Nilotinib boosts the efficacy of anti-PDL1 therapy in colorectal cancer by restoring the expression of MHC-I.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have revolutionized the landscape of cancer treatment, only a minority of colorectal cancer (CRC) patients respond to them. Enhancing tumor immunogenicity by increasing major histocompatibility complex I (MHC-I) surface expression is a promising strategy to boost the antitumor efficacy of ICIs. METHODS: Dual luciferase reporter assays were performed to find drug candidates that can increase MHC-I expression. The effect of nilotinib on MHC-I expression was verified by dual luciferase reporter assays, qRT-PCR, flow cytometry and western blotting. The biological functions of nilotinib were evaluated through a series of in vitro and in vivo experiments. Using RNA-seq analysis, immunofluorescence assays, western blotting, flow cytometry, rescue experiments and microarray chip assays, the underlying molecular mechanisms were investigated. RESULTS: Nilotinib induces MHC-I expression in CRC cells, enhances CD8+ T-cell cytotoxicity and subsequently enhances the antitumor effects of anti-PDL1 in both microsatellite instability and microsatellite stable models. Mechanistically, nilotinib promotes MHC-I mRNA expression via the cGAS-STING-NF-κB pathway and reduces MHC-I degradation by suppressing PCSK9 expression in CRC cells. PCSK9 may serve as a potential therapeutic target for CRC, with nilotinib potentially targeting PCSK9 to exert anti-CRC effects. CONCLUSION: This study reveals a previously unknown role of nilotinib in antitumor immunity by inducing MHC-I expression in CRC cells. Our findings suggest that combining nilotinib with anti-PDL1 therapy may be an effective strategy for the treatment of CRC.

Tumor derived exosomal ENTPD2 impair CD8+ T cell function in colon cancer through ATP-adenosine metabolism reprogramming.

BACKGROUND: Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer. METHODS: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. RESULTS: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. CONCLUSION: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

Knockdown LIMP2 inhibits colorectal cancer cells migration, invasion, and metastasis.

Colorectal cancer (CRC) is a common malignancy worldwide nowadays and liver metastasis is the primary cause of death in patients with CRC. Although lysosomal integral membrane protein 2 (LIMP2) has been reported to play important roles in gastric cancer and prostate cancer, its role in CRC remains unclear. The aim of this study was to investigate the function of LIMP2 in CRC invasion and migration, along with the potential underlying molecular mechanisms. We found that LIMP2 levels were higher in CRC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that high expression of LIMP2 was associated with worse prognosis in CRC patients. Knockdown of LIMP2 significantly inhibited invasion, migration, and wound healing abilities of CRC cells in vitro, and inhibited CRC liver metastasis in vivo. Additionally, LIMP2 knockdown inhibited autophagy in CRC. Therefore, LIMP2 plays an important role in CRC progression. High expression of LIMP2 was associated with worse prognosis in CRC patients. Knockdown LIMP2 can effectively inhibit CRC cell migration and invasion in vitro and prevent liver metastasis in vivo. These findings suggest that LIMP2 may serve as an independent prognostic factor and potential therapeutic target for CRC.

Targeting RPA promotes autophagic flux and the antitumor response to radiation in nasopharyngeal carcinoma.

