Murine Colorectal Cancer Model Research Articles

Administration of adiponectin receptor agonist AdipoRon relieves cancer cachexia by mitigating inflammation in tumour-bearing mice.

Cancer cachexia is a life-threatening, inflammation-driven wasting syndrome that remains untreatable. Adiponectin, the most abundant adipokine, plays an important role in several metabolic processes as well as in inflammation modulation. Our aim was to test whether administration of AdipoRon (AR), a synthetic agonist of the adiponectin receptors, prevents the development of cancer cachexia and its related muscle atrophy. The effect of AR on cancer cachexia was investigated in two distinct murine models of colorectal cancer. First, 7-week-old CD2F1 male mice were subcutaneously injected with colon-26 carcinoma cells (C26) or vehicle (CT). Six days after injection, mice were treated for 5days with AdipoRon (50mg/kg/day; C26+AR) or the corresponding vehicle (CT and C26). Additionally, a genetic model, the ApcMin/+ mouse, that develops spontaneously numerous intestinal polyps, was used. Eight-week-old male ApcMin/+ mice were treated with AdipoRon (50mg/kg/day; Apc+AR) or the corresponding vehicle (Apc) over a period of 12weeks, with C57BL/6J wild-type mice used as controls. In both models, several parameters were assessed in vivo: body weight, grip strength and serum parameters, as well as ex vivo: molecular changes in muscle, fat and liver. The protective effect of AR on cachexia development was observed in both cachectic C26 and ApcMin/+ mice. In these mice, AR administration led to a significant alleviation of body weight loss and muscle wasting, together with rescued muscle strength (P<0.05 for all). In both models, AR had a strong anti-inflammatory effect, reflected by lower systemic interleukin-6 levels (-55% vs. C26, P<0.001 and -80% vs. Apc mice, P<0.05), reduced muscular inflammation as indicated by lower levels of Socs3, phospho-STAT3 and Serpina3n, an acute phase reactant (P<0.05 for all). In addition, AR blunted circulating levels of corticosterone (-46% vs. C26 mice, P<0.001 and -60% vs. Apc mice, P<0.05), the predominant murine glucocorticoid known to induce muscle atrophy. Accordingly, key glucocorticoid-responsive factors implicated in atrophy programmes were-or tended to be-significantly blunted in skeletal muscle by AR. Finally, AR protected against lipid metabolism alterations observed in ApcMin/+ mice, as it mitigated the increase in circulating triglyceride levels (-38%, P<0.05) by attenuating hepatic triglyceride synthesis and fatty acid uptake by the liver. Altogether, these results show that AdipoRon rescued the cachectic phenotype by alleviating body weight loss and muscle atrophy, along with restraining inflammation and hypercorticism in preclinical murine models. Therefore, AdipoRon could represent an innovative therapeutic strategy to counteract cancer cachexia.

The C-type lectin DCIR contributes to the immune response and pathogenesis of colorectal cancer

Development and progression of malignancies are accompanied and influenced by alterations in the surrounding immune microenvironment. Understanding the cellular and molecular interactions between immune cells and cancer cells has not only provided important fundamental insights into the disease, but has also led to the development of new immunotherapies. The C-type lectin Dendritic Cell ImmunoReceptor (DCIR) is primarily expressed by myeloid cells and is an important regulator of immune homeostasis, as demonstrated in various autoimmune, infectious and inflammatory contexts. Yet, the impact of DCIR on cancer development remains largely unknown. Analysis of available transcriptomic data of colorectal cancer (CRC) patients revealed that high DCIR gene expression is associated with improved patients’ survival, immunologically "hot" tumors and high immunologic constant of rejection, thus arguing for a protective and immunoregulatory role of DCIR in CRC. In line with these correlative data, we found that deficiency of DCIR1, the murine homologue of human DCIR, leads to the development of significantly larger tumors in an orthotopic murine model of CRC. This phenotype is accompanied by an altered phenotype of tumor-associated macrophages (TAMs) and a reduction in the percentage of activated effector CD4+ and CD8+ T cells in CRC tumors of DCIR1-deficient mice. Overall, our results show that DCIR promotes antitumor immunity in CRC, making it an attractive target for the future development of immunotherapies to fight the second deadliest cancer in the world.

