of the experimental animals. The radiosensitizing effect of Procitol in combination with radiotherapy turned out to be more substantial than that of Cyclopherone.

Cetuximab enhances the anti-tumor function of macrophages in an IL-6 dependent manner

AimsCetuximab improves the survival of patients with advanced colorectal cancer (CRC). However, how cetuximab affects the tumor microenvironment has not been sufficiently understood. This study was to investigate whether cetuximab could inhibit the pro-tumor function of tumor-associated macrophages (TAMs) by suppressing the EGFR/IL-6 pathway. Main methodsThe azoxymethane/dextran sodium sulfate (AOM/DSS) and tumor xenograft mouse models were used to assess the effect of cetuximab on TAMs. Flow cytometry, Western blotting, RT-qPCR, and ELISA were used to assess the prevalence of M2 and M1 phenotypes. Publicly available datasets of CRC patients were used to assess the relevance of EGFR and IL-6 expression as prognostic indicators. Key findingsThe two mouse models showed that cetuximab could attenuate the pro-tumor function of TAMs and decrease tumor burden. Cetuximab repolarized TAMs from M2-like to M1-like phenotypes, mainly by suppressing the IL-6 expression through NFκB and STAT3 pathways. Analysis of public scRNA-seq data indicated EGFR was mainly expressed on the surface of macrophage infiltration into tumor microenvironment. The public transcriptomics datasets showed that the expression level of IL-6 was positively correlated with EGFR in CRC patients, and PROGgeneV2 analysis indicated that IL-6 and CD206 both predicted poor recurrence-free and overall survival of CRC patients. Furthermore, the inhibition efficacy of cetuximab was significantly attenuated in IL-6 knockout CRC mice model. SignificanceThese results indicate a new macrophage-based molecular mechanism explaining the effect of cetuximab in treatment of colorectal cancer.

Suppression MGP inhibits tumor proliferation and reverses oxaliplatin resistance in colorectal cancer

Matrix Gla protein (MGP), an extracellular matrix protein, has been widely reported to participate in the tumorigenic process and is abnormally expressed in several tumors. However, the role of MGP in colorectal cancer (CRC) remains unknown. Chemotherapy resistance represents a significant limitation in the treatment of CRC. Here, a comprehensive bioinformatics analysis revealed that MGP, which is overexpressed in CRC, might act as one of the critical genes conferring resistance to oxaliplatin (OXA). Furthermore, we found that MGP overexpression in tumor tissue might be correlated with cancer stage and patient prognosis, consistent with the bioinformatics analysis. The upregulation of MGP may act as an independent risk factor for CRC. The knockdown of MGP or inhibition of MGP expression significantly increased the sensitivity of the CRC cell lines to OXA. Suppression of MGP may reverse OXA resistance by upregulating copper transporter 1 (CTR1) and downregulating ATP7A and ATP7B. When used in combination with OXA, the inhibition of MGP reduced cancer cell proliferation, invasion, and migration and increased cell apoptosis in vitro. Suppression of MGP or OXA treatment alone significantly inhibited tumor growth in the CRC mouse model. Additionally, we found that OXA might promote the antitumor immune response in vivo. In summary, our study is the first to provide evidence that MGP expression confers OXA chemotherapy resistance in CRC and provides novel strategies to overcome chemotherapy resistance in CRC.

Lipoproteins LDL versus HDL as nanocarriers to target either cancer cells or macrophages.

In this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL, respectively) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of “Trojan horses” delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass spectometry and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anticancer immune response. Cumulative antitumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient blood can be used as natural nanocarriers allowing cell-specific targeting, paving the way toward more efficient, safer, and personalized use of chemotherapeutic and immunotherapeutic drugs in cancer.

Black Raspberries Suppress Colorectal Cancer by Enhancing Smad4 Expression in Colonic Epithelium and Natural Killer Cells.

