Mouse Model Of Colorectal Cancer Research Articles

Co-inhibition of TGF-β and PD-L1 pathways in a metastatic colorectal cancer mouse model triggers interferon responses, innate cells and T cells, alongside metabolic changes and tumor resistance

ABSTRACT Colorectal cancer (CRC) is the third most prevalent cancer worldwide with a high mortality rate (20–30%), especially due to metastasis to adjacent organs. Clinical responses to chemotherapy, radiation, targeted and immunotherapies are limited to a subset of patients making metastatic CRC (mCRC) difficult to treat. To understand the therapeutic modulation of immune response in mCRC, we have used a genetically engineered mouse model (GEMM), “KPN”, which resembles the human ‘CMS4’-like subtype. We show here that transforming growth factor (TGF-β1), secreted by KPN organoids, increases cancer cell proliferation, and inhibits splenocyte activation in vitro. TGF-β1 also inhibits activation of naive but not pre-activated T cells, suggesting differential effects on specific immune cells. In vivo, the inhibition of TGF-β inflames the KPN tumors, causing infiltration of T cells, monocytes and monocytic intermediates, while reducing neutrophils and epithelial cells. Co-inhibition of TGF-β and PD-L1 signaling further enhances cytotoxic CD8+T cells and upregulates innate immune response and interferon gene signatures. However, simultaneous upregulation of cancer-related metabolic genes correlated with limited control of tumor burden and/or progression despite combination treatment. Our study illustrates the importance of using GEMMs to predict better immunotherapies for mCRC.

SLC26A9 promotes colorectal tumorigenesis by modulating Wnt/β-catenin signaling

Solute carrier family 26 member 9 (SLC26A9) is a member of the Slc26a family of multifunctional anion transporters that functions as a Cl- channel in parietal cells during acid secretion. We explored the role of SLC26A9 in colorectal cancer (CRC) and its related mechanisms through clinical samples from CRC patients, CRC cell lines and mouse models. We observed that SLC26A9 was expressed at low levels in the cytoplasm of adjacent tissues, polyps and adenomas but was significantly increased in colorectal adenocarcinoma. Moreover, increased levels of SLC26A9 were associated with a high risk of disease and poor prognosis. In addition, downregulation of SLC26A9 in CRC cells induced cell cycle arrest and apoptosis but inhibited cell proliferation and xenograft tumor growth both in vitro and in vivo. Mechanistic analysis revealed that SLC26A9 was colocalized with β-catenin in the nucleus of CRC cells. The translocation of these two proteins from the cytoplasm to the nucleus reflected the activation of Wnt/β-catenin signaling, and promoted the transcription of downstream target proteins, including CyclinD1, c-Myc and Snail, but inhibited the expression of cytochrome C (Cyt-c), cleaved Caspase9, cleaved Caspase3 and apoptosis-inducing factor (AIF). CRC is accompanied by alteration of epithelial mesenchymal transition (EMT) markers. Meanwhile, further studies showed that in SW48 cells, overexpressing SLC26A9 was cocultured with the β-catenin inhibitor XAV-939, β-catenin was downregulated, and EMT was reversed. Our study demonstrated SLC26A9 may be responsible for alterations in the proliferative ability and aggressive potential of CRC by regulating the Wnt/β-catenin signaling pathway.

Isolation and high-dimensional flow cytometric analysis of tumor-infiltrating leukocytes in a mouse model of colorectal cancer.

Colorectal cancer (CRC) is a complex and heterogeneous disease characterized by dysregulated interactions between tumor cells and the immune system. The tumor microenvironment plays a pivotal role in cancer initiation as well as progression, with myeloid immune cells such as dendritic cell and macrophage subsets playing diverse roles in cancer immunity. On one hand, they exert anti-tumor effects, but they can also contribute to tumor growth. The AOM/DSS colitis-associated cancer mouse model has emerged as a valuable tool to investigate inflammation-driven CRC. To understand the role of different leukocyte populations in tumor development, the preparation of single cell suspensions from tumors has become standard procedure for many types of cancer in recent years. However, in the case of AOM/DSS-induced colorectal tumors, this is still challenging and rarely described. For one, to be able to properly distinguish tumor-associated immune cells, separate processing of cancerous and surrounding colon tissue is essential. In addition, cell yield, due to the low tumor mass, viability, as well as preservation of cell surface epitopes are important for successful flow cytometric profiling of tumor-infiltrating leukocytes. Here we present a fast, simple, and economical step-by-step protocol for isolating colorectal tumor-associated leukocytes from AOM/DSS-treated mice. Furthermore, we demonstrate the feasibility of this protocol for high-dimensional flow cytometric identification of the different tumor-infiltrating leukocyte populations, with a specific focus on myeloid cell subsets.

