Murine Colon Cancer Cells Research Articles

Photoimmunotherapy using indocyanine green-loaded Codium fragile polysaccharide and chitosan nanoparticles suppresses tumor growth and metastasis

Metastasis and recurrence are the main challenges in cancer treatment. Among various therapeutic approaches, immunotherapy holds promise for preventing metastasis and recurrence. In this study, we evaluated the efficacy of treating primary cancer and blocking metastasis and recurrence with photo-immunotherapeutic nanoparticles, which were synthesized using two types of charged polysaccharides. Codium fragile polysaccharide (CFP), which exhibits immune-stimulating properties and carries a negative charge, was combined with positively charged chitosan to synthesize nanoparticles. Additionally, indocyanine green (ICG), a photosensitizer, was loaded inside these particles and was referred to as chitosan-CFP-ICG (CC-ICG). Murine colon cancer cells (CT-26) internalized CC-ICG, and subsequent 808-nanometer laser irradiation promoted apoptotic/necrotic cell death. Moreover, intratumoral injection of CC-ICG, with 808-nanometer laser irradiation eliminated CT-26 tumors in mice. Rechallenged lung metastases of CT-26 cancer were inhibited by dendritic cell activation-mediated cytotoxic T lymphocyte stimulation in mice cured by CC-ICG. These results demonstrated that CC-ICG is a natural tumor therapeutic with the potential to treat primary tumors and suppress metastasis and recurrence.

MiR-155-targeted IcosL controls tumor rejection

Elevated levels of miR-155 in solid and liquid malignancies correlate with aggressiveness of the disease. In this manuscript, we show that miR-155 targets transcripts encoding IcosL, the ligand for Inducible T-cell costimulator (Icos), thus impairing the ability of T cells to recognize and eliminate malignant cells. We specifically found that overexpression of miR-155 in B cells of Eµ-miR-155 mice causes loss of IcosL expression as they progress toward malignancy. Similarly, in mice where miR-155 expression is controlled by a Cre-Tet-OFF system, miR-155 induction led to malignant infiltrates lacking IcosL expression. Conversely, turning miR-155 OFF led to tumor regression and emergence of infiltrates composed of IcosL-positive B cells and Icos-positive T cells forming immunological synapses. Therefore, we next engineered malignant cells to express IcosL, in order to determine whether IcosL expression would increase tumor infiltration by cytotoxic T cells and reduce tumor progression. Indeed, overexpressing an IcosL-encoding cDNA in MC38 murine colon cancer cells before injection into syngeneic C57BL6 mice reduced tumor size and increased intratumor CD8+ T cell infiltration, that formed synapses with IcosL-expressing MC38 cells. Our results underscore the fact that by targeting IcosL transcripts, miR-155 impairs the infiltration of tumors by cytotoxic T cells, as well as the importance of IcosL on enhancing the immune response against malignant cells. These findings should lead to the development of more effective anticancer treatments based on maintaining, increasing, or restoring IcosL expression by malignant cells, along with impairing miR-155 activity.

Exosomes derived from cancer cells relieve inflammatory bowel disease in mice

Exosome therapy has garnered significant attention due to its natural delivery capabilities, low toxicity, high biocompatibility, and potential for personalised treatment through engineering modifications. Recent studies have highlighted the ability of tumour cell-derived exosomes (TDEs) to interact with immune cells or modify the immune microenvironment to suppress host immune responses, as well as their unique homing ability to parental cells. The core question of this study is whether this immunomodulatory property of TDEs can be utilised for the immunotherapy of inflammatory diseases. In our experiments, we prepared exosomes derived from murine colon cancer cells CT26 (CT26 exo) using ultracentrifugation, characterised them, and conducted proteomic analysis. The therapeutic potential of CT26 exo was evaluated in our dextran sulphate sodium salt (DSS)-induced inflammatory bowel disease (IBD) mouse model. Compared to the control and 293 T exo treatment groups, mice treated with CT26 exo showed a reduction in the disease activity index (DAI) and colon shortening rate, with no noticeable weight loss. Haematoxylin and eosin (H&E) staining of colon paraffin sections revealed reduced inflammatory infiltration and increased epithelial goblet cells in the colons of CT26 exo-treated group. Furthermore, we conducted preliminary mechanistic explorations by examining the phenotyping and function of CD4+ T cells and dendritic cells (DCs) in the colonic lamina propria of mice. The results indicated that the ameliorative effect of CT26 exosomes might be due to their inhibition of pro-inflammatory cytokine secretion by colonic DCs and selective suppression of Th17 cell differentiation in the colon. Additionally, CT26 exo exhibited good biosafety. Our findings propose a novel exosome-based therapeutic approach for IBD and suggest the potential application of TDEs in the treatment of inflammatory diseases.

Characterizing OXPHOS inhibitor-mediated alleviation of hypoxia using high-throughput live cell-imaging

BackgroundHypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell’s mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia.MethodsB16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system.ResultsHypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors.ConclusionsWe successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.

Abstract 3253: Novel gene & protein-carrying vector BC-PIV expressing immunomodulatory molecules potently stimulates anti-tumor immunity against solid tumors in mice

Abstract Background: Cancer immunotherapy such as immune-checkpoint inhibitors or CAR-T cell therapy has emerged as an effective therapeutic modality for some cancers such as hematological malignancies. However, they still show little efficacy for many solid tumors, because of their immune suppressive microenvironment. BC-PIV is a replication-deficient viral vector derived from human parainfluenza virus type 2, that can convey two ectopic genes simuoltaneously and uniquely display ectopic proteins with intact steric structure on the surface of viral particles. Furthermore, BC-PIV itself can induce maturation of dendritic cells with efficient induction of CD80/86. These unique characteristics of BC-PIV make it an attractive tool for improving anti-tumor responses under immunesuppressive milieu of solid tumors. Aim and Methods: To develop novel immunotherapy using BC-PIV, we have introduced various immunomodulatory molecules into BC-PIV and tested their anti-tumor efficacy in vivo. To activate CD4/8 positive T cells, we introduced genes for OX-40L, 4-1BBL, GITRL, CD30L, OX40L/4-1BBL, OX40L/GITRL, or 4-1BBL/GITRL (collectively, TNFRSF ligands) to BC-PIV. All gene products were effectively presented on BC-PIV envelope. For expansion of T, B, and NK cells, or for promoting migration of immune cells, genes for IL-7, CCL19, IL-7/CCL19, IL-7/CCL21, or IL-12 (P40/P35) were introduced. CT-26 murine colon cancer cells (5 × 105) were implanted into syngeneic Balb/c mice at one or both side(s) of their ventral wall. Approximately 107 viral particles derived from BC-PIV carrying immunomodulatory molecules were directly injected into tumors that had grown to 5-7mm in diameter, and the anti-tumor efficacy was assessed by monitoring the tumor size. Results: In the mice treated with BC-PIV carrying TNFRSF ligands, tumor regression was observed at the virus administered site, but also at the non-treated, distant site, possibly due to the Abscopal effect. Moreover, these mice maintained efficient anti-tumor responses and rejected tumors when they were re-challenged with CT-26 cells at 8 months after the initial BC-PIV treatment, showing that BC-PIV/TNFRSF ligands induced sustained anti-tumor immunity. In the BC-PIV/IL-7/CCL19-treated mice, the tumors began to shrink about six days after the single injection of the virus. This effect was even more enhanced by concurrent administration of anti-PD-1 antibody. Conclusions: BC-PIV carrying TNFRSF ligands, cytokines or chemokines induced efficient, sustained anti-tumor immunity and eradicated tumors in in vivo solid tumor model. Unique characteristics of BC-PIV, being able to deliver two genes simultaneously and to display ectopic proteins with intact steric structure on the viral surface, make this vector an attractive, novel platform for next generation cancer immunotherapy. Citation Format: Masayuki Fukumura, Junpei Ohtsuka, Tomomi Ohtsuka, Hideaki Nakajima, Tetsuya Nosaka. Novel gene & protein-carrying vector BC-PIV expressing immunomodulatory molecules potently stimulates anti-tumor immunity against solid tumors in mice [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 3253.

KLK10 derived from tumor endothelial cells accelerates colon cancer cell proliferation and hematogenous liver metastasis formation.

Tumor endothelial cells (TECs), which are thought to be structurally and functionally different from normal endothelial cells (NECs), are increasingly attracting attention as a therapeutic target in hypervascular malignancies. Although colorectal liver metastasis (CRLM) tumors are hypovascular, inhibitors of angiogenesis are a key drug in multidisciplinary therapy, and TECs might be involved in the development and progression of cancer. Here, we analyzed the function of TEC in the CRLM tumor microenvironment. We used a murine colon cancer cell line (CT26) and isolated TECs from CRLM tumors. TECs showed higher proliferation and migration than NECs. Coinjection of CT26 and TECs yielded rapid tumor formation invivo. Immunofluorescence analysis showed that coinjection of CT26 and TECs increased vessel formation and Ki-67+ cells. Transcriptome analysis identified kallikrein-related peptide 10 (KLK10) as a candidate target. Coinjection of CT26 and TECs after KLK10 downregulation with siRNA suppressed tumor formation invivo. TEC secretion of KLK10 decreased after KLK10 downregulation, and conditioned medium after KLK10 knockdown in TECs suppressed CT26 proliferative activity. Double immunofluorescence staining of KLK10 and CD31 in CRLM tissues revealed a significant correlation between poor prognosis and positive KLK10 expression in TECs and tumor cells. On multivariate analysis, KLK10 expression was an independent prognostic factor in disease-free survival. In conclusion, KLK10 derived from TECs accelerates colon cancer cell proliferation and hematogenous liver metastasis formation. KLK10 in TECs might offer a promising therapeutic target in CRLM.

An alternative hybrid lipid nanosystem combining cytotoxic and magnetic properties as a tool to potentiate antitumor effect of 5-fluorouracil

AimsColorectal cancer is the third most frequent type of cancer and the second leading cause of cancer-related deaths worldwide. The majority of cases are diagnosed at a later stage, leading to the need for more aggressive treatments such as chemotherapy. 5-Fluorouracil (5-FU), known for its high cytotoxic properties has emerged as a chemotherapeutic agent. However, it presents several drawbacks such as lack of specificity and short half-life. To reduce these drawbacks, several strategies have been designed namely chemical modification or association to drug delivery systems. Materials and methodsCurrent research was focused on the design, physicochemical characterization and in vitro evaluation of a lipid-based system loaded with 5-FU. Furthermore, aiming to maximize preferential targeting and release at tumour sites, a hybrid lipid-based system, combining both therapeutic and magnetic properties was developed and validated. For this purpose, liposomes co-loaded with 5-FU and iron oxide (II, III) nanoparticles were accomplished. Key findingsThe characterization of the developed nanoformulation was performed in terms of incorporation parameters, mean size and surface charge. In vitro studies assessed in a murine colon cancer cell line confirmed that 5-FU antiproliferative activity was preserved after incorporation in liposomes. In same model, iron oxide (II, III) nanoparticles did not exhibit cytotoxic properties. Additionally, the presence of these nanoparticles was shown to confer magnetic properties to the liposomes, allowing them to respond to external magnetic fields. SignificanceOverall, a lipid nanosystem loading a chemotherapeutic agent displaying magnetic characteristics was successfully designed and physicochemically characterized, for further in vivo applications.

Generation of murine tumor models refractory to αPD-1/-L1 therapies due to defects in antigen processing/presentation or IFNγ signaling using CRISPR/Cas9.

Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.

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.

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

Immune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement. DAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody. VEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models. VEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

Magnetic Resonance Imaging of Macrophage Response to Radiation Therapy.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality which, in conjunction with biopsies, provide a qualitative assessment of tumor response to treatment. Intravenous injection of contrast agents such as fluorine (19F) nanoemulsions labels systemic macrophages, which can, then, be tracked in real time with MRI. This method can provide quantifiable insights into the behavior of tumor-associated macrophages (TAMs) in the tumor microenvironment and macrophage recruitment during therapy. Female mice received mammary fat pad injections of murine breast or colon cancer cell lines. The mice then received an intravenous 19F nanoemulsion injection, followed by a baseline 19F MRI. For each cancer model, half of the mice then received 8 Gy of localized radiation therapy (RT), while others remained untreated. The mice were monitored for two weeks for tumor growth and 9F signal using MRI. Across both cohorts, the RT-treated groups presented significant tumor growth reduction or arrest, contrary to the untreated groups. Similarly, the fluorine signal in treated groups increased significantly as early as four days post therapy. The fluorine signal change correlated to tumor volumes irrespective of time. These results demonstrate the potential of 19F MRI to non-invasively track macrophages during radiation therapy and its prognostic value with regard to tumor growth.

Analysis and identification of phenolic compounds with antiproliferative activity from Chinese olive (Canarium album L.) fruit extract by HPLC-DAD-SPE-TT-NMR.

Chinese olives (Canarium album L.) are rich in phenolic compounds, exhibiting a broad spectrum of potential clinical applications. This study is the first report on the isolation and elucidation of bioactive compounds with high antiproliferative activity from the ethyl acetate fraction of a Chinese olive fruit methanolic extract (CO-EtOAc). We used the WST-1 assay to determine which subfractions of CO-EtOAc had significant antiproliferative activity using the murine colon cancer cell line CT26. Subsequently, the functional compounds were characterized by the hyphenated technique and high-performance liquid chromatography-diode array detector-solid phase extraction-transfer tube-nuclear magnetic resonance (HPLC-DAD-SPE-TT-NMR). Thirteen phenolic constituents were identified from the antiproliferation-enhancing subfractions of CO-EtOAc, including two new compounds, 2,4-didehydrochebulic acid 1,7-dimethyl ester (5) and 1-hydroxybrevifolin (7), which were further purified and found to exhibit marked antiproliferative activity. Chebulic acid dimethyl ester (2), which was isolated from C. album for the first time, also possessed antiproliferative activity.

Sec1 regulates intestinal mucosal immunity in a mouse model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common immune-mediated condition with its molecular pathogenesis remaining to be fully elucidated. This study aimed to deepen our understanding of the role of FUT2 in human IBD, by studying a new surrogate gene Sec1, a neighboring gene of Fut2 and Fut1 that co-encodes the α 1,2 fucosyltransferase in mice. CRISPR/Cas9 was used to prepare Sec1 knockout (Sec1−/−) mice. IBD was induced in mice using 3% w/v dextran sulphate sodium. Small interfering RNA (siRNA) was employed to silence Sec1 in murine colon cancer cell lines CT26.WT and CMT93. IBD-related symptoms, colonic immune responses, proliferation and apoptosis of colon epithelial cells were assessed respectively to determine the role of Sec1 in mouse IBD. Impact of Sec1 on the expression of death receptor 5 (DR5) and other apoptosis-associated proteins were determined. Sec1 knockout was found to be associated with deterioration of IBD in mice and elevated immune responses in the colonic mucosa. Silencing Sec1 in CT26.WT and CMT93 cells led to greater secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α. Cell counting kit 8 (CCK8) assay, flow cytometry and TUNEL detection suggested that Sec1 expression promoted the proliferation of colon epithelial cells, inhibited cell apoptosis, reduced cell arrest in G0/G1 phase and facilitated repair of inflammatory injury. Over-expression of DR5 and several apoptosis-related effector proteins was noticed in Sec1−/− mice and Sec1-silenced CT26.WT and CMT93 cells, supporting a suppressive role of Sec1 in cell apoptosis. Our results depicted important regulatory roles of Sec1 in mouse IBD, further reflecting the importance of FUT2 in the pathogenesis of human IBD.

In Vitro Inhibitory Potential of Different Anthocyanin-Rich Berry Extracts in Murine CT26 Colon Cancer Cells

Anti-oxidant, -inflammatory, and -carcinogenic activities of bioactive plant constituents, such as anthocyanins, have been widely discussed in literature. However, the potential interaction of anthocyanin-rich extracts with routinely used chemotherapeutics is still not fully elucidated. In the present study, anthocyanin-rich polyphenol extracts of blackberry (BB), bilberry (Bil), black currant (BC), elderberry (EB), and their respective main anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside) were investigated concerning their cytotoxic and DNA-damaging properties in murine CT26 cells either alone or in combination with the chemotherapeutic agent SN-38. BB exerted potent cytotoxic effects, while Bil, BC, and EB only had marginal effects on cell viability. Single anthocyanins comprised of the extracts could not induce comparable effects. Even though the BB extract further pronounced SN-38-induced cytotoxicity and inhibited cell adhesion at 100–200 µg/mL, no effect on DNA damage was observed. In conclusion, anti-carcinogenic properties of the extracts on CT26 cells could be ranked BB >> BC ≥ Bil ≈ EB. Mechanisms underlying the potent cytotoxic effects are still to be elucidated since the induction of DNA damage does not play a role.

Tumor-secreted IFI35 promotes proliferation and cytotoxic activity of CD8+ T cells through PI3K/AKT/mTOR signaling pathway in colorectal cancer

BackgroundA large proportion of the patients with cancer do not respond to immunotherapies. Recent studies suggested an important role for tumor-infiltrating cytotoxic T lymphocytes (CTL) in enhancing response to immunotherapy. Here, we aim to identify gene that induce proliferative and cytotoxic states of CD8+ T cells, and to investigate its effect on CAR-T cells against colorectal cancer.MethodsCorrelation between the expression of IFI35 with the activation and cytotoxicity of CD8+ T cells was assessed with TCGA and proteomic databases. Then we constructed murine colon cancer cells over-expressing IFI35 and tested their effect on anti-tumor immunity in both immunodeficient and immunocompetent mouse models. Flow cytometry and immunohistochemistry were performed to assess the immune microenvironment. Western blot analysis was used to identify the potential down-stream signaling pathway regulated by IFI35. We further investigated the efficacy of the rhIFI35 protein in combination with immunotherapeutic treatment.ResultsThe transcriptional and proteomic analysis of the activation and cytotoxicity of CD8+ T cells in human cancer samples demonstrated that IFI35 expression is correlated with increased CD8+ T cell infiltration and predicted a better outcome in colorectal cancer. The number and cytotoxicity of CD8+ T cells were significantly increased in IFI35-overexpressing tumors. Mechanistically, we identified that the IFNγ-STAT1-IRF7 axis stimulated IFI35 expression, and that IFI35-mediated regulation of CD8+ T cell proliferation and cytotoxicity was dependent on PI3K/AKT/mTOR signaling pathway in vitro. Furthermore, IFI35 protein enhanced the efficacy of CAR-T cells against colorectal cancer cells.ConclusionOur findings identify IFI35 as a new biomarker that can enhance the proliferation and function of CD8+ T cells, as well as increase the efficacy of CAR-T cells against colorectal cancer cells.Graphical

Phosphatase-dependent function of PTEN in maintaining mitotic chromatin condensation

The process for genetic transmission takes place in a chromatin environment constituted of histones and non-histone architectural proteins. PTEN, a potent tumor suppressor defective in a large number of human cancers, has been found to interact with various chromatin components, guarding the genome against chromosome instability. Studies have shown that PTEN-deficient cells exhibit a globally defective chromatin environment. However, a comprehensive understanding of how the dynamic process of mitotic chromosome condensation is impacted by the PTEN-deficient epigenetic milieu is needed. Thus, the purpose of this study was to identify aberrations of dynamic mitotic chromatin compression resulting from PTEN dysfunction and to evaluate the importance of PTEN phosphatase activity in maintaining heterochromatin stability. An intrinsic metaphase structure assay was utilized to monitor the structural integrity of chromosomes in murine MC38 colon cancer cells. Following successive cycles of condensation and decondensation, MC38 cells with and without Pten were subjected to analysis of chromosome morphology. Pten-/cells exhibited fewer heterochromatic centers as compared to Pten+/+ cells. Ectopic expression of wild-type Pten (WT) rescued the aberrant heterochromatic morphology in Pten-null cells. In contrast, a lipid phosphatase deficient Pten mutant G129E (GE) failed to correct the Pten-deficient chromatin aberrations. These observations reveal that PTEN plays an essential role in maintaining mitotic chromosome stability and suggest that epigenetic approaches may be implemented to correct mitotic chromatin deregulation in tumors carrying PTEN mutations.

Base-edited live microbiome therapeutics with target-aid -live biotherapeutic products (LBPS) with synergistic effects of anti-PD-1 antibodies in a syngeneic model of mice.

e15104 Background: Base editing without DNA cleaving is a technique that introduces point mutations using enzymes that induce base conversions. Generally, only the point mutation occurs, achieving a high level of precision. Base editing can easily be applied to bacteria, for which DNA cleaving is often lethal. With Bio Palette's core technology of base editing, Target-AID, we can quickly achieve the required therapeutic effects by making pinpoint modifications to bacteria. Immune checkpoint inhibitors (ICIs) targeting CTLA-4, PD-1, or PD-L1 have shown outstanding therapeutic outcomes in various cancers. However, the therapeutic efficacy of ICIs-based immunotherapy has been limited. Recent studies have revealed that the human gut microbiome controls the efficacy of ICIs. Bacterial flagellin derived from Enterococcus spp. has been suggested to enhance immune responses to cancer through activation of TLR5 signaling (1). As a regulatory mechanism of flagellin secretion in Enterococcus spp, FliD, FlgK, FlgN, or FlgM are known as negative regulators. Thus, if the flagellar expression can be enhanced by making the negative regulator functionally deficient, it can be expected to increase the efficacy and response rate of ICIs when combination with a base-edited strain. Methods: As human TLR5 luciferase reporter assay, HEK293T cells were transfected with human TLR5-expression vector. BALB/cAnN mice (6–8 weeks’ old) were injected subcutaneously with 3 x 106 cells for murine colon cancer cell line CT-26. Six days post inoculation, the mice were randomized based on tumor volume into treatment and vehicle control groups (n = 10 per each group). Anti-mouse PD-1 antibody was dosed 5 mg/kg, q3d, iv, for up to 3 weeks either alone or in combination with each of various strains (1 x 109 CFU/mouse, po). Body weight and tumor size were routinely measured until endpoint. Results: To achieve the increased extracellular secretion of Flagellin, we created fliD dysfunctional strain because deletion of the flagellar cap component FliD was expected to promote extracellular secretion of Flagellin monomers. Comparing the wild-type strains we used, E. casseliflavus activated TLR5 compared to E. gallinarum. E. casseliflavus with a stop codon introduced into the fliD gene, BP5040 strain, was found to increase TLR5 activation by approximately 4.9-fold when compared to wild type strain. In vivo experiments indicated that antitumor effect was observed with BP5040 alone. Strikingly, a remarkable antitumor effect was also demonstrated when BP5040 was combined with an anti-PD-1 antibody. Conclusions: To achieve the desired therapeutic effect, we performed base editing without inserting foreign genes. As a result, BP5040 strain is found to be a potential candidate for the treatment of immuno-oncology, which was created using our LBP platform technology.

Augmented interferon regulatory factor 7 axis in whole tumor cell vaccines prevents tumor recurrence by inducing interferon gamma-secreting B cells

ABSTRACT Among cancer immunotherapy, which has received great attention in recent years, cancer vaccines can potentially prevent recurrent tumors by using the exquisite power and specificity of the immune system. Specifically, whole tumor cell vaccines (WTCVs) based on surgically resected tumors have been considered to elicit robust anti-tumor immune responses by exposing various tumor-associated antigens to host immunity. However, most tumors have little immunogenicity because of immunoediting by continuous interactions with host immunity; thus, preparing WTCVs based on patient-derived non-modified tumors cannot prevent tumor onset. Hence, the immunogenicity of tumor cells must be improved for effective WTCVs. In this study, we indicate the importance of the interferon regulatory factor 7 (Irf7) axis, including Irf7 and its downstream factors, within tumor cells in regulating immunogenicity. Indeed, WTCVs that augmented the Irf7 axis have exerted remarkable recurrence-preventive effects when vaccinated after tumor inactivation by radiation. Most notably, vaccination with murine colon cancer cells that enhanced the Irf7 axis prevented the development of challenged tumors in all mice and resulted in a 100% survival rate during the observation period. Furthermore, the mechanism leading to vaccine effectiveness was mediated by interferon-gamma-producing B cells. This study provides novel insights into how to enhance tumor immunogenicity and use WTCVs as recurrence prophylaxis.

Dose-Dependent Effects in Plasma Oncotherapy: Critical In Vivo Immune Responses Missed by In Vitro Studies.

Cold atmospheric plasma (CAP) generates abundant reactive oxygen and nitrogen species (ROS and RNS, respectively) which can induce apoptosis, necrosis, and other biological responses in tumor cells. However, the frequently observed different biological responses to in vitro and in vivo CAP treatments remain poorly understood. Here, we reveal and explain plasma-generated ROS/RNS doses and immune system-related responses in a focused case study of the interactions of CAP with colon cancer cells in vitro and with the corresponding tumor in vivo. Plasma controls the biological activities of MC38 murine colon cancer cells and the involved tumor-infiltrating lymphocytes (TILs). In vitro CAP treatment causes necrosis and apoptosis in MC38 cells, which is dependent on the generated doses of intracellular and extracellular ROS/RNS. However, in vivo CAP treatment for 14 days decreases the proportion and number of tumor-infiltrating CD8+T cells while increasing PD-L1 and PD-1 expression in the tumors and the TILs, which promotes tumor growth in the studied C57BL/6 mice. Furthermore, the ROS/RNS levels in the tumor interstitial fluid of the CAP-treated mice are significantly lower than those in the MC38 cell culture supernatant. The results indicate that low doses of ROS/RNS derived from in vivo CAP treatment may activate the PD-1/PD-L1 signaling pathway in the tumor microenvironment and lead to the undesired tumor immune escape. Collectively, these results suggest the crucial role of the effect of doses of plasma-generated ROS and RNS, which are generally different in in vitro and in vivo treatments, and also suggest that appropriate dose adjustments are required upon translation to real-world plasma oncotherapy.

Abstract 4861: Copanlisib enhances the effectiveness of anti-PD1 therapies for colorectal cancer

Abstract Introduction: Immunotherapies are increasingly being used for patients diagnosed with cancer, however, for metastatic colorectal cancer (CRC), more than 95% of patients have shown little to no clinical benefit to immunotherapy. Previous work from our lab has shown that copanlisib, a PI3K inhibitor, was found to enhance MHC class I expression in Kras mutant CT26 murine colon cancer cells. Here we examine the potential for anti-cancer activity with the combination of copanlisib and anti-PD1 treatments using this model. Methods: The impact of copanlisib on CT26 cells was performed using the WST assay and the expression of MHC class I was assessed using flow cytometry. CT26 flank allografts were generated in Balb/C mice and subsequently treated for 15 days with copanlisib (10 mg/kg), anti-PD1 (0.2 mg, BioXCell) or the combination. An IgG2a antibody (BioXCell) was used as a control. Tumors were measured twice a week using a caliper. Tumors were excised, and prepared for immunohistochemistry for CD8, CD4, perforin, granzyme B, and F480 was performed. Staining was quantified as the number of positive staining cells per 20X field of view (FOV). Results: CT26 viability in response to 200 nM copanlisib was relatively unchanged as compared with untreated controls. Via flow cytometry, a 75% increase in mean fluorescent intensity (MFI) for MHC class I was observed comparing control to copanlisib (p=0.003). In vivo, after 15 days there was no difference in the growth rate of those cancers treated with control versus copanlisib or anti-PD1. A significant reduction in growth rate was observed with the combination of anti-PD1 with copanlisib compared to the other arms (median fold change=3.62; control: 7.49, p=0.002, copanlisib: 8.88, p=0.003; anti-PD1: 8.93, p=0.002). There were no differences in CD8 T cell and perforin expression between the treatment groups. Granzyme B expression was higher in the combination compared (median/FOV= 44) to the control (27, p= 0.01). Additionally, a significant reduction in F480 expression was seen in the combination compared to the other treatment groups (median/FOV=43; control: 139, p<0.001, copanlisib: 110, p<0.001; anti-PD1:149, p<0.001). Conclusions: In conclusion, copanlisib in combination with anti-PD1 demonstrated enhanced anti-tumor activity in Balb/c mice that were injected with CT26 CRC cells. This response was correlated with increased granzyme B expression and a reduction in macrophages with the combination treatment. Further studies will expand on the mechanism of this combination therapy. Citation Format: Alexa E. Schmitz, Kennedy J. Maduscha, Sarbjeet Makkar, Cheri A. Pasch, Rebecca A. DeStefanis, Philip B. Emmerich, Dustin A. Deming. Copanlisib enhances the effectiveness of anti-PD1 therapies for colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4861.