Murine Colon Research Articles

P068 Recapitulation of human colonocytes in situ by human colon organoids in vitro and after orthotopic transplantation

Abstract Background The intestinal epithelium plays a central role in human health and disease, and several chronic inflammatory disorders associate with a weakened epithelial barrier. The organoid model allows the cultivation, expansion and analysis of non-immortalized intestinal epithelial cells and has been instrumental in studying epithelial behavior in homeostasis and disease. Recent advances in human organoid transplantation into mouse and human lay the base for models to study human epithelial cell behavior within the intestinal tissue context, and promise novel therapeutic approaches for diseases such as short bowel syndrome and inflammatory bowel disease. It remained unclear how organoid transplantation into the colon would affect epithelial phenotypes and protein expression, which is key to assess the suitability of this model to study human epithelial cells in situ and safety aspects upon transplantation into human recipients. Methods To address this, we employed state-of-the-art deep visual proteomics to compare human colonic epithelial stem and differentiated cells in vivo, upon transplantation in situ, and to organoids cultured in vitro. Results We find that organoids transplanted into the murine colon closely resemble human intestinal epithelial cells in vivo compared to organoids grown in vitro, indicating that organoid culture induces a transient shift in epithelial phenotypes, which is reversible upon reintroduction into the mucosa. Phenotypic differences between epithelial cells in vitro and in situ/in vivo were largely driven by hallmarks of high proliferation and lower functional differentiation in organoids, likely due to culture conditions. Conclusion Taken together, we demonstrate that transplanted epithelial cells in situ represent a physiological, relevant model for studying functional aspects of mature colonocytes compared to the organoid model. We furthermore show that the proliferative phenotype of organoids in vitro is a transient state driven by culture conditions, which is reversible upon reintroduction into the mucosal environment. This is an important consideration regarding the safety of organoid transplantation into humans as a potential therapeutic strategy.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 μg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

Designing Peptide-Based Nanoinhibitors of Programmed Cell Death Ligand 1 (PD-L1) for Enhanced Chemo-immunotherapy.

The combination of immune checkpoint blockade (ICB) and chemotherapy has shown significant potential in the clinical treatment of various cancers. However, circulating regeneration of PD-L1 within tumor cells greatly limits the efficiency of chemo-immunotherapy and consequent patient response rates. Herein, we report the synthesis of a nanoparticle-based PD-L1 inhibitor (FRS) with a rational design for effective endogenous PD-L1 suppression. The nanoinhibitor is achieved through self-assembly of fluoroalkylated competitive peptides that target PD-L1 palmitoylation. The FRS nanoparticles provide efficient protection and delivery of functional peptides to the cytoplasm of tumors, showing greater inhibition of PD-L1 than nonfluorinated peptidic inhibitors. Moreover, we demonstrate that FRS synergizes with chemotherapeutic doxorubicin (DOX) to boost the antitumor activities via simultaneous reduction of PD-L1 abundance and induction of immunogenic cell death in murine colon tumor models. The nano strategy of PD-L1 regulation present in this study is expected to advance the development of ICB inhibitors and overcome the limitations of conventional ICB-assisted chemo-immunotherapy.

A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles.

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction

The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy invivo. To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

Nanoscale Covalent Organic Framework with Staggered Stacking of Phthalocyanines for Mitochondria-Targeted Photodynamic Therapy.

Phthalocyanine photosensitizers (PSs) have shown promise in fluorescence imaging and photodynamic therapy (PDT) of malignant tumors, but their practical application is limited by the aggregation-induced quenching (AIQ) and inherent photobleaching of PSs. Herein, we report the synthesis of a two-dimensional nanoscale covalent organic framework (nCOF) with staggered (AB) stacking of zinc-phthalocyanines (ZnPc), ZnPc-PI, for fluorescence imaging and mitochondria-targeted PDT. ZnPc-PI isolates and confines ZnPc PSs in the rigid nCOF to reduce AIQ, improve photostability, enhance cellular uptake, and increase the level of reactive oxygen species (ROS) generation via mitochondrial targeting. ZnPc-PI shows efficient tumor accumulation, which allowed precise tumor imaging and nanoparticle tracking. With high cellular uptake and tumor accumulation, intrinsic mitochondrial targeting, and enhanced ROS generation, ZnPc-PI exhibits potent PDT efficacy with >95% tumor growth inhibition on two murine colon cancer models without causing side effects.