Mechanic evaluation of Wu-Mei-Pill on colitis-associated colorectal cancer: An integrated transcriptomics, metabolomics, and experimental validation study

BackgroundChronic intestinal inflammatory diseases play a crucial role in the onset of colorectal cancer (CRC). Effectively impeding the progression of colitis-associated colorectal cancer (CAC) can be instrumental in hindering CRC development. Wu-Mei-Pill (WMP), a formulation comprising various herbal extracts, is clinically employed for CAC treatment, yet the underlying mechanism of WMP's efficacy in CAC remains unclear. Our study firstly demonstrated the effects and mechanisms of WMP on transcriptional and metabolic levels based on integrated transcriptomics and untargeted metabolomics and relative experimental validations. Materials and methodsA CAC mouse model was established through a single injection of azoxymethane (AOM) followed by intermittent dextran sodium sulfate (DSS) intervention, with subsequent WMP administration. Initially, the therapeutic impact of WMP on the CAC model was assessed by observing survival rate, body weight change, colon length, tumor number, tumor load, and pathological changes in the colon tissue of CAC mice post-WMP intervention. Subsequently, differential genes and metabolites in the colorectal tissue of CAC mice following WMP intervention were identified through transcriptomics and non-targeted metabolomics. Finally, the influence of WMP on the peroxisome proliferator activated receptor (PPAR) pathway, Wnt pathway, and CC motif chemokine ligand 3 (CCL3)/ CC motif chemokine receptor 1 (CCR1) axis in CAC mice was verified through western blot, immunofluorescence, and ELISA based on the results of transcriptomics and non-targeted metabolomics. ResultsWMP intervention enhanced survival, alleviated body weight loss, shortened colon length, tumor occurrence, and pathological changes in the colorectal tissue of CAC mice, such as glandular damage, tumourigenesis, and inflammatory cell infiltration. Transcriptomic and non-targeted metabolomic results revealed that WMP intervention up-regulated the expression of key regulatory mechanisms of fatty acid oxidation PPAR pathway-related genes (Pparg, Ppara, Cpt1a, and Acadm) and metabolites (L-carnitine and L-palmitoylcarnitine). Additionally, it down-regulated Wnt pathway-related genes (Wnt3, Axin2, Tcf7, Mmp7, Lgr5, Wnt5a, Fzd6, Wnt7b, Lef1, and Fzd10 etc.) and pro-inflammatory related genes (Il1b, Il6, Il17a, Ccl3, and Ccr1 etc.). Experimental validation demonstrated that WMP up-regulated PPAR pathway-related proteins [PPARγ, PPARα, carnitine palmitoyltransferase 1A (CPT1A), and acyl-CoA dehydrogenase medium chain (ACADM)] in the colorectal tissue of CAC mice. It also down-regulated Wnt pathway-related proteins [β-catenin, T-cell factor (TCF), lymphoid enhancer-binding factor (LEF), and matrix metallopeptidase 7 (MMP7)], inhibited the nuclear translocation of the key transcription factor β-catenin in the Wnt pathway, and suppressed epithelial-to-mesenchymal transition (EMT) activation induced by the Wnt pathway (up-regulated E-cadherin and down-regulated Vimentin). Furthermore, WMP intervention reduced pro-inflammatory factors [interleukin (IL)-6, IL-1β, and IL-17A] and decreased CCL3/CCR1 axis factors, including CCL3 protein levels and diminished F4/80+CCR1+ positive expressed cells. ConclusionWMP significantly inhibits CAC tumorigenesis by up-regulating PPARα-mediated fatty acid oxidation, inhibiting the Wnt signaling pathway-mediated EMT, and suppressing CCL3/CCR1-mediated inflammatory responses.