BACKGROUND: Autophagy is involved in nasopharyngeal carcinoma (NPC) radioresistance. Replication protein A 1 (RPA1) and RPA3, substrates of the RPA complex, are potential therapeutic targets for reversing NPC radioresistance. Nevertheless, the role of RPA in autophagy is not adequately understood. This investigation was performed to reveal the cytotoxic mechanism of a pharmacologic RPA inhibitor (RPAi) in NPC cells and the underlying mechanism by which RPAi-mediated autophagy regulates NPC radiosensitivity. METHODS AND RESULTS: We characterized a potent RPAi (HAMNO) that was substantially correlated with radiosensitivity enhancement and proliferative inhibition of in vivo and in NPC cell lines in vitro. We show that the RPAi induced autophagy at multiple levels by inducing autophagic flux, AMPK/mTOR pathway activation, and autophagy-related gene transcription by decreasing glycolytic function. We hypothesized that RPA inhibition impaired glycolysis and increased NPC dependence on autophagy. We further demonstrated that combining autophagy inhibition with chloroquine (CQ) treatment or genetic inhibition of the autophagy regulator ATG5 and RPAi treatment was more effective than either approach alone in enhancing the antitumor response of NPC to radiation. CONCLUSIONS: Our study suggests that HAMNO is a potent RPAi that enhances radiosensitivity and induces autophagy in NPC cell lines by decreasing glycolytic function and activating autophagy-related genes. We suggest a novel treatment strategy in which pharmacological inhibitors that simultaneously disrupt RPA and autophagic processes improve NPC responsiveness to radiation.

Pyrimethamine inhibits cell growth by inducing cell senescence and boosting CD8+ T-cell mediated cytotoxicity in colorectal cancer.

BACKGROUND: The emergence of nonresponse or resistance to traditional chemotherapeutic agents is one of the main challenges of colorectal cancer (CRC) therapies. Thus, novel therapeutic drugs that can improve the clinical outcomes of CRC patients are urgently needed. The purpose of this study was to investigate the effects and mechanisms of pyrimethamine in CRC. METHODS AND RESULTS: In this study, we assessed the role of pyrimethamine on CRC cell growth by cell counting kit-8 and colony formation assays. Cell cycle distribution and cellular senescence were determined by flow cytometry and senescence-associated ß-galactosidase staining respectively. RNA-seq analysis and western blotting were used to investigate the potential pathways of pyrimethamine in CRC development. Moreover, animal experiments were performed to evaluate the effect of pyrimethamine in vivo. Our results demonstrated that pyrimethamine could inhibit cell growth by inducing S phase arrest followed by cellular senescence in CRC cells, and the p38MAPK-p53 axis was probably involved in that effect. In addition, pyrimethamine could also boost CD8+ T-cell mediated cytotoxicity and exert antitumor activity in vivo. CONCLUSION: These results indicated that pyrimethamine may be a promising candidate agent for CRC treatment.

Evacetrapib Elicits Antitumor Effects on Colorectal Cancer by Inhibiting the Wnt/ß-Catenin Signaling Pathway and Activating the JNK Signaling Pathway.

Despite advances in colorectal cancer (CRC) treatment, most advanced CRC patients who experience disease progression after chemotherapy, targeted therapy, and immunotherapy face a situation in which there is no available medicine. Thus, new therapeutic drugs for CRC are urgently needed. Studies have shown that cholesteryl ester transfer protein (CETP) has a vital role in tumor development and is a possible target for CRC therapy. We found that Evacetrapib, a CETP inhibitor, suppressed CRC cell growth by inhibiting the Wnt/ß-catenin signaling pathway and activating the c-Jun NH2-terminal kinase (JNK) signaling pathway in CRC. Therefore, Evacetrapib displays an anti-cancer effect and is a possible option for treating CRC.

Comparison of the binding interactions of 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicides with humic acid: Insights from multispectroscopic techniques, DFT and 2D-COS-FTIR.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor is one of the important herbicides to solve the problem of weed control. With the widespread and continued use of HPPD inhibitor (HPPDi) herbicides, it may inevitably put pressure on the environment. Humic acid (HA) can effectively interact with pesticides through sorption or covalent bond formation and promote the degradation of pesticides, which can reduce the risk of pesticides in the environment. In the present study, the interactions of four HPPDi herbicides (sulcotrione, tembotrione, topramezone and mesotrione) with HA were reported and comparative assessment of the binding using multispectral technology, density functional theory (DFT) calculation and two-dimensional correlation spectroscopy (2D-COS). Time-resolved measurements and the Stern-Volmer constant at different temperature verified that HPPDi can bind with HA through the static quenching mechanism. From the thermodynamic parameters, the interaction force between HA and sulcotrione, tembotrione, topramezone and mesotrione was provided by electrostatic force. DFT, binding constant and three-dimensional (3D) fluorescence peak variation all indicated that the order of the binding ability of the four HPPDi and HA was mesotrione > tembotrione > sulcotrione > topramezone. According to dynamic light scattering (DLS), pH 7 is most conducive to the formation of HA-HPPDi complexes. Fourier transform infrared spectroscopy (FTIR) and 2D-COS showed that HA combined with HPPDi through aromatic C-H, CO and C-X, and the first binding group to HA was almost all CO. Sulcotrione, tembotrione, topramezone and mesotrione quench the endogenous fluorescence of HA by a static quenching mechanism and bind to HA through electrostatic interaction to form a complex. These results provide important insights into the combination of environmental pollutants with HA.