Synthesis and biological evaluation of 99mTc-tricarbonyl complexes of C-3 carboxy derivatives of fluoroquinolones in infection and tumor animal models

Fluoroquinolones (FQ) are clinically used antibacterial agents. Antibacterial fluoroquinolone derivatives have been shown to exhibit cytotoxic properties in tumor cells. In this work, C3-carboxy fluoroquinolones derivatives were radiolabeled with the diagnostic radionuclide technetium-99 m and were evaluated as infection- and tumor-targeted agents. First, ciprofloxacin and enrofloxacin were converted to 3-hydrazides and then to the respective pyridine hydrazones (L1 and L2). Secondly, 3-carboxamide of enrofloxacin was converted to dipicolylamine (L3), and picolylaminoacetate (L4) tridentate chelators. The respective rhenium-tricarbonyl complexes of L1-L4 were synthesized and characterized by spectroscopic methods and were used as non-radioactive standards to characterize the analogous 99mTc-radiotracers. The 99mTc-tricarbonyl complexes were synthesized and their lipophilicity and stability were evaluated in vitro. The most stable tracers 99mTcL3, 99mTcL4, were incubated with E. coli cells and CT-26 murine colon adenocarcinoma cells to assess their cellular uptake. Also, the 99mTc-tracers were injected in mice, infected with E. coli, as well as in mice, bearing CT-26 tumors.

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.

Ansofaxine hydrochloride inhibits tumor growth and enhances Anti-TNFR2 in murine colon cancer model.

Introduction: As psychoneuroimmunology flourishes, there is compelling evidence that depression suppresses the anti-tumor immune response, promotes the progression of cancer, and inhibits the effectiveness of cancer immunotherapy. Recent studies have reported that antidepressants can not only alleviate the depressant condition of cancer patients, but also strengthen the anti-tumor immunity, thus suppressing tumors. Tumor necrosis factor receptor 2 (TNFR2) antagonistic antibodies (Anti-TNFR2) targeting tumor-infiltrating regulatory T cells (Tregs) has achieved great results in preclinical studies, and with a favorable toxicity profile than existing immunotherapies, and is expected to become a new generation of more effective treatment strategies. Understanding the effects of combination therapy with antidepressants and Anti-TNFR2 may help design new strategies for cancer immunotherapy. Methods: We treated CT26, HCT116, MCA38 and SW620 colon cancer cells with fluoxetine (0-50 µM), ansofaxine hydrochloride (0-50 µM) and amitifadine hydrochloride (0-150 µM) to examine their effects on cell proliferation and apoptosis. We explored the antitumor effects of ansofaxine hydrochloride in combination with or without Anti-TNFR in subcutaneously transplanted CT26 cells in tumor-bearing mouse model. Antitumor effects were evaluated by tumor volume. NK cell, M1 macrophage cell, CD4+ T cell, CD8+ T cell, exhausted CD8+ T and regulatory T cell (Tregs) subtypes were measured by flow cytometry. 5-hydroxytryptamine, dopamine and norepinephrine levels were measured by ELISA. Results: Oral antidepression, ansofaxine hydrochloride, enhanced peripheral dopamine levels, promoted CD8+T cell proliferation, promoted intratumoral infiltration of M1 and NK cells, decreased the proportion of tumor-infiltrating exhausted CD8+T cells, and strengthened anti-tumor immunity, thereby inhibiting colon cancer growth. In combination therapy, oral administration of ansofaxine hydrochloride enhanced the efficacy of Anti-TNFR2, and produced long-term tumor control in with syngeneic colorectal tumor-bearing mice, which was attributable to the reduction in tumor-infiltrating Treg quantity and the recovery of CD8+ T cells function. Discussion: In summary, our data reveal the role of ansofaxine hydrochloride in modulating the anti-tumor immunity. Our results support that exhausted CD8+T is an important potential mechanism by which ansofaxine hydrochloride activates anti-tumor immunity and enhances anti-tumor effects of anti-TNFR2.