Abstract 4044: Potentiated antitumor activity of poly-γ-glutamic acid combined with anti PD-L1 blockade on murine models of colorectal cancer

Abstract Objective: Immune checkpoint inhibitors(ICIs) have been shown to be key role in cancer therapy. Especially, antibodies targeting programmed cell death ligand1(PD-L1) promoted T cells to activate immune response against cancer cells. However, the current ICIs still have insignificant to motivate effective antitumor immunity by blocking PD-1/PD-L1 pathway and the regulatory network of PD-L1 by cytokines in the tumor microenvironment remains to be uncovered. Previously, we have reported the anti-cancer effects of combination treatment with a natural product, poly-γ-glutamic acid(γ-PGA), and PD-1 blockade. Now, we show that combination therapy with anti PD-L1 and γ-PGA can overcome the limits of ICIs and enhance anticancer effect in murine colorectal cancer. Design: In vivo efficacy studies were performed by intraperitoneal injection of antibody targeting PD-L1 and oral administration of γ-PGA in C57BL/6 model transplanted with mouse tumor cell lines. Systemic changes in immunophenotype was measured by tumor volume size and body weight. The splenocyte was isolated from mice and the populations of myeloid-driven suppressor cells(MDSCs) and CD4/CD8 T cells were analyzed by FACS. Also, the significant cytokines activated by T- cell, IFN-γ and IL-2, were measured by ELISA. Results and Conclusions: In murine model of colorectal cancer, γ-PGA and anti PD-L1 blockade combination therapy significantly improved anti-tumor activity compared with single agents. In addition, tumoral infiltration of CD8+ T cells and IFN-γ productions were increased by γ-PGA, and its antitumoral activity was attenuated by antibody-mediated depletion of these cells, indicating that g-PGA suppressed tumor growth in a CD8 T cell-dependent manner. Therefore, our results suggest that γ-PGA promote ICI-mediated antitumor responses by the transformation of "cold tumors" into “hot tumors” Citation Format: Jae-Pyung Jang, Chan-Hee Yoo, Kyung-Soo Hahm, Do Young Lee, Young-Chul Park, Jae Chul Choi. Potentiated antitumor activity of poly-γ-glutamic acid combined with anti PD-L1 blockade on murine models of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4044.

Abstract 1688: Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability

Abstract Programmed death-ligand 1 (PD-L1), one of the immunosuppressive molecules, overexpresses in multiple cancers and is critical for their immune escape. We previously showed that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability; however, the role of SRC-1 in CRC immune escape is unclear. Here, we demonstrated that SRC-1 was positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibited PD-L1 expression in both human and murine CRC cells and retarded murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration and antitumor activity of effector CD8+ T cells. Genetic ablation of SRC-1 in mice also decreased PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, our findings highlight a crucial role of SRC-1 in facilitating CRC immune escape and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment. Citation Format: Yilin Hong, Qiang Chen, Zinan Wang, Yong Zhang, Bei Li, Xu Kong, Pingli Mo, Nengming Xiao, Jianming Xu, Yunbin Ye, Chundong Yu. Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1688.