Innate immune cells in the tumor microenvironment have been proposed to control the transition from benign to malignant stages. In many cancers, increased infiltration of natural killer (NK) cells associates with good prognosis. Although the mechanisms that enable NK cells to restrain colorectal cancer (CRC) are unclear, the current study suggests the involvement of Smad4. We found suppressed Smad4 expression in circulating NK cells of untreated metastatic CRC patients. Moreover, NK cell-specific Smad4 deletion promoted colon adenomas in DSS-treated ApcMin/+ mice and adenocarcinomas in AOM/DSS-treated mice. Other studies have shown that Smad4 loss or weak expression in colonic epithelium associates with poor survival in CRC patients. Therefore, targeting Smad4 in both colonic epithelium and NK cells could provide an excellent opportunity to manage CRC. Toward this end, we showed that dietary intervention with black raspberries (BRBs) increased Smad4 expression in colonic epithelium in patients with FAP or CRC and in the two CRC mouse models. Also, benzoate metabolites of BRBs, such as hippurate, upregulated Smad4 and Gzmb expression that might enhance the cytotoxicity of primary human NK cells. Of note, increased levels of hippurate is a metabolomic marker of a healthy gut microbiota in humans, and hippurate also has antitumor effects. In conclusion, our study suggests a new mechanism for the action of benzoate metabolites derived from plant-based foods. This mechanism could be exploited clinically to upregulate Smad4 in colonic epithelium and NK cells, thereby delaying CRC progression.

An Antibody-Tumor Necrosis Factor Fusion Protein that Synergizes with Oxaliplatin for Treatment of Colorectal Cancer.

We have cloned and characterized a novel fusion protein (Sm3E-TNF), consisting of the mAb, S 6m3E, in single-chain Fv fragment format, fused to murine TNF. The protein, which was expressed in mammalian cells and purified as a noncovalent stable homotrimer, bound to the cognate carcinoembryonic antigen (CEA) and retained TNF activity. A quantitative biodistribution experiment, performed in immunocompetent mice with CT26 colon carcinomas transfected with human CEA, revealed that Sm3E-TNF was able to preferentially accumulate in the tumors with excellent selectivity (tumor:blood ratio = 56:1, 24 hours after intravenous administration). The fusion protein mediated a rapid hemorrhagic necrosis of a large portion of the tumor mass, but a rim survived and eventually regrew. Surprisingly, the combination of Sm3E-TNF with 5-fluorouracil led to a reduction of therapeutic activity, while a combination with oxaliplatin led to a prolonged stabilization, with complete tumor eradication in 40% of treated mice. These therapy results were confirmed in a second immunocompetent mouse model of colorectal cancer (CEA-transfected C51 tumors) and provide a rationale for the possible clinical use of oxaliplatin in combination with fully human antibody-TNF fusions.

Fucoidan-coated nanoparticles target radiation-induced P-selectin to enhance chemoradiotherapy in murine colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related death for both men and women, highlighting the need for new treatment strategies. Advanced disease is often treated with a combination of radiation and cytotoxic agents, such as DNA damage repair inhibitors and DNA damaging agents. To optimize the therapeutic window of these multimodal therapies, advanced nanomaterials have been investigated to deliver sensitizing agents or enhance local radiation dose deposition. In this study, we demonstrate the feasibility of employing an inflammation targeting nanoscale metal-organic framework (nMOF) platform to enhance CRC treatment. This novel formulation incorporates a fucoidan surface coating to preferentially target P-selectin, which is over-expressed or translocated in irradiated tumors. Using this radiation stimulated delivery strategy, a combination PARP inhibitor (talazoparib) and chemotherapeutic (temozolomide) drug-loaded hafnium and 1,4-dicarboxybenzene (Hf-BDC) nMOF was evaluated both in vitro and in vivo. Significantly, these drug-loaded P-selectin targeted nMOFs (TT@Hf-BDC-Fuco) show improved tumoral accumulation over multiple controls and subsequently enhanced therapeutic effects. The integrated radiation and nanoformulation treatment demonstrated improved tumor control (reduced volume, density, and growth rate) and increased survival in a syngeneic CRC mouse model. Overall, the data from this study support the continued investigation of radiation-priming for targeted drug delivery and further consideration of nanomedicine strategies in the clinical management of advanced CRC.