GTP binding protein 2 maintains the quiescence, self-renewal, and chemoresistance of mouse colorectal cancer stem cells via promoting Wnt signaling activation

Colorectal cancer (CRC) is one of the most common cancers and the second most deadly cancer across the globe. Colorectal cancer stem cells (CCSCs) fuel CRC growth, metastasis, relapse, and chemoresistance. A complete understanding of the modulatory mechanisms of CCSC biology is essential for developing efficacious CRC treatment. In the current study, we characterized the expression and function of GTP binding protein 2 (GTPBP2) in a chemical-induced mouse CRC model. We found that GTPBP2 was expressed at a higher level in CD133+CD44+ CCSCs compared with other CRC cells. Using a lentivirus-based Cas9/sgRNA system, GTPBP2 expression was ablated in CRC cells in vitro. GTPBP2 deficiency caused the following effects on CCSCs: 1) Significantly accelerating proliferation and increasing the proportions of cells at G1, S, and G2/M phase; 2) Impairing resistance to 5-Fluorouracil; 3) Weakening self-renewal but not impacting cell migration. In addition, GTPBP2 deficiency remarkably decreased β-catenin expression while increasing β-catenin phosphorylation in CCSCs. These effects of GTPBP2 were present in CCSCs but not in other CRC cell populations. The Wnt agonist SKL2001 completely abolished these changes in GTPBP2-deficient CCSCs. When GTPBP2-deficient CCSCs were implanted in nude mice, they exhibited consistent changes compared with GTPBP2-expressing CCSCs. Collectively, this study indicates that GTPBP2 positively modulates Wnt signaling to reinforce the quiescence, self-renewal, and chemoresistance of mouse CCSCs. Therefore, we disclose a novel mechanism underlying CCSC biology and GTPBP2 could be a therapeutic target in future CRC treatment.

The alleviating effect of Akkermansia muciniphila PROBIO on AOM/DSS-induced colorectal cancer in mice and its regulatory effect on gut microbiota

Emerging evidence indicates that Akkermansia muciniphila (AKK) may be a potential probiotic agent for ameliorating colorectal cancer (CRC). Thus, we utilized the azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced mouse model of colorectal cancer to evaluate the intervention effect of AKK PROBIO. The results showed that AKK PROBIO significantly improved mouse colon health, decreased the serum pro-inflammatory cytokines levels, as well as downregulated the mRNA levels of p50, p52, p65, and IκBβ, upregulated the expressions of Bid, Bim, and caspase-9 in the colon. In addition, AKK PROBIO modulated the gut microbiota composition, as characterized by the increased abundance of Muribaculaceae and Akkermansia and the decreased levels of Bacteroides, and Parasutterella. Notably, the high-dosage group was better than that of low-dosage. In conclusion, AKK PROBIO has the potential to modulate colorectal cancer by alleviating intestinal inflammation through the NF-κB pathway and promoting cell apoptosis, thereby intervening in the development of colorectal tumors.

Intestinal Microbiota Modulates the Antitumor Effect of Oncolytic Virus Vaccines in Colorectal Cancer.

Immunotherapies, such as oncolytic viruses, have become powerful cancer treatments, but only some patients with cancer can benefit from them, especially those with advanced-stage cancer, and new therapeutic strategies are needed to facilitate extended survival. The intestinal microbiota may contribute to colorectal cancer (CRC) carcinogenesis and the response to immunotherapy. However, whether and how the intestinal microbiota modulates the effects of oncolytic virus vaccines (OVVs) in CRC remain to be investigated. We generated an MC38-gp33 CRC mouse model and treated it with OVV-gp33 in early and advanced stages. Probiotics, fecal microbiota transplantation (FMT), and antibiotics (ABX) were administered to regulate the microbial composition of CRC mice at an advanced stage. The tumor growth rate and survival time of the mice were recorded; 16S rDNA sequencing was used to analyze the microbial composition and flow cytometry was used to detect T-cell subset activity. OVV-gp33 treatment inhibited tumor growth and prolonged survival in the early stage of CRC butdid not have a significant effect on the advanced stage of CRC. Moreover, 16S rDNA sequence analysis and flow cytometry showed significant differences in intestinal microbiota composition, microbial metabolites, and T-cell subsets in early and advanced-stage CRC. Probiotic and FMT treatment significantly enhanced the antitumor effect of OVV in the advanced stage of CRC with an increased abundance of activated CD8+ T cells and a decreased ratio of Treg cells, while depletion of the microbiota by ABX eliminated the antitumor activity of OVV with decreased CD8+ T-cell activation and upregulated Treg cells. These results indicate that the intestinal microbiota and microbial metabolites play an important role in the antitumor effect of OVV in CRC. Furthermore, altering the intestinal microbiota composition can modulate the antitumor and immunomodulatory effects of OVV in CRC.