Abstract 5271: Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer

Abstract Purpose: Microsatellite-stable (MSS) colorectal cancer is resistant for immune therapy, necessitating the exploration of novel modalities for its conquest. Recent revelations have revealed the genetic upregulation of HIF-1α as a potential activator of CD8+ T-cells, enhancing an anti-tumor effect. Pseudohypoxia, characterized by normoxic upregulation of Hypoxia-Inducible Factor (HIF-1α), is known to be induced by iron chelators, including HIF-PHD inhibitors. In this study, we aimed to examine whether pseudohypoxia by HIF-PHD inhibitor can enhance immune response and elucidate the underlying mechanism. Method: Colon 26, SW480 and HT29 were used as murine and human MSS colorectal cancer model. Human and murine CD8+ T-cells were represented by Jurkat cells and isolated splenic CD8+ T-cells, respectively. HIF-PHD inhibitors Roxadustat and Vadadustat were used in the study, and a subcutaneous Colon 26 tumor model was established in BALB/c WT and nude mice. Hypoxic culture conditions were maintained for 48 hours at 2% O2. Results: Roxadustat and Vadadustat did not suppress the proliferation of cancer cells under conditions marked by HIF-1α upregulation. Roxadustat and Vadadustat suppress the colon 26 subcutaneous tumor growth in WT mice, the effect was cancelled in the nude mice. The treatment regimen was associated with an augmentation in the total number and effector-like subtypes of CD8+ T-cells (Tex) infiltrating the tumor. Combination therapy involving Roxadustat and PD-1 antibody synergistically inhibited tumor growth and was accompanied by an increase in PD-1lo, Ly108-, TIM-3+ CD8+ T-cells within the tumor microenvironment. Immunohistochemistry revealed that Interleukin-2 (IL-2) expression of murine spleen was stronger in Roxadustat than control group. Interestingly, HIF-PHD inhibitors induced the secretion of IL-2 from isolated CD3+ T-cells of murine spleens in ELISA, a phenomenon uniquely observed under pseudo-hypoxic conditions created by HIF-PH inhibitors, rather than actual hypoxia. This result indicates that pseudo-hypoxia may induce more robust hypoxic response than real hypoxia. Conclusion: The induction of pseudohypoxia by HIF-PHD inhibitors activates the tumor immune response for MSS colorectal cancer via induction of splenic IL-2. Citation Format: Toshiaki Ohara, Yuehua Chen, Yusuke Hamada, Seitaro Nishimura, Hotaka Kawai, Kazuhiro Noma, Hiroshi Tazawa, Toshiyoshi Fujiwara, Akihiro Matsukawa. Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5271.

Targeting two radiation-induced immunosuppressive pathways to improve the efficacy of normofractionated radiation therapy in a preclinical colorectal cancer model

Purpose We have previously demonstrated in a murine colorectal cancer model that normofractionated RT (normoRT: 18 × 2 Gy) induced MDSC infiltration and PD-L1 expression, while hypofractionated RT (hypoRT: 3 × 8 Gy) induced Treg. Here, we wanted to assess whether the association of normoRT with treatments that target two radiation-induced immunosuppressive pathways (MDSC and PD-L1) could improve tumor control. Materials and methods Subcutaneous tumors were induced using colon tumor cells (CT26) in immunocompetent mice (BALB/c) and were treated with RT alone (18 × 2 Gy or 3 × 8 Gy), or concomitantly with 5-Fluorouracil (5FU) (10 mg/kg) to deplete MDSC, and/or anti-PD-L1 (10 mg/kg). We assessed the impact of these combinations on tumor growth and immune cells infiltration by flow cytometry. In addition, we performed tumor rechallenge experiments and IFN-γ ELISpots to study the long-term memory response. Results Even though tumor growth was significantly delayed in the RT + 5FU compared to 5FU and untreated groups (p < .05), there was no significant difference between RT + 5FU (CRT) and RT alone. The rate of MDSC increased significantly 1 week after the end of normoRT (8.09% ± 1.03%, p < .05) and decreased with the addition of 5FU (3.39% ± 0.69%, p < .05). PD-L1 expressing tumor cells were increased after treatment. Adding anti-PD-L1 significantly delayed tumor growth, achieved the highest complete response rate, and induced a long-lasting protective specific anti-tumor immunity. Conclusions These results tend to demonstrate the interest of inhibiting two radiation-induced immunosuppressive mechanisms. In patients, the combination of normoRT with 5FU is already the standard of care in locally advanced rectal cancer. Adding an anti-PD-L1 to this treatment could show promising results.

A Novel Protozoa Parasite-Derived Protein Adjuvant Is Effective in Immunization with Cancer Cells to Activate the Cancer-Specific Protective Immunity and Inhibit the Cancer Growth in a Murine Model of Colorectal Cancer.

Cancer-specific CD8+ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8+ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable (either treated with mitomycin C or irradiated by X-ray) MC38 CRC cells in combination with rGRA6Nt adjuvant and received a challenge implantation of replication-capable MC38 tumor cells, those mice markedly inhibited the growth of the implanted tumors in association with a two-fold increase in CD8+ T cell density within the tumors. In addition, CD8+ T cells of the immunized mice secreted significantly increased amounts of granzyme B, a key mediator of the cytotoxic activity of CD8+ T cells, and IFN-γ in response to MC38 CRC cells in vitro when compared to the T cells from unimmunized mice. Notably, the protective effects of the immunization were specific to MC38 CRC cells, as the immunized mice did not exhibit a significantly inhibited growth of EL4 lymphoma tumors. These results indicate that rGRA6Nt is a novel and effective protein adjuvant when used in immunizations with non-replicable cancer cells to potently activate the protective immunity specifically against the cancer cells employed in the immunization.

KRAS allelic imbalance drives tumour initiation yet suppresses metastasis in colorectal cancer in vivo.

Oncogenic KRAS mutations are well-described functionally and are known to drive tumorigenesis. Recent reports describe a significant prevalence of KRAS allelic imbalances or gene dosage changes in human cancers, including loss of the wild-type allele in KRAS mutant cancers. However, the role of wild-type KRAS in tumorigenesis and therapeutic response remains elusive. We report an in vivo murine model of colorectal cancer featuring deletion of wild-type Kras in the context of oncogenic Kras. Deletion of wild-type Kras exacerbates oncogenic KRAS signalling through MAPK and thus drives tumour initiation. Absence of wild-type Kras potentiates the oncogenic effect of KRASG12D, while incidentally inducing sensitivity to inhibition of MEK1/2. Importantly, loss of the wild-type allele in aggressive models of KRASG12D-driven CRC significantly alters tumour progression, and suppresses metastasis through modulation of the immune microenvironment. This study highlights the critical role for wild-type Kras upon tumour initiation, progression and therapeutic response in Kras mutant CRC.

Photoactive imaging and therapy for colorectal cancer using a CEA-Affimer conjugated Foslip nanoparticle.

Theranostic nanoparticles hold promise for simultaneous imaging and therapy in colorectal cancer. Carcinoembryonic antigen can be used as a target for these nanoparticles because it is overexpressed in most colorectal cancers. Affimer reagents are synthetic proteins capable of binding specific targets, with additional advantages over antibodies for targeting. We fabricated silica nanoparticles using a water-in-oil microemulsion technique, loaded them with the photosensitiser Foslip, and functionalised the surface with anti-CEA Affimers to facilitate fluorescence imaging and photodynamic therapy of colorectal cancer. CEA-specific fluorescence imaging and phototoxicity were quantified in colorectal cancer cell lines and a LS174T murine xenograft colorectal cancer model. Anti-CEA targeted nanoparticles exhibited CEA-specific fluorescence in the LoVo, LS174T and HCT116 cell lines when compared to control particles (p < 0.0001). No toxicity was observed in LS174T cancer mouse xenografts or other organs. Following photo-irradiation, the anti-CEA targeted particles caused significant cell death in LoVo (60%), LS174T (90%) and HCT116 (70%) compared to controls (p < 0.0001). Photodynamic therapy (PDT) at 24 h in vivo showed a 4-fold reduction in tumour volume compared to control mouse xenografts (p < 0.0001). This study demonstrates the efficacy of targeted fluorescence imaging and PDT using Foslip nanoparticles conjugated to anti-CEA Affimer nanoparticles in in vitro and in vivo colorectal cancer models.