Piperlongumine Alleviates Mouse Colitis and Colitis-Associated Colorectal Cancer.

Colorectal cancer is one of the most common and lethal cancers in the world. An important causative factor of colorectal cancer is ulcerative colitis. In this study, we investigated the therapeutic effects of piperlongumine (PL) on the dextran sulfate sodium (DSS)-induced acute colitis and azoxymethane (AOM)/DSS-induced colorectal cancer mouse models. Our results showed that PL could inhibit the inflammation of DSS-induced mouse colitis and reduce the number of large neoplasms (diameter >2 mm) of AOM/DSS-induced mouse colorectal cancer by downregulation of proinflammatory cytokines cyclooxygenase-2 and interleukin-6 and epithelial-mesenchymal transition-related factors, β-catenin, and snail expressions, but fail to improve the colitis symptoms and to decrease the incidence of colonic neoplasms and the number of small neoplasms (diameter <2 mm). These data suggested that PL might be an effective agent in treating colitis and colorectal cancer.

Long-term model of colitis-associated colorectal cancer suggests tumor spread mechanism and nature of cancer stem cells.

Although chemical-induced animal models of colorectal cancer (CRC) suggest a lot about the disease, more efforts are required to establish metastasis models. Azoxymethane (AOM) and dextran sodium sulfate (DSS)-treated (AOM/DSS) Crl:CD-1 mice were sacrificed after 10 or 20 weeks in our previous study, and most colon tumors exhibited intramucosal adenocarcinomas. Our observations were extended until 30 weeks to study a colitis-associated advanced CRC mouse model, and explore whether linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) immunostaining-positive cells were involved in the progressive course of colitis-associated CRC as cancer stem cells. AOM/DSS mice were sacrificed at 10, 20 and 30 weeks after AOM administration. Following the histopathological analysis, immunohistochemical staining was performed for the following markers: CD34, podoplanin, β-catenin, E-cadherin, Ki67, Bmi1 and pSmad2/3L-Thr. Compared with AOM/DSS mice at 10 and 20 weeks, submucosal tumor infiltration and tumor invasion into vessels were markedly increased at 30 weeks. In the parts of colon tumors from AOM/DSS mice, particularly in mice at 30 weeks, the positive signal of E-cadherin was clearly reduced in the cell membranes. The percentage of Ki67-positive tumor cells in mucosal areas of AOM/DSS mice was higher than that in the sites of submucosal infiltration. In mucosal areas of colon tumors, pSmad2/3L-Thr-positive cells were scattered among tumor cells. At sites of submucosal infiltration and vessel invasion of these tumors, pSmad2/3L-Thr-positive cells were also observed among tumor cells. In colon tumors from AOM/DSS mice at 30 weeks, the percentage of pSmad2/3L-Thr-positive cells among the nuclear β-catenin-positive tumor cells was higher than that among the cytoplasmic β-catenin-positive tumor cells. For both non-neoplastic and neoplastic epithelial cells, pSmad2/3L-Thr-positive cells exhibited immunohistochemical co-localization with Bmi1. The present study developed an advanced CRC mouse model that exhibited tumor infiltration into the submucosa and invasion into vessels. The present study re-confirmed the theory that pSmad2/3L-Thr-positive cells may be cancer stem cells.

O-GlcNAcylation Links Nutrition to the Epigenetic Downregulation of UNC5A during Colon Carcinogenesis.

Simple SummaryNutritional disorders represent major risk factors for colorectal cancer according to mechanisms of action that are still insufficiently known. The aim of our study was to investigate the putative involvement of nutrition in the epigenetic downregulation of the tumor suppressor genes of the UNC5 (Uncoordinated 5) family during colonic carcinogenesis and to understand its molecular relays. Herein, we provided evidence that the consumption of a High Carbohydrate Diet worsens colon carcinogenesis in mice and is correlated with the downregulation of several members of the UNC5 family whose UNC5A (Uncoordinated 5A). Mechanistically, we identified the nutritional sensor O-GlcNAcylation as one of the molecular relays that regulate the recruitment of the PRC2 complex onto the UNC5A promoter to repress its transcription.While it is now accepted that nutrition can influence the epigenetic modifications occurring in colorectal cancer (CRC), the underlying mechanisms are not fully understood. Among the tumor suppressor genes frequently epigenetically downregulated in CRC, the four related genes of the UNC5 family: UNC5A, UNC5B, UNC5C and UNC5D encode dependence receptors that regulate the apoptosis/survival balance. Herein, in a mouse model of CRC, we found that the expression of UNC5A, UNC5B and UNC5C was diminished in tumors but only in mice subjected to a High Carbohydrate Diet (HCD) thus linking nutrition to their repression in CRC. O-GlcNAcylation is a nutritional sensor which has enhanced levels in CRC and regulates many cellular processes amongst epigenetics. We then investigated the putative involvement of O-GlcNAcylation in the epigenetic downregulation of the UNC5 family members. By a combination of pharmacological inhibition and RNA interference approaches coupled to RT-qPCR (Reverse Transcription-quantitative Polymerase Chain Reaction) analyses, promoter luciferase assay and CUT&RUN (Cleavage Under Target & Release Using Nuclease) experiments, we demonstrated that the O-GlcNAcylated form of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2) represses the transcription of UNC5A in human colon cancer cells. Collectively, our data support the hypothesis that O-GlcNAcylation could represent one link between nutrition and epigenetic downregulation of key tumor suppressor genes governing colon carcinogenesis including UNC5A.

Antitumor, Immunomodulatory and Antiangiogenic Efficacy of Medicinal Mushroom Extract Mixtures in Advanced Colorectal Cancer Animal Model.

Due to frequent drug resistance and/or unwanted side-effects during conventional and targeted cancer treatments, development of multi-target therapies is an important research field. Medicinal mushrooms’ isolated specific compounds and mushroom extracts have been already proven as non-toxic multi-target inhibitors of specific oncogenic pathways, as well as potent immunomodulators. However, research on antitumor effects of multiple-species extract mixtures was limited so far. The aim of this study was therefore, a study of medicinal mushroom preparations AGARIKON.1 and AGARIKON PLUS on colorectal cell lines in vitro and colorectal mice model in vivo. We found a significant antiproliferative and pro-apoptotic effect of tested medicinal mushroom preparations on colorectal (HCT-116, SW620) tumor cell lines, while the effect on human fibroblast cell line (WI-38) was proliferative emphasizing a specificity towards tumor cell lines. We further investigated the effect of the medicinal mushroom preparations AGARIKON.1 and AGARIKON PLUS in various combinations with conventional cytostatic drug 5-fluorouracil in the advanced metastatic colorectal cancer mouse model CT26.WT. AGARIKON.1 and AGARIKON PLUS exhibited immunostimulatory and antiangiogenic properties in vivo which resulted in significantly increased survival and reduction in tumor volume. The antitumor effects of AGARIKON.1 and AGARIKON PLUS, with or without 5-fluorouracil, are based on M1 macrophage polarization enhancement, inhibition of M2 and tumor-associated macrophage (TAM) polarization, effects on T helper cell Th1/Th2/Th17 cytokine profiles, direct inhibition of CT26.WT tumor growth, inhibition of vascular endothelial growth factors (VEGF) and metalloproteinases 2 and 9 (MMP-2 and MMP-9) modulation. The administration of AGARIKON.1 and AGARIKON PLUS did not show genotoxic effect. This data provides good basis for an expanded translational study.

Ribosome Biogenesis Alterations in Colorectal Cancer.

Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

Paroxetine combined with fluorouracil plays a therapeutic role in mouse models of colorectal cancer with depression through inhibiting IL-22 expression to regulate the MAPK signaling pathway.

The objective of the present study was to observe the therapeutic effect of paroxetine combined with fluorouracil on mice with colorectal cancer (CRC) complicated with depression and to explore its mechanism of action. Using chronic mild stress and xenograft tumor methods to model CRC complicated with depression, 60 BALB/c mice were randomly divided into control, tumor model, tumor depression model, tumor depression antidepressant, tumor depression chemotherapy and tumor depression antidepressant plus chemotherapeutic drug groups. Changes in mouse sucrose preference and forced swimming tests were tracked. Changes in tumor volume and weight were compared, the tumor inhibition rate was calculated, Ki-67 expression in tumor tissues was detected using immunohistochemistry and IL-22 levels in peripheral blood were detected using ELISAs. Additionally, protein expression levels of IL-22, Bcl-2, Bax, caspase-3, p38, phosphorylated (p)-p38, ERK, p-ERK, JNK and p-JNK in tumor tissue were detected using western blotting. Following treatment with paroxetine and chemotherapy drugs, the sucrose preference index was increased, autonomic behavior dysfunction was alleviated and tumor growth was significantly inhibited. Furthermore, the expression levels of Ki-67 and apoptosis-related proteins, Bax and caspase-3, increased in tumor tissues, anti-apoptosis protein Bcl2 expression levels decreased significantly, IL-22 levels in the blood and tumor tissues were reduced and p-p38, p-ERK and p-JNK proteins were significantly reduced. It was concluded that paroxetine combined with chemotherapy drugs improved depressive behavior and promoted the survival state in a mouse model of CRC and depression, possibly through inhibiting IL-22 expression to regulate the activity of the MAPK signaling pathway.

The Fragile X Mental Retardation Protein Regulates RIPK1 and Colorectal Cancer Resistance to Necroptosis.

Background & AimsThe fragile X mental retardation protein (FMRP) affects multiple steps of the mRNA metabolism during brain development and in different neoplastic processes. However, the contribution of FMRP in colon carcinogenesis has not been investigated.MethodsFMR1 mRNA transcript and FMRP protein expression were analyzed in human colon samples derived from patients with sporadic colorectal cancer (CRC) and healthy subjects. We used a well-established mouse model of sporadic CRC induced by azoxymethane to determine the possible role of FMRP in CRC. To address whether FMRP controls cancer cell survival, we analyzed cell death pathway in CRC human epithelial cell lines and in patient-derived colon cancer organoids in presence or absence of a specific FMR1 antisense oligonucleotide or siRNA.ResultsWe document a significant increase of FMRP in human CRC relative to non-tumor tissues. Next, using an inducible mouse model of CRC, we observed a reduction of colonic tumor incidence and size in the Fmr1 knockout mice. The abrogation of FMRP induced spontaneous cell death in human CRC cell lines activating the necroptotic pathway. Indeed, specific immunoprecipitation experiments on human cell lines and CRC samples indicated that FMRP binds receptor-interacting protein kinase 1 (RIPK1) mRNA, suggesting that FMRP acts as a regulator of necroptosis pathway through the surveillance of RIPK1 mRNA metabolism. Treatment of human CRC cell lines and patient-derived colon cancer organoids with the FMR1 antisense resulted in up-regulation of RIPK1.ConclusionsAltogether, these data support a role for FMRP in controlling RIPK1 expression and necroptotic activation in CRC.

Establishment of an Endoscopy-Guided Minimally Invasive Orthotopic Mouse Model of Colorectal Cancer.

Simple SummaryOpen orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these limitations, this study aimed to evaluate the establishment of an endoscopy-guided minimally invasive model without laparotomy. Different concentrations of the murine CRC cell lines CT26 and MC38 were endoscopically injected into the colorectal wall of BALB/C and C57BL/6J mice, respectively. Consistent tumor growth with the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. Analysis of the learning curve demonstrated that this model is easy to learn and quick to establish. It enables intra-individual follow-up endoscopies, and features tumors to study mechanisms of metastasis and the interaction with the immune system. The application of specific cell lines and concentrations enables a controlled local tumor growth and metastatic formation within short observation periods.Open orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these drawbacks, this study aimed to evaluate the establishment of a minimally invasive model using murine colonoscopy. CT26 and MC38 CRC cells of different concentrations were injected into BALB/C and C57BL/6J mice, respectively. Follow-up endoscopies were performed to assign an endoscopic score to tumor growth. Gross autopsy, histologic and immuno-histochemical evaluation, and immune scoring were performed. To describe the learning curve of the procedures, a performance score was given. Local tumor growth with colorectal wall infiltration, luminal ulceration, the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. The tumors showed cytoplasmic immuno-staining for CK20. Compared to the MC38/C57BL/6J model, tumorigenicity and immunogenicity of the CT26/BALB/C model were higher. Tumor volume correlated with the endoscopic score. This endoscopy-guided orthotopic mouse model is easy to learn and quick to establish. It features early metastasis and enables the study of interactions with the immune system. When specific cell concentrations and cell lines are applied, controlled local tumor growth and metastasis can be achieved within short observation periods.

The Wnt-β-Catenin-IL-10 Signaling Axis in Intestinal APCs Protects Mice from Colitis-Associated Colon Cancer in Response to Gut Microbiota.

Loss of immune tolerance to gut microflora is inextricably linked to chronic intestinal inflammation and colitis-associated colorectal cancer (CAC). The LRP5/6 signaling cascade in APCs contributes to immune homeostasis in the gut, but whether this pathway in APCs protects against CAC is not known. In the current study, using a mouse model of CAC, we show that the LRP5/6-β-catenin-IL-10 signaling axis in intestinal CD11c+ APCs protects mice from CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of LRP5/6 in CD11c+ APCs in mice (LRP5/6ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of proinflammatory factors and decreased expression of the immunosuppressive cytokine IL-10. This condition could be improved in LRP5/6ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor-promoting inflammatory factors in CD11c+ APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin specifically in CD11c+ APCs or in vivo administration of IL-10 protected LRP5/6ΔCD11c mice from CAC by suppressing the expression of inflammatory factors. In summary, in this study, we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in resolving chronic intestinal inflammation and protecting against CAC in response to the commensal flora.

Interplay between APC and ALDH1B1 in a newly developed mouse model of colorectal cancer

BackgroundColorectal cancer (CRC) is a leading cause of cancer mortality worldwide. Mutations in the adenomatous polyposis coli (APC) gene are pivotal in colorectal tumorigenesis. Recently, we demonstrated that aldehyde dehydrogenase 1B1 (ALDH1B1) knockdown dramatically reduced colon tumor growth in a mouse xenograft model. The purpose of the present preliminary study is to examine the effect of loss of ALDH1B1 in CRC development in an inducible colon-specific Apc mouse model. MethodsApcW/FCdx2ERT2-Cre mice develop uni-allelic inactivation of Apc specifically in colon epithelial cells following tamoxifen treatment. Aldh1b1−/− KO mice were crossed with ApcW/FCdx2ERT2-Cre mice. Six-month-old male ApcW/FCdx2ERT2-Cre/Aldh1b1−/−, and ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ mice were treated with tamoxifen (50 mg/kg, i.p.) for three consecutive days. ApcW/F/Aldh1b1−/− and ApcW/F/Aldh1b1+/+ mice were treated with corn oil (i.e., tamoxifen vehicle control) for three consecutive days. Eighteen days later, mice were sacrificed and their colons examined microscopically, macroscopically and histologically for the presence of adenoma. ResultsAll ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ and ApcW/FCdx2ERT2-Cre/Aldh1b1−/− mice treated with tamoxifen developed colorectal adenoma. The ApcW/FCdx2ERT2-Cre/Aldh1b1−/− mice showed a significant decrease in the total volume of all ileal and colonic adenomas, and decreased incidence of large colonic adenoma compared to ApcW/FCdx2ERT2-Cre/Aldh1b1+/+ mice. Immunohistochemical analysis of p53 and β-catenin showed a trend toward decreased expression score in colonic adenomas of ApcW/FCdx2ERT2-Cre/Aldh1b1−/− mice. ConclusionThe present preliminary study suggests that deletion of ALDH1B1 may protect against the full development of colorectal cancer. Further mechanistic studies are required to elucidate how ALDH1B1 contributes for colorectal cancer.

Alteration of fecal microbiota by fucoxanthin results in prevention of colorectal cancer in AOM/DSS mice.

Fucoxanthin (Fx), a marine carotenoid found in edible brown algae, is well known for having anticancer properties. The gut microbiota has been demonstrated as a hallmark for colorectal cancer progression in both humans and rodents. However, it remains unclear whether the gut microbiota is associated with the anticancer effect of Fx. We investigated the chemopreventive potency of Fx and its effect on gut microbiota in a mouse model of inflammation-associated colorectal cancer (by azoxymethane/dextran sulfate sodium treatment). Fx administration (30 mg/kg bw) during a 14 week period significantly inhibited the multiplicity of colorectal adenocarcinoma in mice. The number of apoptosis-like cleaved caspase-3high cells increased significantly in both colonic adenocarcinoma and mucosal crypts. Fx administration significantly suppressed Bacteroidlales (f_uc; g_uc) (0.3-fold) and Rikenellaceae (g_uc) (0.6-fold) and increased Lachnospiraceae (g_uc) (2.2-fold), compared with those of control mice. Oral administration of a fecal suspension obtained from Fx-treated mice, aimed to enhance Lachnospiraceae, suppress the number of colorectal adenocarcinomas in azoxymethane/dextran sulfate sodium-treated mice with a successful increase in Lachnospiraceae in the gut. Our findings suggested that an alteration in gut microbiota by dietary Fx might be an essential factor in the cancer chemopreventive effect of Fx in azoxymethane/dextran sulfate sodium-treated mice.

Toll-like receptor 4 prevents AOM/DSS-induced colitis-associated colorectal cancer in Bacteroides fragilis gnotobiotic mice.

Bacteroides fragilis (BF) plays a critical role in developing and maintaining the mammalian immune system. We previously found that BF colonization could prevent inflammation and tumor formation in a germ-free (GF) colitis-associated colorectal cancer (CAC) mouse model. The role of Toll-like receptor 4 (TLR4) in CAC development has not been clearly elucidated in BF mono-colonized gnotobiotic mice. The wild-type (WT) and TLR4 knockout (T4K) germ-free mice were raised with or without BF colonization for 28 days (GF/WT, GF/T4K, BF/WT, and BF/T4K) and then CAC was induced under azoxymethane (AOM)/dextran sulfate sodium (DSS) administration. The results showed that tumor formation and tumor incidence were significantly inhibited in the BF/WT group compared to those observed in the GF/WT group. However, the tumor prevention effect was not observed in the BF/T4K group unlike in the BF/WT group. Moreover, the CAC histological severity of the BF/WT group was ameliorated, but more severe lesions were found in the GF/WT, GF/T4K, and BF/T4K groups. Immunohistochemistry showed decreased cell proliferation (PCNA, β-catenin) and inflammatory markers (iNOS) in the BF/WT group compared to those in the BF/T4K group. Taken together, BF mono-colonization of GF mice might prevent CAC via the TLR4 signal pathway.