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.

Macrophage membrane-coated self-assembled curcumin nanoparticle missile for the treatment of colorectal cancer

Curcumin is a natural compound with various anti-cancer properties, but its poor solubility and low bioavailability limit its clinical application. In this study, we developed a novel nanoparticle system based on curcumin self-assembly, polydopamine modification and macrophage membrane coating (Cur-PDA@CM) to overcome these challenges. The Cur-PDA@CM nanoparticles exhibited high drug loading, enhanced stability, excellent photothermal effects and powerful anti-tumor efficacy. Moreover, the macrophage membrane endows the nanoparticles with active targeting ability to tumor tissues. We evaluated the anti-tumor efficacy of Cur-PDA@CM in vitro and in vivo using a murine colon carcinoma model. The results showed that Cur-PDA@CM could effectively inhibit tumor growth and induce apoptosis by releasing curcumin, generating reactive oxygen species and polarizing tumor-associated macrophages towards the M1 phenotype. The combination of Cur-PDA@CM with near-infrared irradiation further enhanced the therapeutic outcomes by facilitating drug release and photothermal therapy. The Cur-PDA@CM system represents a promising platform for delivering natural products with poor solubility and harnessing their full anti-cancer potential.

Hypoxia-activated glutamine antagonist prodrug combined with combretastatin A4 nanoparticles for tumor-selective metabolic blockade

6-Diazo-5-oxo-L-norleucine (DON) is a potent glutamine antagonist with toxic side effects; in order to reduce these effects, multiple prodrugs have been designed. However, there are currently no reports of a DON prodrug with a defined mechanism to achieve high tumor selectivity. To improve the selective toxicity of DON to tumor cells while reducing systemic toxicity, a hypoxia-activated prodrug, termed HDON, was designed. HDON achieved remarkable tumor suppression of 76.4 ± 5.2% without leading to weight loss in an H22 murine liver cancer model with high hypoxia. Moreover, to augment the therapeutic efficacy of HDON, combretastatin A4 nanoparticles were used to aggravate tumor hypoxia of MC38 murine colon cancer and 4T1 murine breast cancer, activate HDON to DON, and stimulate a robust anti-tumor immune response while selectively killing in tumor cells in vivo, achieving significantly elevated tumor suppression rates of 98.3 ± 3.4% and 98.1 ± 3.1%, with cure rates of 80.0% and 20.0%, respectively.

Carbon Ion and Photon Radiation Therapy Show Enhanced Antitumoral Therapeutic Efficacy With Neoantigen RNA-LPX Vaccines in Preclinical Colon Carcinoma Models