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

Supramolecular nanoparticles based on elastin-like peptides modified capsid protein as drug delivery platform with enhanced cancer chemotherapy efficacy

Cancer, a prevalent disease posing significant threats to human health and longevity, necessitates effective therapeutic interventions. Chemotherapy has emerged as a primary strategy following surgical procedures for combating most malignancies. Despite the considerable efficacy of conventional chemotherapeutic agents against cancer cells, their utility is hindered by profound challenges such as multidrug resistance and deleterious toxic side effects, thereby limiting their systemic application. To tackle these challenges, we have devised a promising nanomedicine platform based on a plant virus. Specifically, we have selected the cowpea melanoma mottled virus (CCMV) as our nano-delivery system owing to its monodisperse and homogeneous size, as well as its intrinsic ability for controlled self-assembly. Leveraging the potential of this platform, we have engineered CCMV-based nanoparticles functionalized with elastin-like peptides (ELPs) at their N-terminal region. The target protein, CP-ELP, was expressed via E.coli, enabling encapsulation of the model drug DOX upon structural domain modification of the protein. The resulting nanoparticles exhibit uniform size distribution, facilitating efficient internalization by tumor cells and subsequent intracellular drug release, leading to enhanced antitumor efficacy. In addition, EVLP@DOX nanoparticles were found to activate immune response of tumor microenvironment in vivo, which further inhibiting tumor growth. Our designed nanoparticles have also demonstrated remarkable therapeutic effectiveness and favorable biological safety profiles in both murine melanoma and colorectal cancer models.

Murine models of colorectal cancer: the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colitis-associated cancer.

Colorectal cancer (CRC) is the third most common cancer. It is a heterogeneous disease, including both hereditary and sporadic types of tumors. CRC results from complex interactions between various genetic and environmental factors. Inflammatory bowel disease is an important risk factor for developing CRC. Despite growing understanding of the CRC biology, preclinical models are still needed to investigate the etiology and pathogenesis of the disease, as well as to find new methods of treatment and prevention. The purpose of this review is to describe existing murine models of CRC with a focus on the models of colitis-associated CRC. This manuscript could be relevant for experimental biologists and oncologists. We checked PubMed and Google from 01/2018 to 05/2023 for reviews of CRC models. In addition, we searched PubMed from 01/2022 to 01/2023 for articles using the azoxymethane (AOM)/dextran sulfate sodium (DSS) CRC model. Existing murine models of CRC include spontaneous, genetically engineered, transplantation, and chemically induced models. For the study of colitis-associated cancer (CAC), the AOM/DSS model is predominantly used. This model is very similar in histological and molecular characteristics to the human CAC, and is highly reproducible, inexpensive, and easy to use. Despite its popularity, the AOM/DSS model is not standardized, which makes it difficult to analyze and compare data from different studies. Each model demonstrates particular advantages and disadvantages, and allows to reproduce different subtypes or aspects of the pathogenesis of CRC.

SCF and IL-33 regulate mouse mast cell phenotypic and functional plasticity supporting a pro-inflammatory microenvironment

Mast cells (MCs) are multifaceted innate immune cells often present in the tumor microenvironment (TME). Several recent findings support their contribution to the transition from chronic inflammation to cancer. However, MC-derived mediators can either favor tumor progression, inducing the spread of the tumor, or exert anti-tumorigenic functions, limiting tumor growth. This apparent controversial role likely depends on the plastic nature of MCs that under different microenvironmental stimuli can rapidly change their phenotype and functions. Thus, the exact effect of unique MC subset(s) during tumor progression is far from being understood. Using a murine model of colitis-associated colorectal cancer, we initially characterized the MC population within the TME and in non-lesional colonic areas, by multicolor flow cytometry and confocal microscopy. Our results demonstrated that tumor-associated MCs harbor a main connective tissue phenotype and release high amounts of Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α. This MC phenotype correlates with the presence of high levels of Stem Cell Factor (SCF) and IL-33 inside the tumor. Thus, we investigated the effect of SCF and IL-33 on primary MC cultures and underscored their ability to shape MC phenotype eliciting the production of pro-inflammatory cytokines. Our findings support the conclusion that during colonic transformation a sustained stimulation by SCF and IL-33 promotes the accumulation of a prevalent connective tissue-like MC subset that through the secretion of IL-6 and TNF-α maintains a pro-inflammatory microenvironment.

P21 Prevents the Exhaustion of CD4+ T Cells Within the Antitumor Immune Response Against Colorectal Cancer

Background and aimsT cells are crucial for the antitumor response against colorectal cancer (CRC). T cell reactivity to CRC is nevertheless limited by T cell exhaustion. However, molecular mechanisms regulating T cell exhaustion are only poorly understood. MethodsWe investigated the functional role of cyclin-dependent kinase 1a (Cdkn1a or p21) in CD4+ T cells using murine CRC models. Furthermore, we evaluated the expression of p21 in patients with stage I–IV CRC. In vitro co-culture models were used to understand the effector function of p21-deficient CD4+ T cells. ResultsWe observed that the activation of cell cycle regulator p21 is crucial for CD4+ T cell cytotoxic function and that p21-deficiency in Th1 cells leads to increased tumor growth in murine CRC. Similarly, low p21 expression in CD4+ T cells infiltrated into tumors of CRC patients is associated with reduced cancer-related survival. In mouse models of CRC, p21-deficient Th1 cells show signs of exhaustion, where an accumulation of effector/effector memory T cells and CD27/CD28 loss are predominant. Immune reconstitution of tumor-bearing Rag1-/- mice using ex vivo treated p21-deficient T cells with Palbociclib, an inhibitor of CDK4/6, restored cytotoxic function and prevented exhaustion of p21-deficient CD4+ T cells as a possible concept for future immunotherapy of human disease. ConclusionOur data reveal the importance of p21 in controlling cell cycle and preventing exhaustion of Th1 cells. Furthermore, we unveil the therapeutic potential of CDK inhibitors such as Palbociclib to reduce T cell exhaustion for future treatment of patients with colorectal cancer.

Global proteomic identifies multiple cancer-related signaling pathways altered by a gut pathobiont associated with colorectal cancer

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG’s capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways.

Cancer Cell Membrane Nanodiscs for Antitumor Vaccination.

Cell membrane-based nanovaccines have demonstrated attractive features due to their inherently multiantigenic nature and ability to be formulated with adjuvants. Here, we report on cellular nanodiscs fabricated from cancer cell membranes and incorporated with a lipid-based adjuvant for antitumor vaccination. The cellular nanodiscs, with their small size and discoidal shape, are readily taken up by antigen-presenting cells and drain efficiently to the lymph nodes. Due to its highly immunostimulatory properties, the nanodisc vaccine effectively stimulates the immune system and promotes tumor-specific immunity. Using a murine colorectal cancer model, strong control of tumor growth is achieved in both prophylactic and therapeutic settings, particularly in combination with checkpoint blockades. Considerable therapeutic efficacy is also observed in treating a weakly immunogenic metastatic melanoma model. This work presents a new paradigm for the design of multiantigenic nanovaccines that can effectively activate antitumor immune responses and may be applicable to a wide range of cancers.

CD44-targeted nanoparticles for co-delivery of docetaxel and an Akt inhibitor against colorectal cancer