Radiofrequency Combined with Intratumoral Immunotherapy: Preclinical Results and Safety in Metastatic Colorectal Carcinoma.

Radiofrequency ablation (RFA) of cancer induces an anti-tumor immunity, which is insufficient to prevent recurrences. In mice, RFA-intratumoral immunotherapy by granulocyte-macrophage colony-stimulating factor (GM-CSF) and Bacillus Calmette-Guerin resulted in complete metastases regression. Infectious risk in human needs replacement of live vaccines. Intratumoral purified protein derivatives (PPD) have never been tested in digestive cancers, and the safety of intratumoral immunotherapy after RFA has not yet been validated in human models. We investigated the therapeutic efficacy of combined radiofrequency ablation (RFA) and intratumoral immunotherapy (ITI) using an immune-muco-adherent thermogel (IMT) in a mouse model of metastatic colorectal cancer (CRC) and the safety of this approach in a pig model. Intratumoral stability of the immunogel was assessed using magnetic resonance imaging (MRI) and bioluminescent imaging. Seventy-four CT26 tumor-bearing female BALB/c mice were treated with RFA either alone or in combination with intratumoral IMT. Regression of distant metastasis and survival were monitored for 60 days. Six pigs that received liver radiofrequency and intralesional IMT injections were followed for 15 days. Experimental gel embolisms were treated using an intravascular approach. Pertinent rheology of IMT was confirmed in tumors, by the signal stability during 3 days in MRI and 7 days in bioluminescence imaging. In mice, the abscopal effect of RFA-intratumoral immunotherapy resulted in regression of distant lesions completed at day 16 vs. a volume of 350 ± 99.3 mm3 in the RFA group at day 25 and a 10-fold survival rate at 60 days. In pigs, injection of immunogel in the liver RFA area was safe after volume adjustment without clinical, hematological, and liver biology disorder. Flow cytometry showed an early increase in CD3 TCRγδ+T cells at D7 (p < 0.05) and a late decrease in CD29+-CD8 T cells at D15 (p < 0.05), reflecting the inflammation status changes. Systemic GM-CSF release was not detectable. Experimental caval and pulmonary thermogel embolisms were treated by percutaneous catheterism and cold serum infusion. RFA-intratumoral immunotherapy as efficient and safe mini-invasive interventional oncology is able to improve ablative treatment of colorectal liver metastases.

Modulation of intestinal metabolites by calorie restriction and its association with gut microbiota in a xenograft model of colorectal cancer

BackgroundColorectal cancer (CRC) is a common malignant tumor, and its occurrence and development are closely related to dysbiosis of gut microbes. Previously, we found calorie restriction altered the composition of the microbial community in a colorectal cancer mouse model and inhibited in vivo growth of CRC cells. Here, we aim to further investigate alteration in the intestinal metabolites and explore the interplay between gut microbiota and intestinal metabolites upon calorie restriction.MethodsHuman colorectal cancer HCT116 cells were used to establish a colorectal cancer xenograft mouse model. The changes of intestinal metabolites in the ad libitum group and calorie restriction group were investigated through untargeted metabolomics analysis. The integrative analysis of gut microbiota and metabolites to elucidate the associations between gut microbiota and intestinal metabolites.ResultsCompared with the mice in the ad libitum group, mice upon calorie restriction exhibited downregulation of Isoleucyl-Valine, and upregulation of D-Proline, 1-Palmitoylphosphatidylcholine, and 4-Trimethylammoniobutanoic acid. Additionally, an integrative analysis of gut microbiota and metabolites revealed that Lactobacillus, Parabacteroides and rC4-4 genus were upregulated in the calorie restriction group and positively correlated with D-Proline, 4-Trimethylammoniobutanoic acid or 1-Palmitoylphosphatidylcholine, while negatively correlated with Isoleucyl-Valine. In contrast, the Nitrospirae and Deferribacteres phylum exhibited opposite trends.ConclusionCalorie restriction affects the abundance of gut microbes such as Nitrospirae phylum and Lactobacillus genus in mouse model of colorectal cancer, leading to changes in the metabolites such as D-Proline、Isoleucyl-Valine, which contributes to the suppression of in vivo growth of CRC by calorie restriction.