The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice.

Bacillus subtilis (B. subtilis) spore can serve as an ideal vehicle for expressing heterologous antigens, and elicit specific immune responses by oral administration. In previous studies, we successfully constructed the recombinant B. subtilis spores expressing cysteine protease of Clonorchis sinensis (C. sinensis, B.s-CsCP), and confirmed that oral administration of B.s-CsCP could elicit good protective immune responses in mice. In this study, Gram staining was used to observe the morphology of B.s-CsCP in different form, and the storage of liquid spores and lyophilized spores at different temperatures was compared. The mice were orally immunized with three different doses of spores (2×108, 1×109, and 5×109 CFU/day) for three times in total at biweekly interval. Then, antibody levels of mice were measured, the safety of spores was evaluated, and the changes of gut microbiota after oral gavage of spores (1×109 dose) were investigated. Results showed that B. subtilis was a typical Gram-positive bacterium, and its spore had good resistance to chemical dye. Liquid B. subtilis spores resuspended in sterile water could be stored for a long time at 4 °C or below, while lyophilized spores could be well stored even at RT and better at lower temperatures. Oral administration of B. subtilis spores to mice could stimulate both local mucosal and systemic immune responses in a dose-dependent manner without toxic side effects. Besides, beneficial bacteria producing butyrate such as Odoribacter were increased, while potential pathogens such as Escherichia-Shigella were decreased in mice intestine. Therefore, our work further confirmed that B. subtilis spores expressing CsCP could be a promising oral vaccine against C. sinensis with the advantages of stability, safety, easy storage, and promotion of intestinal health.Key Points• Recombinant CsCP B. subtilis spores could be easily preserved in either liquid or freeze-dried state.• Oral immunization of recombinant spores in mice could increase both local and system immune levels in a dose-dependent manner.• Oral administration of recombinant spores increased the number of beneficial bacteria and reduced the number of harmful bacteria in the intestinal tract of mice.

Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection.

Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.

Clonorchis sinensis lysophospholipase inhibits TGF-ß1-induced expression of pro-fibrogenic genes through attenuating the activations of Smad3, JNK2, and ERK1/2 in hepatic stellate cell line LX-2.

Liver fibrosis is a wound healing response associated with chronic liver injury. Hepatic stellate cells (HSCs) activation is a key event in the development of liver fibrosis. Since helminths have the ability to live for decades in the host by establishing an adaptive relationship in the interplay with its hosts, we hypothesize that whether Clonochis sinensis LysophospholipaseA (CsLysoPLA), a component of excretory/secretory proteins, can attenuate the fibrogenic response by inhibiting activation of LX-2 cells, thereby balancing the pro-fibrotic and anti-fibrotic response during the Clonochis sinensis (C. sinensis) infection. In the present study, LX-2 cells were stimulated with CsLysoPLA in the presence of TGF-ß1, and the expressions of collagen type I (COL1A1), α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 (MMP2) were decreased. In addition, CsLysoPLA significantly inhibited the proliferation and migration of LX-2 cells stimulated by TGF-ß1. Pretreatment of LX-2 cells with CsLysoPLA attenuated the phosphorylation of Smad3 as well as JNK2 and ERK1/2 in response to the stimulation of TGF-ß1. For the first time, our results showed an anti-fibrogenic effect of CsLysoPLA by attenuating the response of LX-2 cells to TGF-ß1 through inhibiting the activations of Smad3, ERK1/2, and JNK2.

Oral delivery of Bacillus subtilis spore expressing enolase of Clonorchis sinensis in rat model: induce systemic and local mucosal immune responses and has no side effect on liver function.

Caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensis, human clonorchiasis remains a major public health problem in China. In previous study, we had expressed enolase from C. sinensis (CsENO) on the surface of Bacillus subtilis spore and the recombinant spore induced a pronounced protection in terms of reduced worm burden and eggs per gram feces, suggesting B. subtilis spore as an ideal vehicle for antigen delivery by oral treatment and CsENO as a promising vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgG and IgA levels both in serum and in intestinal mucus from rats orally administrated with B. subtilis spore surface expressing CsENO by ELISA. Lysozyme levels in serum and in intestinal mucus were analyzed too. In addition, IgA-secreting cells in intestine epithelium of the rats were detected by immunohistochemistry assay. The intestinal villi lengths of duodenum, jejunum, and ileum were also measured. Rats orally treated with B. subtilis spore or normal saline were used as controls. Our results showed that, compared with the control groups, oral administration of B. subtilis spore expressing CsENO induced both systemic and local mucosal immune response. The recombinant spores also enhanced non-specific immune response in rats. The spores had no side effect on liver function. Moreover, it might facilitate food utilization and digestion of the rats. Our work will pave the way to clarify the involved mechanisms of protective efficacy elicited by B. subtilis spore expressing CsENO and encourage us to carry out more assessment trails of the oral treated spore to develop vaccine against clonorchiasis.

Prothioconazole Stress Reduces Bacterial Richness and Alters Enzyme Activity in Soybean Rhizosphere.

Prothioconazole (PTC) is currently a popular triazole fungicide. In recent years, as the use of PTC has increased, there has been growing concern about its environmental and toxicological effects. Here, we studied the effect of PTC on the growth of soybean plants and further analyzed the enzyme activity and microbial community of rhizosphere soil after PTC treatment through 16S rRNA gene high-throughput sequencing and fungal ITS. Changes in structural diversity and species richness were measured using Simpson's diversity index, Shannon's diversity index and the Chao1 and ACE algorithms. The statistical t-test was applied to test whether the index values were significantly different between the two groups. The results showed that the contents of malondialdehyde (MDA) and H2O2 increased after the recommended dose of PTC, indicating that PTC has a strong toxic effect on plant growth, thus affecting the healthy growth of plants. In the presence of PTC, the species richness of fungi and bacteria decreased in all three soil types (black soil, yellow earth and red earth), and the community structure also changed significantly (the p-values were all less than 0.05). Proteobacteria, Actinomycetota, Bacteroidota and Acidobacteriota were the main bacteria, and the abundance of Acidobacteriota and Chloroflexi increased. The dominant fungal communities were Ascomycota and Mortierellomycota. The increased abundance of potentially beneficial microorganisms, such as Sphingomonadaceae, suggested that plants may be resistant to PTC stress by recruiting beneficial microorganisms. PICRUSt analysis showed that the metabolism-related functions and membrane transport pathway of rhizosphere bacterial community were inhibited after PTC stress. Spearman correlation analysis revealed a weak correlation between key fungal taxa and rhizosphere variables in the presence of PTC. Therefore, compared with those in the fungal community, the bacterial community was more likely to help plants resist PTC stress, indicating that these key fungal groups may indirectly help soybean growth under PTC stress by affecting the bacterial community.

Bazedoxifene attenuates dextran sodium sulfate-induced colitis in mice through gut microbiota modulation and inhibition of STAT3 and NF-κB pathways.

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract for which treatment options remain limited. In this study, we used a dual-luciferase-based screening of an FDA-approved drug library, identifying Bazedoxifene (BZA) as an inhibitor of the NF-κB pathway. We further investigated its therapeutic effects in a dextran sodium sulfate (DSS)-induced colitis model and explored its impact on gut microbiota regulation and the underlying molecular mechanisms. Our results showed that BZA significantly reduced DSS-induced colitis symptoms in mice, evidenced by decreased colon length shortening, lower histological scores, and increased expression of intestinal mucosal barrier-associated proteins, such as Claudin 1, Occludin, Zo-1, Mucin 2 (Muc2), and E-cadherin. Used independently, BZA showed therapeutic effects comparable to those of infliximab (IFX). In addition, BZA modulated the abundance of gut microbiota especially Bifidobacterium pseudolongum, and influenced microbial metabolite production. Crucially, BZA's alleviation of DSS-induced colitis in mice was linked to change in gut microbiota composition, as evidenced by in vivo gut microbiota depletion and fecal microbiota transplantation (FMT) mice model. Molecularly, BZA inhibited STAT3 and NF-κB activation in DSS-induced colitis in mice. In general, BZA significantly reduced DSS-induced colitis in mice through modulating the gut microbiota and inhibiting STAT3 and NF-κB activation, and its independent use demonstrated a therapeutic potential comparable to IFX. This study highlights gut microbiota's role in IBD drug development, offering insights for BZA's future development and its clinical applications.

HIF-1α Mediates Immunosuppression and Chemoresistance in Colorectal Cancer by Inhibiting CXCL9, -10 and -11.

Uncertainty exists regarding the mechanisms by which hypoxia-inducible factors (HIFs) control CD8+T-cell migration into tumor microenvironments. Here, we found that HIF-1α knockdown or overexpression resulted in increased or decreased CXCL9, -10, and -11 expression in vitro, respectively. Gene Set Variation Analysis revealed that elevated HIF-1α levels correlated with a poor prognosis, severe pathological stage, and an absence of CD8+ T cells in the tumor microenvironment in colorectal cancer (CRC) patients. HIF-1α was inversely associated with pathways beneficial to anti-tumor immunotherapy and cytokine/chemokine function. In vivo, inhibiting HIF-1α or its upstream regulator BIRC2 significantly suppressed tumor growth and promoted CD8+ T-cell infiltration. CXCR3 neutralizing antibodies reversed these effects, implicating the involvement of CXCL9, -10, and -11/CXCR3 axis. The presence of HIF-1α weakened the upregulation of CXCL9, -10, and -11 by bleomycin and doxorubicin. Combining HIF-1α inhibition with bleomycin promoted CD8+ T-cell infiltration and tumor suppression in vivo. Moreover, doxorubicin could upregulate CXCL9, -10 and -11 by suppressing HIF-1α. Our findings highlight the potential of HIF-1α inhibition to improve CRC microenvironments and increase chemotherapy sensitivity.

Effects of HPPD inhibitor herbicides on soybean root exudates: A combination study of multispectral technique and 2D-COS analysis.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides are widely used in modern agriculture. Plant root exudates (REs) play an important role in the adsorption, degradation, migration and transformation of pesticides in soil. In the present study, the structural affinity and interaction mechanism between four HPPD inhibitors (HPPDi) and soybean REs were investigated via multispectral technologies and two-dimensional correlation analysis (2D-COS). UV-vis absorption and fluorescence spectra showed that mesotrione, tembotrione, sulcotrione and topramezone effectively quench the intrinsic fluorescence of soybean REs through static quenching. The binding constant Ka revealed that the binding ability of HPPDi to soybean REs takes the following order: mesotrione > tembotrione > sulcotrione > topramezone. According to the thermodynamic parameters, the main interaction force between tembotrione, sulcotrione, topramezone and soybean REs is electrostatic interaction, while the main interaction force is a hydrogen bond or van der Waals force between mesotrione and soybean REs. The conformational changes of REs were attributed to HPPDi by 3D spectral evaluation. FTIR spectroscopy and 2D-COS analysis suggested that soybean REs mainly formed stable complexes with HPPDi through functional groups such as carbonyl, carboxyl, methoxy and nitrate, and the first binding groups were carbonyl and carboxyl. These results provide helpful information for the adsorption and desorption process of environmental pollutants on the surface of plants and soil.

Jun-APOE-LRP1 axis promotes tumor metastasis in colorectal cancer.

Apolipoprotein E (apoE) has previously been reported to play vital roles in tumor progression. However, the impact of apoE on colorectal cancer (CRC) metastasis remains largely unexplored. This study aimed to investigate the role of apoE in CRC metastasis and to identify the transcription factor and receptor of apoE involved in regulation of CRC metastasis. Bioinformatic analyses were conducted to examine the expression pattern and prognosis of apolipoproteins. APOE-overexpressing cell lines were utilized to explore the effects of apoE on proliferation, migration and invasion of CRC cells. Additionally, the transcription factor and receptor of apoE were screened via bioinformatics, and further validated through knockdown experiments. We discovered that the mRNA levels of APOC1, APOC2, APOD and APOE were higher in lymphatic invasion group, and a higher apoE level indicated poorer overall survival and progression-free interval. In vitro studies demonstrated that APOE-overexpression did not affect proliferation but promoted the migration and invasion of CRC cells. We also reported that APOE-expression was modulated by the transcription factor Jun by activating the proximal promoter region of APOE, and APOE-overexpression reversed the metastasis suppression of JUN knockdown. Furthermore, bioinformatics analysis suggested an interaction between apoE and low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 was highly expressed in both the lymphatic invasion group and the APOEHigh group. Additionally, we found that APOE-overexpression upregulated LRP1 protein levels, and LRP1 knockdown attenuated the metastasis-promoting function of APOE. Overall, our study suggests that the Jun-APOE-LRP1 axis contributes to tumor metastasis in CRC.

Low-dose radiotherapy combined with dual PD-L1 and VEGFA blockade elicits antitumor response in hepatocellular carcinoma mediated by activated intratumoral CD8+ exhausted-like T cells.

Atezolizumab (anti-PD-L1) combined with bevacizumab (anti-VEGFA) is the first-line immunotherapy for advanced hepatocellular carcinoma (HCC), but the number of patients who benefit from this regimen remains limited. Here, we combine dual PD-L1 and VEGFA blockade (DPVB) with low-dose radiotherapy (LDRT), which rapidly inflames tumors, rendering them vulnerable to immunotherapy. The combinatorial therapy exhibits superior antitumor efficacy mediated by CD8+ T cells in various preclinical HCC models. Treatment efficacy relies upon mobilizing exhausted-like CD8+ T cells (CD8+ Tex) with effector function and cytolytic capacity. Mechanistically, LDRT sensitizes tumors to DPVB by recruiting stem-like CD8+ Tpex, the progenitor exhausted CD8+ T cells, from draining lymph nodes (dLNs) into the tumor via the CXCL10/CXCR3 axis. Together, these results further support the rationale for combining LDRT with atezolizumab and bevacizumab, and its clinical translation.

Potential roles of serum ATPase and AMPase in predicting diagnosis of colorectal cancer patients.

Colorectal cancer (CRC) is a common gastrointestinal cancer with high incidence and mortality rates. CRC may be associated with regulation of circulating nucleotides. This study aimed to evaluate the serum levels of nucleotide-metabolizing enzymes (ATPase and AMPase) in patients with CRC and to explore the clinical diagnostic value of these enzymes. The gene set variation analysis (GSVA) score of the ATP-adenosine signature was calculated using tumor samples from The Cancer Genome Atlas (TCGA). ATP-adenosine signaling plays a central role in CRC progression. A total of 135 subjects, including 87 patients with CRC and 48 healthy controls, were included. The serum levels of ATPase and AMPase in the CRC group were significantly higher than those in the control group (P < 0.05). Furthermore, ATP and AMP hydrolysis levels significantly increased in the advanced CRC group (P < 0.05). ATP and AMP hydrolysis was decreased by the ENTPDase inhibitors (POM-1 and ARL67156) and CD73 inhibitor (APCP). The sensitivities of ATPase and AMPase were 95.4% and 75.9%, respectively, which were higher than those of CEA (67.8%) and CA19-9 (72.4%). The specificities of ATPase and AMPase were 69.9% and 73.9%, respectively, which were higher than that of CA19-9 (47.8%). The combination of CEA, ATPase, and AMPase demonstrated high sensitivity (92.0%) and specificity (87.0%). Collectively, ATPase and AMPase activities are upregulated in CRC with considerable diagnostic significance. The combination of CEA, ATPase, and AMPase may provide a novel approach for CRC screening.

CD8+CXCR5+T cells infiltrating hepatocellular carcinomas are activated and predictive of a better prognosis.

CD8+ T cells are thought to be the primary cytotoxic lymphocytes exerting antitumor effects. However, few studies have focused on the antitumor effects of CD8+ T cell-mediated humoral immunity or on interactions between CD8+ T cells and B cells in hepatocellular carcinoma (HCC). We found that the frequency of IL-21-producing CD8+CXCR5+ T cells was higher in HCC tumor tissue than in peritumoral tissue or peripheral blood from the same patients or in blood from healthy donors. Moreover, CD8+CXCR5+ T cells migrated in response to supernatants from primary HCC (HCC-SN) cells, and HCC-SN cells also powerfully induced CXCR5 expression in CD8+ T cells and IL-21 expression in CD8+CXCR5+ T cells. CD8+CXCR5+ T cells from HCC patients, but not those from healthy individuals, stimulated CD19+ B cells to differentiate into IgG-producing plasmablasts. These findings reveal that CD8+CXCR5+ T cells strongly infiltrate HCC tumors, and their infiltration is predictive of a better prognosis. Surprisingly, moreover, CD8+CXCR5+ T cells produced IL-21, which induced B cells to differentiate into IgG-producing plasmablasts and to play a key role in humoral immunity in HCC.

Tumor-derived exosomal HMGB1 fosters hepatocellular carcinoma immune evasion by promoting TIM-1+ regulatory B cell expansion.

BACKGROUND: Regulatory B (Breg) cells represent one of the B cell subsets that infiltrate solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the phenotype, function and clinical relevance of Breg cells in human hepatocellular carcinoma (HCC) are presently unknown. METHODS: Flow cytometry analyses were performed to determine the levels, phenotypes and functions of TIM-1+Breg cells in samples from 51 patients with HCC. Kaplan-Meier plots for overall survival and disease-free survival were generated using the log-rank test. TIM-1+Breg cells and CD8+ T cells were isolated, stimulated and/or cultured in vitro for functional assays. Exosomes and B cells were isolated and cultured in vitro for TIM-1+Breg cell expansion assays. RESULTS: Patients with HCC showed a significantly higher TIM-1+Breg cell infiltration in their tumor tissue compared with the paired peritumoral tissue. The infiltrating TIM-1+Breg cells showed a CD5highCD24-CD27-/+CD38+/high phenotype, expressed high levels of the immunosuppressive cytokine IL-10 and exhibited strong suppressive activity against CD8+ T cells. B cells activated by tumor-derived exosomes strongly expressed TIM-1 protein and were equipped with suppressive activity against CD8+ T cells similar to TIM-1+Breg cells isolated from HCC tumor tissue. Moreover, the accumulation of TIM-1+Breg cells in tumors was associated with advanced disease stage, predicted early recurrence in HCC and reduced HCC patient survival. Exosome-derived HMGB1 activated B cells and promoted TIM-1+Breg cell expansion via the Toll like receptor (TLR) 2/4 and mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: Our results illuminate a novel mechanism of TIM-1+Breg cell-mediated immune escape in HCC and provide functional evidence for the use of these novel exosomal HMGB1-TLR2/4-MAPK pathways to prevent and to treat this immune tolerance feature of HCC.