New strategies to develop drug-loaded nanocarriers with improved therapeutic efficacy are needed for cancer treatment. Herein we report a novel drug-delivery nanosystem comprising encapsulation of the chemotherapeutic drug docetaxel (DTX) and recombinant fusion of a small peptide inhibitor of Akt kinase within an elastin-like recombinamer (ELR) vehicle. This combined approach is also precisely targeted to colorectal cancer cells by means of a chemically conjugated DNA aptamer specific for the CD44 tumor marker. This 53 nm dual-approach nanosystem was found to selectively affect cell viability (2.5 % survival) and proliferation of colorectal cancer cells in vitro compared to endothelial cells (50 % survival), and to trigger both apoptosis- and necrosis-mediated cell death. Our findings also show that the nanohybrid particles remain stable under physiological conditions, trigger sustained drug release and possess an adequate pharmacokinetic profile after systemic intravenous administration. In vivo assays showed that these dual-approach nanohybrids significantly reduced the number of tumor polyps along the colorectal tract in a murine colorectal cancer model. Furthermore, systemic administration of advanced nanohybrids induced tissue recovery by improving the morphology of gastrointestinal crypts and the tissue architecture. Taken together, these findings indicate that our strategy of an advanced dual-approach nanosystem allows us to achieve successful controlled release of chemotherapeutics in cancer cells and may have a promising potential for colorectal cancer treatment.

Fasting-mimicking diet drives antitumor immunity against colorectal cancer by reducing IgA-producing cells.

As a safe, feasible, and inexpensive dietary intervention, fasting-mimicking diet (FMD) exhibits excellent antitumor efficacy by regulating metabolism and boosting antitumor immunity. A better understanding of the specific mechanisms underlying the immunoregulatory functions of FMD could help improve and expand the clinical application of FMD-mediated immunotherapeutic strategies. In this study, we aimed to elucidate the role of metabolic reprogramming induced by FMD in activation of antitumor immunity against colorectal cancer (CRC). Single-cell RNA sequencing (scRNA-seq) analysis of intratumoral immune cells revealed that tumor-infiltrating IgA+ B cells were significantly reduced by FMD treatment, leading to the activation of antitumor immunity and tumor regression in murine CRC models. Mechanistically, FMD delayed tumor growth by repressing B cell class switching to IgA. Therefore, FMD-induced reduction of IgA+ B cells overcame the suppression of CD8+ T cells. The immunoregulatory and antitumor effects of FMD intervention were reversed by IgA+ B cell transfer. Moreover, FMD boosted fatty acid oxidation (FAO) to trigger RUNX3 acetylation, thus inactivating Cα gene transcription and IgA class switching. IgA+ B cell expansion was also impeded in patients placed on FMD, while B cell expression of CPT1A, the rate-limiting enzyme of FAO, was increased. Furthermore, CPT1A expression was negatively correlated with both IgA+ B cells and IgA secretion within CRC. Together, these results highlight that FMD holds great promise for treating CRC. Furthermore, the degree of IgA+ B cell infiltration and FAO-associated metabolic status are potential biomarkers for evaluating FMD efficacy.

Carnobacterium maltaromaticum boosts intestinal vitamin D production to suppress colorectal cancer in female mice.

Carnobacterium maltaromaticum was found to be specifically depleted in female patients with colorectal cancer (CRC). Administration of C.maltaromaticum reduces intestinal tumor formation in two murine CRC models in a female-specific manner. Estrogen increases the attachment and colonization of C.maltaromaticum via increasing the colonic expression of SLC3A2 that binds to DD-CPase of this bacterium. Metabolomic and transcriptomicprofiling unveils the increased gut abundance of vitamin D-related metabolites and the mucosal activation of vitamin D receptor (VDR) signaling in C.maltaromaticum-gavaged mice in a gut microbiome- and VDR-dependent manner. Invitro fermentation system confirms the metabolic cross-feeding ofC.maltaromaticum with Faecalibacterium prausnitzii to convert C.maltaromaticum-produced 7-dehydrocholesterol into vitamin D for activating the host VDR signaling. Overall, C.maltaromaticum colonizes the gut in an estrogen-dependent manner and acts along with other microbes to augment the intestinal vitamin D production to activate the host VDR for suppressing CRC.

Low molecular weight heparin synergistically enhances the efficacy of adoptive and anti-PD-1-based immunotherapy by increasing lymphocyte infiltration in colorectal cancer

BackgroundImmunotherapy, including adoptive cell therapy (ACT) and immune checkpoint inhibitors (ICIs), has a limited effect in most patients with colorectal cancer (CRC), and the efficacy is further limited in patients...