Personalized liposome-formulated mRNA vaccines (RNA-LPX) are a new powerful tool in cancer immunotherapy. In preclinical tumor models, RNA-LPX are known to achieve potent results when combined together with conventional X-ray radiotherapy (XRT). Densely ionizing radiation used in carbon-ion radiotherapy (CIRT) may induce distinct effects in combination with immunotherapy, compared to sparsely ionizing X-rays. Within this study, we investigate the potential of CIRT and isoeffective doses of XRT to mediate tumor growth inhibition and survival in murine colon adenocarcinoma models in conjunction with neoantigen (neoAg)-specific RNA-LPX vaccines, encoding both MHC class I and II-restricted tumor-specific neoantigens. We characterize tumor immune infiltrates and antigen-specific T cell responses by flow cytometry and IFNγ ELISpot respectively. RNA-LPX vaccines significantly potentiate radiotherapy-mediated tumor growth inhibition. CIRT and XRT alone marginally prime neoAg-specific T cell responses in the tumor, but not in the blood or spleen of the mice. The infiltration and cytotoxicity of neoAg-specific T cells is strongly driven by RNA-LPX vaccines and accompanied by reduced expression of the inhibitory markers PD-1 and Tim-3 on these cells. NeoAg RNA-LPX vaccines shows similar overall therapeutic efficacy in combination with both CIRT and XRT, even if the physical radiation dose is lower for carbon ions than X-rays. We hence conclude that the combination of CIRT and neoAg RNA-LPX is a promising strategy for treatment of radioresistant tumors.

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.

Stable IL‐2 Nano‐Assembly for Improved Anti‐Tumor Effect

AbstractInterleukin 2 (IL‐2) is the first approved immune therapeutic drug to treat cancer, however various off‐target effects and intolerable dose‐related toxicities induced by high‐dose intravenous infusion regimens result in a large proportion of patients require dose reduction, preventing the widespread adoption of this treatment. To address these issues, a novel IL‐2 nano‐assembly formulation (nano‐IL‐2) is developed using distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐n‐[methoxy (polyethylene glycol)‐2000] (PEG2000‐DSPE), which is durably stable due to the high binding affinity (10–8 m level) of two components and hardly induces vascular leak or inflamed injury at the injection site. Besides, nano‐IL‐2 exerts excellent solubility, lymph‐targeting property, prolonged and stable serum IL‐2 concentration ranges, and much lower toxicities compared to commercial formulation. Monotherapy of nano‐IL‐2 shows optimal capability to control the growth of murine melanoma and colon cancer. Collectively, the present study provides a novel design strategy for lymph‐targeting IL‐2 formulation which is more suitable for subcutaneous administration with higher safety concern.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

High efficacy of particle beam therapies against tumors under hypoxia and prediction of the early stage treatment effect using 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography

ObjectiveCompared with radiation therapy using photon beams, particle therapies, especially those using carbons, show a high relative biological effectiveness and low oxygen enhancement ratio. Using cells cultured under normoxic conditions, our group reported a greater suppressive effect on cell growth by carbon beams than X-rays, and the subsequent therapeutic effect can be predicted by the cell uptake amount of 3'-deoxy-3'-[18F]fluorothymidine (18F-FLT) the day after treatment. On the other hand, a hypoxic environment forms locally around solid tumors, influencing the therapeutic effect of radiotherapy. In this study, the influence of tumor hypoxia on particle therapies and the ability to predict the therapeutic effect using 18F-FLT were evaluated.MethodsUsing a murine colon carcinoma cell line (colon 26) cultured under hypoxic conditions (1.0% O2 and 5.0% CO2), the suppressive effect on cell growth by X-ray, proton, and carbon irradiation was evaluated. In addition, the correlation between decreased 18F-FLT uptake after irradiation and subsequent suppression of cell proliferation was investigated.ResultsTumor cell growth was suppressed most efficiently by carbon-beam irradiation. 18F-FLT uptake temporarily increased the day after irradiation, especially in the low-dose irradiation groups, but then decreased from 50 h after irradiation, which is well correlated with the subsequent suppression on tumor cell growth.ConclusionsCarbon beam treatment shows a strong therapeutic effect against cells under hypoxia. Unlike normoxic tumors, it is desirable to perform 18F-FLT positron emission tomography 2–3 days after irradiation for early prediction of the treatment effect.Graphical

Inflammation-Related Immune-Modulatory SLURP1 Prevents the Proliferation of Human Colon Cancer Cells, and Its Delivery by Salmonella Demonstrates Cross-Species Efficacy against Murine Colon Cancer.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans.