Mouse Model Of Colorectal Cancer Research Articles

Human Soluble TRAIL Secreted by Modified Lactococcus lactis Bacteria Promotes Tumor Growth in the Orthotopic Mouse Model of Colorectal Cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of sensitive cancer cells, including colorectal cancer (CRC). Due to its short biological half-life after intravenous administration and related clinical ineffectiveness, novel formulations of TRAIL need to be developed. Here we propose Lactococcus lactis bacteria as a vehicle for local delivery of human soluble TRAIL (hsTRAIL) in CRC. The use of common probiotics targeting guts as carriers for TRAIL could ensure its sustained release at the tumor site and extend the duration of its activity. We have already engineered hsTRAIL-secreting L.lactis bacteria and showed their effectiveness in elimination of human CRC cells in vitro and in vivo in a mouse subcutaneous model. Here, L.lactis(hsTRAIL+) were administered by gastric gavage to SCID mice with orthotopically developed HCT116 tumor in cecum, in monotherapy or in combination with metformin (MetF), already shown to enhance the hsTRAIL anti-tumor activity in subcutaneous CRC model. Oral administration of L.lactis(hsTRAIL+) resulted in significant progression of HCT116 tumors and shortening of the colon crypts. Secretion of hsTRAIL in the colon was accompanied by infiltration of the primary tumor with M2-macrophages, while MetF promoted transient colonization of the gut by L.lactis. Our study indicates that L.lactis bacteria after oral administration enable delivery of biologically active hsTRAIL to colon, however its potential therapeutic effect in CRC treatment is abolished by its pro-tumorigenic signalling, leading to the recruitment of M2-macrophages and tumor growth promotion.

Interleukin-17 directly stimulates tumor infiltrating Tregs to prevent cancer development.

Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.

HDAC6 inhibitor ACY-1215 enhances STAT1 acetylation to block PD-L1 for colorectal cancer immunotherapy

The search for effective combination therapy with immune checkpoint inhibitors (ICI) has become important for cancer patients who do not respond to the ICI well. Histone deacetylases (HDACs) inhibitors have attracted wide attention as anti-tumor agents. ACY-1215 is a selective inhibitor of HDAC6, which can inhibit the growth of a variety of tumor. We previously revealed that HDAC family is highly expressed in colorectal cancer specimens and mouse models. In this study, ACY-1215 was combined with anti-PD1 to treat tumor-bearing mice associated with colorectal cancer. ACY-1215 combined with anti-PD1 effectively inhibited the colorectal tumor growth. The expression of PD-L1 in tumor of mice were inhibited by ACY-1215 and anti-PD1 combination treatment, whereas some biomarkers reflecting T cell activation were upregulated. In a co-culture system of T cells and tumor cells, ACY-1215 helped T cells to kill tumor cells. Mechanically, HDAC6 enhanced the acetylation of STAT1 and inhibited the phosphorylation of STAT1, thus preventing STAT1 from entering the nucleus to activate PD-L1 transcription. This study reveals a novel regulatory mechanism of HDAC6 on non-histone substrates, especially on protein acetylation. HDAC6 inhibitors may be of great significance in tumor immunotherapy and related combination strategies.

Novel chromium (III)-based compound for inhibition of oxaliplatin-resistant colorectal cancer progression.

Colorectal cancer (CRC) ranks as the third leading cause of cancer-related mortality worldwide. The current standard of care includes systemic chemotherapy with cytotoxic agents, offering palliative relief for severe CRC cases and serving as the primary therapy for metastatic recurrence. However, the development of chemoresistance poses a substantial obstacle in the realm of chemotherapy. This study delved into the potential of a novel chromium (III)-based compound, hexaacetotetraaquadihydroxochromium (III) diiron (III) nitrate, for CRC treatment. The therapeutic promise of this innovative chromium (III)-based compound was explored by utilizing LoVo colon cancer cells and an in-vivo mouse model of CRC. Various dosages of the compound were administered to LoVo parental cells and LoVo oxaliplatin-resistant cells. Findings unveiled that a concentration of 2000 μg/mL of the chromium (III) compound significantly inhibited mesenchymal transition and the migratory and invasive properties of LoVo oxaliplatin-resistant cells. This novel chromium (III)-based compound also demonstrated similar efficacy in other different CRC cell lines. The tumor growth was in the in-vivo mouse model was reduced by this compound. Moreover, the chromium (III)-based compound induced apoptosis by triggering the endoplasmic reticulum (ER) stress pathway in LoVo oxaliplatin-resistant cells. This study illuminates the capacity of the novel chromium (III)-based compound to impede the progression and growth of chemotherapy-resistant CRC. This discovery instills confidence in the potential of this compound as a therapeutic agent for CRC, even in the face of drug resistance. It holds the promise of serving as a valuable asset in the future treatment of chemotherapy-resistant CRC.

Anti-Prokineticin1 Suppresses Liver Metastatic Tumors in a Mouse Model of Colorectal Cancer with Liver Metastasis.

Multidisciplinary treatment for colorectal cancer (CRC) has undergone significant advances, and molecularly targeted drugs have substantially improved patient prognosis. However, one problem with current molecularly targeted therapeutics is that they must be used in combination with anticancer agents. New molecular targeted therapies that can be used alone are needed. We have previously identified prokineticin1 (PROK1) factor as a therapeutic potential target for CRC. PROK1 factor is involved in the angiogenesis of tissues surrounding CRC tumors. Additionally, PROK1 receptors 1 and 2 are expressed in CRC cell lines, playing roles in cell proliferation via an autocrine mechanism and in the signaling system. In this study, a liver metastasis mouse model was developed using human colorectal cancer cell lines, and mice were divided into anti-PROK1 antibody administration and control groups. Mice were treated intraperitoneally with antibodies or phosphate-buffered saline (control) every three days. The number, size, and cell proliferation ability of metastatic lesions were analyzed. Our results suggested that the number, size, and cancer cell proliferation ability of metastatic lesions decreased, and the survival time significantly increased in the antibody-treated group compared to those in the control group. Thus, the anti-PROK1 antibody therapy suppressed the cell proliferation ability of liver metastatic lesions in a CRC mouse model, suggesting its potential as a novel treatment strategy.

Cationic micelle delivery of a multi-epitope vaccine candidate derived from tumor-associated antigens, causing regression in established CT26 colorectal tumors in mice.

Among all the cancers, colorectal cancer (CRC) has the third mortality rank in both genders. Cancer vaccines have shown promising results in boosting patients' immune systems to fight cancer. Using the IEDB database, we predicted mouse MHC-I (H2-Ld) binding epitopes from four tumor-associated antigens (APC, KRAS, TP53, and PIK3CA) and designed a multi-epitope vaccine. We expressed the candidate vaccine and encapsulated it into the cationic micelle with polyethyleneimine conjugated to oleic acid as its building blocks. We studied tumor inhibition effect, cytokine production, and lymphocyte proliferation in the mouse CRC model after vaccination. Our finding illustrated significant tumor growth inhibition in mouse models treated with the candidate nanovaccine. Besides the significant release of IFN-γ and IL-4 by immunized mouse spleen T-lymphocytes, T-cell proliferation assay results confirmed effective immune response after the vaccination. These results demonstrate the potential therapeutic effects of nanovaccines and could be a possible approach to CRC immunotherapy.

An exploration of the effect of Chinese herbal compound on the occurrence and development of large intestine cancer and intestinal flora

This study was conducted to observe the effect of Chinese herbal compound on the treatment of colon cancer using AOM/DSS-induced C57BL/6J colon cancer mice and to validate potential influence on intestinal flora of mice. A colorectal cancer (CRC) mouse model was built with a total of 50 C57BL/6J mice that were induced by administrating AOM/DSS. These experimental animals were split up into 5 groups, a control group, a model group, and low-, medium- and high-dose Chinese herbal compound groups. All mice were given Chinese herbal compound treatment, and the colon tissues of each group were harvested with the length measured and the number of colon polyps accounted. The Ki-67 expression in the colon tissues was detected via immuno-histochemistry. Relative quantification of the expression of genes and proteins was determined through qPCR and WB assays. Contents of IL-6, TNF-α, IFN-γ, and IL-10 in serum and colon tissues of mice were determined by ELISA. An additional 16S rRNA sequencing analysis was implemented for the identification of mouse intestinal flora. The results suggested that all low-, medium- or high-dose Chinese herbal compound could markedly inhibit the shortening of colon length and significant number reduction of colon polyps in the model group. The relative expression of genes and proteins (PCNA, Muc16, and MMP-9) associated with proliferation in mouse colon tissues were inhibited. In addition, compared with the model group, the contents of IL-6, TNF-α, and IFN-γ in serum and colon tissues were substantially decreased in the high-dose Chinese herbal compound group, thereby reducing the structure damage in colon tissues and the infiltration degree of inflammatory cells. Besides, the expression of TLR4/MyD88/NF-κB protein was markedly decreased. The 16S rRNA sequencing analysis demonstrated that mice in the model group had decreased intestinal flora diversity, and there were significant changes in flora abundance and amino acid metabolism between the control group and the model group. Taken together, the treatment of Chinese herbal compound against CRC in this study might be regulated by the TLR4/MyD88/NF-κB signaling pathway, and the imbalance in intestinal flora was also closely related to CRC occurrence.

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Dendritic Cell-Based In Situ Nanovaccine for Reprogramming Lipid Metabolism to Boost Tumor Immunotherapy.

Cancer vaccines have been considered to be an alternative therapeutic strategy for tumor therapy in the past decade. However, the popularity and efficacy of cancer vaccines were hampered by tumor antigen heterogeneity and the impaired function of cross-presentation in the tumor-infiltrating dendritic cells (TIDCs). To overcome these challenges, we engineered an in situ nanovaccine (named as TPOP) based on lipid metabolism-regulating and innate immune-stimulated nanoparticles. TPOP could capture tumor antigens and induce specific recognition by TIDCs to be taken up. Meanwhile, TPOP could manipulate TIDC lipid metabolism and inhibit de novo synthesis of fatty acids, thus improving the ability of TIDCs to cross-present by reducing their lipid accumulation. Significantly, intratumoral injection of TPOP combined with pretreatment with doxorubicin showed a considerable therapeutic effect in the subcutaneous mouse colorectal cancer model and melanoma model. Moreover, in combination with immune checkpoint inhibitors, such TPOP could markedly inhibit the growth of distant tumors by systemic antitumor immune responses. This work provides a safe and promising strategy for improving the function of immune cells by manipulating their metabolism and activating the immune system effectively for in situ cancer vaccines.

Colon-targeted S100A8/A9-specific peptide systems ameliorate colitis and colitis-associated colorectal cancer in mouse models.

The link between chronic inflammation and cancer development is well acknowledged. Inflammatory bowel disease including ulcerative colitis and Crohn's disease frequently promotes colon cancer development. Thus, control of intestinal inflammation is a therapeutic strategy to prevent and manage colitis-associated colorectal cancer (CRC). Recently, gut mucosal damage-associated molecular patterns S100A8 and S100A9, acting via interactions with their pattern recognition receptors (PRRs), especially TLR4 and RAGE, have emerged as key players in the pathogenesis of colonic inflammation. We found elevated serum levels of S100A8 and S100A9 in both colitis and colitis-associated CRC mouse models along with significant increases in their binding with PRR, TLR4, and RAGE. In this study we developed a dual PRR-inhibiting peptide system (rCT-S100A8/A9) that consisted of TLR4- and RAGE-inhibiting motifs derived from S100A8 and S100A9, and conjugated with a CT peptide (TWYKIAFQRNRK) for colon-specific delivery. In human monocyte THP-1 and mouse BMDMs, S100A8/A9-derived peptide comprising TLR4- and RAGE-interacting motif (0.01, 0.1, 1 μM) dose-dependently inhibited the binding of S100 to TLR4 or RAGE, and effectively inhibited NLRP3 inflammasome activation. We demonstrated that rCT-S100A8/A9 had appropriate drug-like properties including in vitro stabilities and PK properties as well as pharmacological activities. In mouse models of DSS-induced acute and chronic colitis, injection of rCT-S100A8/A9 (50 μg·kg-1·d-1, i.p. for certain consecutive days) significantly increased the survival rates and alleviated the pathological injuries of the colon. In AOM/DSS-induced colitis-associated colorectal cancer (CAC) mouse model, injection of rCT-S100A8/A9 (50 μg·kg-1·d-1, i.p.) increased the body weight, decreased tumor burden in the distal colon, and significantly alleviated histological colonic damage. In mice bearing oxaliplatin-resistant CRC xenografts, injection of rCT-S100A8/A9 (20 μg/kg, i.p., every 3 days for 24-30 days) significantly inhibited the tumor growth with reduced EMT-associated markers in tumor tissues. Our results demonstrate that targeting the S100-PRR axis improves colonic inflammation and thus highlight this axis as a potential therapeutic target for colitis and CRC.

Expression of FIBCD1 by intestinal epithelial cells alleviates inflammation-driven tumorigenesis in a mouse model of colorectal cancer

BackgroundColorectal cancer (CRC) ranks as the third most prevalent cancer globally, highlighting the pressing need to address its development. Inflammation plays a crucial role in augmenting the risk of CRC and actively contributes to all stages of tumorigenesis. Consequently, targeting early inflammatory responses in the intestinal tract to restore homeostasis holds significant potential for preventing and treating CRC. Fibrinogen C domain-containing 1 (FIBCD1), a chitin-binding transmembrane protein predominantly found on human intestinal epithelial cells (IECs), has garnered attention in previous research for its ability to effectively suppress inflammatory responses and promote tissue homeostasis at mucosal barriers.MethodsIn this study, we investigated the role of FIBCD1 in CRC development using transgenic mice that mimic human expression of FIBCD1 at the intestinal mucosal barrier. To model aspects of CRC, we employed the azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. Additionally, we examined the expression pattern of FIBCD1 in surgical specimens obtained from human CRC patients by immunohistochemical methods. By accessing public data repositories, we further evaluated FIBCD1 expression in colon adenocarcinoma and explored survival outcomes associated with FIBCD1 expression.ResultsHere, we demonstrate that FIBCD1 substantially impacts CRC development by significantly reducing intestinal inflammation and suppressing colorectal tumorigenesis in mice. Furthermore, we identify a soluble variant of FIBCD1 that is significantly increased in feces during acute inflammation. Finally, we demonstrate increased expression of FIBCD1 by immunohistochemistry in human CRC specimens at more developed tumor stages. These results are further supported by bioinformatic analyses of publicly available repositories, indicating increased FIBCD1 expression in tumor tissues, where higher expression is associated with unfavorable prognosis.ConclusionCollectively, these findings suggest that FIBCD1 influences early inflammatory responses in the AOM/DSS model, leading to a reduction in tumor size and burden. The increased expression of FIBCD1 in human CRC samples raises intriguing questions regarding its role in CRC, positioning it as a compelling candidate and novel molecular target for future research.

One Mutation to Rule Them All: Mutant KRAS Controls Tumor Intrinsic and Microenvironment Signaling.

In a recent study published in Cancer Discovery, Hsu and colleagues employ an elegant combination of single-cell and bulk RNA sequencing experiments from mouse and human colorectal cancer samples, patient-derived organoids, two-dimensional in vitro systems, and in vivo validation in genetically engineered colorectal cancer mouse models to investigate how mutant KRAS (KRAS*) impacts the tumor microenvironment. They identify a molecular signaling cascade downstream of KRAS* that activates a specific program of lipid-rich cancer-associated fibroblasts, promoting tumor angiogenesis and progression. These findings may lead to new therapeutic strategies for patients with colorectal cancer with KRAS*.

HSPA8 Activates Wnt/β-Catenin Signaling to Facilitate BRAF V600E Colorectal Cancer Progression by CMA-Mediated CAV1 Degradation.

BRAF V600E attracts wide attention in the treatment of colorectal cancer (CRC) as stratifying and predicting a refractory classification of CRC. Recent evidence indicates that Wnt/β-catenin signaling is broadly activated and participates in the refractoriness of BRAF V600E CRC, but the underlying molecular mechanism needs to be elucidated. Here, heat shock 70 kDa protein 8 (HSPA8), an essential regulator in chaperone-mediated autophagy (CMA), is identified as a potential therapeutic target for advanced BRAF V600E CRC. These results show that HSPA8 is transcriptionally upregulated in BRAF V600E CRC, which promotes CMA-dependent degradation of caveolin-1 (CAV1) to release β-catenin into the nucleus and thus activates the Wnt/β-catenin pathway, contributing to metastasis and progression of BRAF V600E CRC. Of note, HSPA8 directly interacts with the KIFSN motif on CAV1, the interaction can be enhanced by p38 MAPK-mediated CAV1 S168 phosphorylation. Furthermore, pharmacological targeting HSPA8 by VER155008 exhibits synergistic effects with BRAF inhibitors on CRC mouse models. In summary, these findings discover the important role of the HSPA8/CAV1/β-catenin axis in the development of refractory BRAF V600E CRC and highlight HSPA8 as a predictive biomarker and therapeutic target in clinical practice.

Combination of Anti-PD-1 and Electroacupuncture Induces a Potent Antitumor Immune Response in Microsatellite-Stable Colorectal Cancer.

Programmed death receptor-1 (PD-1) inhibitors are ineffective against microsatellite-stable (MSS) colorectal cancer. Electroacupuncture (EA) has oncosuppressive and immunomodulatory properties. Here, we investigated the antitumor effects of EA and explored the feasibility of EA combined with anti-PD-1 in MSS colorectal cancer. Results showed that EA exerted its antitumor effect in an intensity-specific manner, and moderate-intensity EA (1.0 mA) induced maximal tumor inhibition. EA enhanced antitumor immune responses by increasing lymphocytes and granzyme B (GzmB) levels, as well as activating the stimulator of IFN genes (STING) pathway. EA combined with anti-PD-1 showed superior efficacy compared with either monotherapy in multiple MSS colorectal cancer mouse models. Single-cell RNA sequencing revealed that cotreatment reprogrammed the tumor immune microenvironment (TIME), as characterized by enhancement of cytotoxic functions. Mechanically, we found that the potentiated effect of EA was dependent upon the STING pathway. Collectively, EA reshapes the TIME of MSS colorectal cancer and sensitizes tumors to anti-PD-1 in a STING pathway-dependent manner. These results provide a mechanistic rationale for using EA as an immunomodulatory strategy to improve the clinical efficacy of anti-PD-1 in MSS colorectal cancer. EA is safe, well-tolerated, and feasible for clinical translation as a promising strategy for treating MSS colorectal cancer.

Mechanism of Qizhen decoction-mediated maturation of DC cells to activate the IL-12/JAK2/STAT4 pathway to sensitise PD-1 inhibitors in the treatment of colorectal cancer

Ethnopharmacological relevanceTraditional Chinese medicine has been utilized to treat colorectal cancer (CRC). Qizhen decoction (QZD), a potential compound prescription of traditional Chinese medicine, possesses multiple biological activities. It has been used to treat CRC in clinical practice and has been proven to be effective. Aim of the studyTo investigate the impact of QZD supported by intestinal flora in combination with PD-1 inhibitor on colorectal cancer, and to elucidate the mechanism by which QZD enhances the sensitivity of PD-1 inhibitor against colorectal cancer. Materials and methodsObservation of Intestinal Flora Mediating the Effect of QZD Combined with PD-1 Inhibitor in the Treatment of Colorectal Cancer. We used Flow cytometry and qPCR to detect the effect of QZD combined with PD-1 inhibitor on the activation of effector T cells in a wild mouse model of colorectal cancer. In wild and germ-free mouse models, the differences in inflammatory factors, pathological change, body mass, colorectal length, and tumour load were observed. In the study of the mechanism of QZD combined with PD-1 inhibitor in the treatment of colorectal cancer, the study evaluated the abundance of Akkermansia, the phenotypes of effector T cells and DC cells, as well as inflammatory factors in each group of mice to determine whether Akkermansia played a role in activating DC cells. Based on the JAK2/TYK2/STAT4 pathway, the mechanism of PD-1 inhibitor sensitisation by QZD in colorectal cancer was further investigated. ResultsWe found that QZD combined with PD-1 inhibitor could improve the therapeutic effect on colorectal cancer by inducing more critical immune functions. QZD promotes increased Akkermansia abundance in the gut. Akkermansia promotes maturation of DC cells, and mature DC cells activate the IL-12/JAK2/STAT4 pathway, which significantly activates effector T cells. Akkermansia is key to QZD combined with PD-1 inhibitor-mediated immunity exerting a therapeutic effect on colorectal cancer. ConclusionThe mechanism of action of the QZD sensitizing PD-1 inhibitor is to promote the maturation of DC cells to release IL-12 and activate the JAK2/STAT4 pathway to induce effector T cell activation by increasing the abundance of Akkermansia.

Heart failure stimulates colorectal tumour growth through gut microbial alterations of specific populations

Abstract Background/purpose The field of reverse cardio-oncology has received growing attention in the last years, since epidemiological studies showed that patients with HF are at increased risk of developing cancer. In addition, HF can stimulate tumour growth directly in distant organs, including colon cancer. The exact mechanisms underlying this phenomenon, however, remain largely unknown. The connection between the microbiome and colorectal cancer (CRC) has been studied extensively and microbial dysbiosis in CRC is linked to cancer progression. Recent studies have also shown that HF patients display microbial dysbiosis. We hypothesized that microbial dysbiosis might contribute to HF-induced CRC tumour growth. Methods C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16s rRNA sequencing, and pooled for faecal microbial transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane (AOM)/Dextran sodium sulphate (DSS). The CRC mice were transplanted with faeces from MI or sham mice. Disease progression was monitored using the disease severity score and endoscopy. After ten weeks mice were sacrificed, and tumour number and proliferation were quantified. All experimental procedures were performed in accordance with the European Union guidelines (CCD_8141, the Netherlands, LA_1400637, Belgium). Results MI-induced HF resulted in a decrease in microbial diversity (Shannon diversity p=0.018), characterized by a decrease in short chain fatty acid-producing bacteria, including the genera Blautia (p<0.01) and Roseburia (p<0.01). In addition, caecal butyrate levels were decreased in MI mice (p=0.029). These changes were largely comparable to microbial changes observed in HF patients and CRC patients. Interestingly, faecal transplantation with faeces from MI mice resulted in faster growth of CRC tumours in mice treated with AOM/DSS. This was demonstrated by a higher endoscopy score (7.9 ±2.5 vs. 10 ±2.4, p=0.038), indicative of worse disease progression, increased tumour counts (22 ±5.3 vs. 29 ±6, p<0.01), and a higher percentage of Ki67 positive cells in tumour tissue (p=0.012), relative to AOM/DSS treated mice transplanted with faeces from sham mice. Sequencing data revealed several differences in the microbial composition of mice transplanted with faeces from MI mice compared to mice transplanted with faeces from sham mice. Conclusion Our results demonstrate that HF induces microbial dysbiosis in mice. In addition, we show, for the first time, using a faecal transplantation model, that HF-induced microbial dysbiosis is causally involved in CRC tumour growth in a mouse model of CRC. Targeting the microbiome may present a tool to mitigate HF associated co-morbidities, especially cancer.Graphical abstract

MiR-597-5p inhibits carcinogenesis and macrophage recruitment in colitis-related colorectal cancer via reducing the expression of CXCL5

ABSTRACT Despite being the subject of multiple cancer studies, nothing is known about miR-597-5p’s role in colitis-associated colorectal cancer (CAC). We intend to explore how miR-597-5p influences the growth and development of CAC. In order to construct a CAC model, mice were stimulated with azoxymethane (AOM)/dextran sulfate sodium (DSS). The in situ hybridization (ISH) and quantitative real-time polymerase chain reaction (qRT-PCR) was used for the detection of miR-597-5p expression. The protein expression of CXCL5 was determined by western blotting, immunohistochemistry and enzyme-linked immuno sorbent assay (ELISA). The histologic colitis score and hematoxylin and eosin (HE) staining were used to evaluate degree of damage to colonic tissues. The proportion of macrophages detected in colon tumors was also measured using flow cytometry. The transwell test was employed to assess macrophage migration. It was found that the miR-597-5p and its target CXCL5 had a negative correlation. MiR-597-5p expression was decreased, while CXCL5 expression was raised in CAC tissues. In AOM/DSS-induced mice, miR-597-5p deficiency in intestinal epithelial cells resulted in decreasing colon length as well as increasing tumor numbers and histologic colitis score, which was reversed by CXCL5 inhibition. MiR-597-5p deficiency facilitated macrophage recruitment in AOM/DSS-induced mice and promoted macrophage migration in vitro, which were reversed by CXCL5 inhibition. Deficiency of miR-597-5p aggravated macrophage recruitment and tumorigenesis in a mouse CAC model, suggesting that miR-597-5p agonists may have an anti-inflammatory therapeutic effect in inflammatory bowel diseases and reduce the risk of developing CAC.

Assessment of Novel Mesothelin-Specific Human Antibody Domain VH-Fc Fusion Proteins-Based PET Agents.

Mesothelin (MSLN) is a tumor-associated antigen found in a variety of cancers and is a target for imaging and therapeutic applications in MSLN-expressing tumors. We have developed high affinity anti-MSLN human VH domain antibodies, providing alternative targeting vectors to conventional IgG antibodies that are associated with long-circulating half-lives and poor penetration of tumors, limiting antitumor activity in clinical trials. Based on two newly identified anti-MSLN VH binders (3C9, 2A10), we generated VH-Fc fusion proteins and modified them for zirconium-89 radiolabeling to create anti-MSLN VH-Fc PET tracers. The focus of this study was to assess the ability of PET-imaging to compare the in vivo performance of anti-MSLN VH-Fc fusion proteins (2A10, 3C9) targeting different epitopes of MSLN vs IgG1 (m912; a clinical benchmark antibody with an overlapped epitope as 2A10) for PET imaging in a mouse model of colorectal cancer (CRC). The anti-MSLN VH-Fc fusion proteins were successfully modified and radiolabeled with zirconium-89. The resulting MSLN-targeted PET-imaging agents demonstrated specific uptake in the MSLN-expressing HCT116 tumors. The in vivo performance of the MSLN-targeted PET-imaging agents utilizing VH-Fc showed more rapid and greater accumulation and deeper penetration within the tumor than the full-length IgG1 m912-based PET-imaging agent. Furthermore, PET imaging allowed us to compare the pharmacokinetics of epitope-specific VH domain-based PET tracers. Overall, these data are encouraging for the incorporation of PET imaging to assess modified VH domain structures to develop novel anti-MSLN VH domain-based therapeutics in MSLN-positive cancers as well as their companion PET imaging agents.

Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

Tumor‑associated macrophages (TAMs) are pivotal components in colorectal cancer (CRC) progression, markedly influencing the tumor microenvironment through their polarization into the pro‑inflammatory M1 or pro‑tumorigenic M2 phenotypes. Recent studies have highlighted that the Grb2‑associated binder 2 (Gab2) is a critical gene involved in the development of various types of tumor, including CRC. However, the precise role of Gab2 in mediating TAM polarization remains incompletely elucidated. In the present study, it was discovered that Gab2 was highly expressed within CRC tissue TAMs, and was associated with a poor prognosis of patients with CRC. Functionally, it was identified that the tumor‑conditioned medium (TCM) induced Gab2 expression, facilitating the TAMs towards an M2‑like phenotype polarization. Of note, the suppression of Gab2 expression using shRNA markedly inhibited the TCM‑induced expression of M2‑associated molecules, without affecting M1‑type markers. Furthermore, the xenotransplantation model demonstrated that Gab2 deficiency in TAMs inhibited tumor growth in the mouse model of CRC. Mechanistically, Gab2 induced the M2 polarization of TAMs by regulatingthe AKT and ERK signaling pathways, promoting CRC growth and metastasis. In summary, the present study study elucidates that decreasing Gab2 expression hinders the transition of TAMs towards the M2 phenotype, thereby suppressing the growth of CRC. The exploration of the regulatory mechanisms of Gab2 in TAM polarization may enhance the current understanding of the core molecular pathways of CRC development and may thus provide a foundation for the development of novel immunotherapeutic strategies targeted against TAMs.

CLMP is a tumor suppressor that determines all-trans retinoic acid response in colorectal cancer

CAR-like membrane protein (CLMP) is a tight junction-associated protein whose mutation is associated with congenital short bowel syndrome (CSBS), but its functions in colorectal cancer (CRC) remain unknown. Here, we demonstrate that CLMP is rarely mutated but significantly decreased in CRC patients, and its deficiency accelerates CRC tumorigenesis, growth, and resistance to all-trans retinoic acid (ATRA). Mechanistically, CLMP recruits β-catenin to cell membrane, independent of cadherin proteins. CLMP-mediated β-catenin translocation inactivates Wnt(Wingless and INT-1)/β-catenin signaling, thereby suppressing CRC tumorigenesis and growth in ApcMin/+, azoxymethane/dextran sodium sulfate (AOM/DSS), and orthotopic CRC mouse models. As a direct target of Wnt/β-catenin, cytochrome P450 hydroxylase A1 (CYP26A1)-an enzyme that degrades ATRA to a less bioactive retinoid-is upregulated by CLMP deficiency, resulting in ATRA-resistant CRC that can be reversed by administering CYP26A1 inhibitor. Collectively, our data identify the anti-CRC role of CLMP and suggest that CYP26A1 inhibitor enable to boost ATRA's therapeutic efficiency.