Keystone pathobionts associated with colorectal cancer promote oncogenic reprograming.

Fusobacterium nucleatum (Fn) and enterotoxigenic Bacteroides fragilis (ETBF) are two pathobionts consistently enriched in the gut microbiomes of patients with colorectal cancer (CRC) compared to healthy counterparts and frequently observed for their direct association within tumors. Although several molecular mechanisms have been identified that directly link these organisms to features of CRC in specific cell types, their specific effects on the epithelium and local immune compartment are not well-understood. To fill this gap, we leveraged single-cell RNA sequencing (scRNA-seq) on wildtype mice and mouse model of CRC. We find that Fn and ETBF exacerbate cancer-like transcriptional phenotypes in transit-amplifying and mature enterocytes in a mouse model of CRC. We also observed increased T cells in the pathobiont-exposed mice, but these pathobiont-specific differences observed in wildtype mice were abrogated in the mouse model of CRC. Although there are similarities in the responses provoked by each organism, we find pathobiont-specific effects in Myc-signaling and fatty acid metabolism. These findings support a role for Fn and ETBF in potentiating tumorigenesis via the induction of a cancer stem cell-like transit-amplifying and enterocyte population and the disruption of CTL cytotoxic function.

A150 DEVELOPMENT OF MIMETIC MATRICES AS INSTRUCTIVE ORGANOIDS MICROENVIRONMENT FOR THE STUDY OF COLONIC DISEASES

Abstract Background The functional complexity of the colonic epithelium is dependent on its interaction with the microenvironment involving gradients of soluble factors, extracellular matrix (ECM) proteins and stiffness. The influence of dynamic changes in the ECM has been observed in tumours, with variations in protein expression and rigidity. Their biomolecular impacts on epithelial cell behaviour are less studied due to the structural complexity of the matrix. Organoids offers a novel approach for the study of ECM biodynamics, as they require a matrix (Matrigel) to develop. Previous work showed that it is possible to grow organoids in hydrogels other than Matrigel. To evaluate the effect of matrix composition alteration on cancer initiation, we developed biofunctionalized composite hydrogels that mimic the ECM changes (chemical and physical) observed in the BmpR1a-deficient telocytes Foxl1 + (Bmpr1a△Foxl1+) mouse model of colorectal cancer (CRC) initiation. Aims The development of mimetic matrices, in association with organoids, would enable us to uncover the impact of matrix deregulation on colonic cells behaviour in pathogenesis process. Methods These hydrogels were made of alginate and 8-arm polyethylene glycol- vinyl sulfone (PEG) macromers bearing cysteine residues (CYS) and peptides. Four peptides derived from fibronectin, laminin 111, collagen I and collagen IV were used to mimic the ECM changes in the Bmpr1a△Foxl1+ colon. The grafting of CYS on alginate or peptides on PEG macromers was first characterized by thiol quantification assays. The non-grafted peptides were also analysed by uHPLC. Stiffness of the hydrogels was then determined by dynamic mechanical analysis. Finally, normal mouse organoids were seeded on hydrogels and evaluated for survival by live/dead labeling and proliferation by Alamar blue assays. Matrigel was used as a control. Results Chemical characterization revealed that CYS were grafted onto the alginate chain at around 190 μmol/g alginate. The PEG macromers were functionalized with four peptides at concentration varying from 0.25 to 1.5 mM. Rigidity of the hydrogels depends on the concentration of alginate used where lower concentration leads to soft matrix (0.5 kPa, homeostasis) and high concentration produced a stiff matrix (4 kPa, diseased). Organoids seeded in these mimetic composite hydrogels survived and proliferated in comparison to alginate alone. Conclusions Our results indicate that we can develop mimetic composite hydrogels where colon organoids can survive and thrive. This is a breakthrough model for defining the roles of ECM mechanical and biochemical stimuli in epithelial behaviour during the initiation and development of CRC. Funding Agencies CIHR

Sex differences in colonic gene expression and fecal microbiota composition in a mouse model of obesity-associated colorectal cancer

This study investigated the sex-specific correlation between obesity and colorectal cancer emphasizing a more pronounced association in males. Estrogen, chromosomal genes, and gut bacteria were assessed in C57BL6/J male, female and ovariectomized (OVX) female mice, subjected to either a low-fat diet (LFD) or high-fat diet (HFD) for 14 weeks. Induction of colon tumor involved azoxymethane (10 mg/kg) administration, followed by three cycles of dextran sulfate sodium. Male mice on HFD exhibited higher final body weight and increased colon tumors compared to females. Colonic mucin 2 expression was significantly higher in females. HFD-modulated differentially expressed genes numbered 290 for males, 64 for females, and 137 for OVX females. Only one up-regulated gene (Gfra3) overlapped between females and OVX females, while two down-regulated genes (Thrsp and Gbp11) overlapped between males and OVX females. Genes up-regulated by HFD in males were linked to cytokine-cytokine interaction, HIF-1 signaling pathway, central carbon metabolism in cancer. Sex-specific changes in gut microbial composition in response to HFD were observed. These findings suggest a male-specific vulnerability to HFD-induced colon tumor formation, implicating key genes and colonic bacteria in colon tumorigenesis.

Fumarate Hydratase Enhances the Therapeutic Effect of PD-1 Antibody in Colorectal Cancer by Regulating PCSK9.

Despite the notable achievements of programmed death 1 (PD-1) antibodies in treating various cancers, the overall efficacy remains limited in the majority of colorectal cancer (CRC) cases. Metabolism reprogramming of tumors inhibits the tricarboxylic acid (TCA) cycle, leading to down-regulation of fumarate hydratase (FH), which is related to poor prognosis in CRC patients. By establishing a tumor-bearing mouse model of CRC with Fh1 expression deficiency, we confirmed that the therapeutic effect of PD-1 antibodies alone was suboptimal in mice with low Fh1 expression, which was improved by combination with a protein invertase subtilisin/kexin 9 (PCSK9) inhibitor. Mechanistically, FH binds to Ras-related nucleoprotein (RAN), which inhibits the nuclear import of the PCSK9 transcription factor SREBF1/2, thus reducing the expression of PCSK9. This leads to increased clonal expansion of CD8+ T cells while the number of Tregs remains unchanged, and the expression of PD-L1 does not change significantly, thus enhancing the immunotherapy response. On the contrary, the expression of PCSK9 increased in CRC cells with low FH expression, which antagonized the effects of immunotherapy. Overall, CRC patients with low FH expression may benefit from combinatorial therapy with PD-1 antibodies and PCSK9 inhibitors to enhance the curative effect.

Targeting MEK/COX-2 axis improve immunotherapy efficacy in dMMR colorectal cancer with PIK3CA overexpression.

PIK3CA mutation or overexpression is associated with immunotherapy resistance in multiple cancer types, but is also paradoxically associated with benefit of COX-2 inhibition on patient survival of colorectal cancer (CRC) with mismatch repair deficiency (dMMR). This study examined whether and how PIK3CA status affected COX-2-mediated tumor inflammation and immunotherapy response of dMMR CRC. Murine colon cancer cells MC38, CT26, and CT26-Mlh1-KO were used to construct PIK3CA knockdown and overexpression models to mimic dMMR CRC with PIK3CA dysregulation, and xenograft models were used to evaluate how PIK3CA regulate COX-2 expression, CD8+ T cells infiltration, tumor growth, and therapy response to anti-PD-L1 treatment using immunocompetent mice. Western blot was carried out to delineate the signaling pathways in human and mouse cancer cells, and immunohistochemical analysis together with bioinformatics analysis using human patient samples. PIK3CA upregulates COX-2 expression through MEK/ERK signaling pathway independent of AKT signaling to promote tumor inflammation and immunosuppression. PIK3CA knockdown profoundly reduced CT26 tumor growth in a CD8+ T cell-dependent manner, while PIK3CA overexpression significantly inhibited CD8+ T cells infiltration and promoted tumor growth. Furthermore, MEK or COX-2 inhibition augmented the anti-tumor activity of anti-PD-L1 immunotherapy on dMMR CRC mouse models, accompanied with increased CD8+ T cells infiltration and activated tumor microenvironment. Our results identified that the PIK3CA hyperactivation in dMMR CRC upregulated COX-2 through MEK signaling, which inhibited CD8+ T cells infiltration and promoted tumor growth, together led to immunotherapy resistance. COX-2 or MEK inhibition may relieve therapy resistance and promote therapy efficacy of anti-PD-1/PD-L1 immunotherapy for treating dMMR CRC with PIK3CA overexpression or activating mutation.

The effect of the timing of iron administration on the outcome of photodynamic therapy using photolon in the CT26 colorectal cancer mouse model

The effect of the timing of iron administration on the outcome of photodynamic therapy using photolon in the CT26 colorectal cancer mouse model

A biomimetic nanocarrier facilitates glucose consumption and reactive oxide species accumulation in enzyme therapy for colorectal cancer

Glucose oxidase (GOx)-based enzyme therapeutics are potential alternatives for colorectal cancer (CRC) treatment via glucose consumption and accumulation of hydrogen peroxide (H2O2). Given that H2O2 can be eliminated by cytoprotective autophagy, autophagy inhibitors that can interrupt autolysosome-induced H2O2 elimination are promising combination drugs of GOx. Here, we developed a multifunctional biomimetic nanocarrier for effective co-delivery of an autophagy inhibitor-chloroquine phosphate (CQP) and GOx to exert their synergistic effect by irreversibly upregulating intracellular reactive oxygen species (ROS) levels. Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were used to encapsulate both GOx and CQP using a W/O/W multi-emulsion method. Calcium phosphate (CaP) was used to “fix” CQP to GOx in the internal water phase, where it served as a pH-sensitive unit to facilitate intracellular drug release. Folic acid-modified red blood cell membranes (FR) were used to camouflage the GOx/CQP/CaP encapsulated PLGA NPs (referred to as PLGA/GCC@FR). In an AOM/DSS-induced CRC mouse model, PLGA/GCC@FR exhibited improved antitumor effects, in which the number of tumor nodes were only a quarter of that in the free drug combination group. The enhanced therapeutic effects of PLGA/GCC@FR were attributed to the prolonged tumor retention which was verified by both dynamic in vivo imaging and drug biodistribution. This multifunctional biomimetic nanocarrier facilitated combined enzyme therapeutics by depleting glucose and augmenting intracellular ROS levels in tumor cells, which exerted a synergistic inhibitory effect on tumor growth. Therefore, this study proposed a novel strategy for the enhancement of combined enzyme therapeutics, which provided a promising method for effective CRC treatment.

Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection.

Farnesoid X receptor mediates macrophage-intrinsic responses to suppress colitis-induced colon cancer progression.

Bile acids (BAs) affect the intestinal environment by ensuring barrier integrity, maintaining microbiota balance, regulating epithelium turnover, and modulating the immune system. As a master regulator of BA homeostasis, farnesoid X receptor (FXR) is severely compromised in patients with inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). At the front line, gut macrophages react to the microbiota and metabolites that breach the epithelium. We aim to study the role of the BA/FXR axis in macrophages. This study demonstrates that inflammation-induced epithelial abnormalities compromised FXR signaling and altered BAs' profile in a mouse CAC model. Further, gut macrophage-intrinsic FXR sensed aberrant BAs, leading to pro-inflammatory cytokines' secretion, which promoted intestinal stem cell proliferation. Mechanistically, activation of FXR ameliorated intestinal inflammation and inhibited colitis-associated tumor growth, by regulating gut macrophages' recruitment, polarization, and crosstalk with Th17 cells. However, deletion of FXR in bone marrow or gut macrophages escalated the intestinal inflammation. In summary, our study reveals a distinctive regulatory role of FXR in gut macrophages, suggesting its potential as a therapeutic target for addressing IBD and CAC.

Anti-angiogenic and antitumor effects of anlotinib combined with bevacizumab for colorectal cancer

BackgroundThe progression and metastasis of tumors are typically accompanied by angiogenesis. Crucially, vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) play a significant role in tumor-associated angiogenesis. In this study, the aim was to investigate the antitumor effect of combining bevacizumab (Bev) with anlotinib (An) on colorectal cancer (CRC). MethodsThe CCK-8 assay, EdU assay, and Annexin V staining were conducted to evaluate the proliferation and apoptosis of CRC cells in vitro. The migration capability of CRC cells and HUVECs was assessed using the Transwell assay. Additionally, the tube formation capability of HUVECs was investigated. Furthermore, the antitumor and antiangiogenic effects were evaluated in the BALB/c mice model using immunohistochemistry, TUNEL staining, and 18F-FDG PET/CT imaging. Finally, we analyzed the inhibitory effect of Bev and/or An on related signaling effectors through western blotting. ResultsThe in vivo CRC mice model revealed that the combination of Bev + An significantly suppressed tumor formation and angiogenesis. Bev + An inhibited tumor glucose metabolism and increased the median survival period in tumor-bearing mice. Mechanistically, the expressions of VEGF, VEGFR2, PDGFR, and FGFR, as well as the phosphorylation levels of AKT, were inhibited after Bev+An treatment. In conclusion, the dual vertical targeting of VEGF and VEGFR in the CRC mice model strongly inhibited tumor growth and angiogenesis, with the suppression of the AKT signaling pathway playing a partial role.

The mechanistic study of cancer-associated adipocytes promoting colorectal cancer peritoneal metastasis via FKBP5/UCP1-mediated lipid metabolism reprogramming.

178 Background: Colorectal cancer (CRC) is a prevalent malignant tumor globally, with peritoneal metastasis as a significant late-stage dissemination pathway, severely impacting patient prognosis. Recent research increasingly suggests the critical role of lipid metabolism within the tumor microenvironment in tumor progression. Cancer associated adipocytes (CAAs), a vital component, contribute to tumor progression, yet the exact molecular mechanisms remain unclear. We aim to explore the role of CAAs in CRC peritoneal metastasis and their specific regulatory mechanisms. Methods: In this study, we analyzed clinical samples from 312 CRC patients to assess the expression levels of FKBP5 and UCP1 proteins in tumor-adjacent tissues. Purified exosomes from co-cultured adipocytes and tumor cells, along with exosomes from ordinary tumor cells, underwent miRNA profiling via sequencing to evaluate exosomal miR-200a-3p expression. We established FKBP5 stable knockdown (FKBP5-/-) transgenic mouse models, organotypic co-culture models, and patient-derived xenograft (PDX) models. Through experiments involving metabolomics, mass spectrometry, immunoprecipitation, western blotting, and qPCR, we investigated the role of the miR-200a-3p/FKBP5/PPARγ/UCP1 signaling pathway in CAAs in CRC peritoneal metastasis. Results: After co-culturing adipocytes and tumor cells, we observed brown adipocyte differentiation, resulting in significant downregulation of FKBP5 protein expression. FKBP5 exhibited a strong association with UCP1 expression. Tumor cells highly expressed miR-200a-3p, released via exosomes into the tumor microenvironment and internalized by adipocytes. Within adipocytes, miR-200a-3p orchestrated metabolic reprogramming via the FKBP5/PPARγ/UCP1 pathway, inducing brown adipocyte differentiation, generating CAAs. CAAs released FFAs, promoting tumor peritoneal metastasis by activating tumor cell surface G-protein coupled receptors (GPCRs). Conversely, miR-200a-3p inhibition in tumor cells yielded opposing effects. In a spontaneous mouse model of colorectal cancer using azoxymethane (AOM)/dextran sulfate sodium salt (DSS), FKBP5-/- mice exhibited increased brown adipocyte content in the tumor microenvironment, along with notably higher spontaneous colorectal tumor numbers and sizes compared to wild-type mice. Conclusions: This study elucidates the pivotal role of CAAs in the peritoneal metastasis of CRC, confirming the regulatory role of the FKBP5/UCP1 pathway-mediated lipid metabolism reprogramming in this process. These findings offer a fresh perspective for further exploration of the molecular mechanisms underlying CRC peritoneal metastasis, and provide a theoretical foundation for the identification of novel therapeutic targets.