Corrigendum to "Generation of murine tumour-reactive T cells by co-culturing murine pancreatic cancer organoids and peripheral blood lymphocytes".

[This corrects the article DOI: 10.1016/j.bbrep.2022.101365.].

Generation of murine tumour-reactive T cells by co-culturing murine pancreatic cancer organoids and peripheral blood lymphocytes.

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at a late stage and becomes resistant to several treatments. Significant clinical effects have been reported for cancer immunotherapies on a subset of patients diagnosed with epithelial cancers. Cancer organoid co-culture with autologous peripheral blood lymphocytes offers an innovative immunotherapeutic approach that is increasingly being tested, although there is a lack of cutting-edge platforms enabling the investigation of cancer-T cell interactions for individual patients. In this study, a pancreatic cancer organoid culture from a genetically engineered pancreatic cancer murine model was established and co-cultured with autologous peripheral blood lymphocytes to induce a tumour-specific T cell response to pancreatic cancer. Co-culturing autologous peripheral blood lymphocytes with cancer organoids can be an effective strategy to enrich tumour-reactive T cells from the peripheral blood of murine models; this approach could potentially be transferred to humans. Co-culture of peripheral blood lymphocytes and cancer organoids could provide an unbiased approach to evaluating the sensitivity of tumour cells to T cell-mediated priming on an individual patient level.

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing's sarcoma cells.

Ewing's sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewing's sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

Development of X-ray contrast agents using single nanometer-sized gold nanoparticles and lactoferrin complex and their application in vascular imaging.

The technology to accurately image the morphology of tumor vessels with X-ray contrast agents is important to clarify mechanisms underlying tumor progression and evaluate the efficacy of chemotherapy. However, in clinical practice, iodine-based contrast agents present problems such as short blood retention owing to a high clearance ability and insufficient X-ray absorption capacity when compared with other high atomic number elements. To resolve these issues, gold nanoparticles (AuNPs), with a high atomic number, have attracted a great deal of attention as contrast agents for angiography, and have been employed in small animal models. Herein, we developed novel contrast agents using AuNPs and captured changes in tumor vessel morphology with time using X-ray computed tomography (CT). First, glutathione-supported single nanometer-sized AuNPs (sAu/GSH) (diameter, 2.2 nm) were fabricated using tetrakis(hydroxymethyl)phosphonium chloride as a reducing agent. The sAu/GSH particles were intravenously injected into mice, remained in vessels for a few minutes, and were then excreted by the kidneys after 24 h, similar to the commercial contrast agent iopamidol. Next, the Au/GSH and lactoferrin (sAu/GSH-LF) (long axis size, 17.3 nm) complex was produced by adding lactoferrin to the sAu/GSH solution under the influence of a condensing agent. On intravenously administering sAu/GSH-LF to mice, the blood retention time was 1-3 h, which was considerably longer than that observed with iopamidol and sAu/GSH. Moreover, we succeeded in imaging morphological changes in identical tumor vessels for several days using X-ray CT with sAu/GSH-LF.

Heterogeneous Drug Efficacy of an Antibody-Drug Conjugate Visualized Using Simultaneous Imaging of Its Delivery and Intracellular Damage in Living Tumor Tissues.

Anticancer drug efficacy varies because the delivery of drugs within tumors and tumor responses are heterogeneous; however, these features are often more homogenous in vitro. This difference makes it difficult to accurately determine drug efficacy. Therefore, it is important to use living tumor tissues in preclinical trials to observe the heterogeneity in drug distribution and cell characteristics in tumors. In the present study, to accurately evaluate the efficacy of an antibody-drug conjugate (ADC) containing a microtubule inhibitor, we established a cell line that expresses a fusion of end-binding protein 1 and enhanced green fluorescent protein that serves as a microtubule plus-end-tracking protein allowing the visualization of microtubule dynamics. This cell line was xenografted into mice to create a model of living tumor tissue. The tumor cells possessed a greater number of microtubules with plus-ends, a greater number of meandering microtubules, and a slower rate of microtubule polymerization than the in vitro cells. In tumor tissues treated with fluorescent dye-labeled ADCs, heterogeneity was observed in the delivery of the drug to tumor cells, and microtubule dynamics were inhibited in a concentration-dependent manner. Moreover, a difference in drug sensitivity was observed between in vitro cells and tumor cells; compared with in vitro cells, tumor cells were more sensitive to changes in the concentration of the ADC. This study is the first to simultaneously evaluate the delivery and intracellular efficacy of ADCs in living tumor tissue. Accurate evaluation of the efficacy of ADCs is important for the development of effective anticancer drugs.

Quantitative analyses of amount and localization of radiosensitizer gold nanoparticles interacting with cancer cells to optimize radiation therapy.

Previous studies showed that gold nanoparticles (AuNPs) are useful radiosensitizers which optimize radiation therapy under low-dose radiation. However, the mechanisms of AuNP radiosensitization, including the amount and localization of the AuNPs interacting with cancer cells, has not yet been quantified. To answer these questions, we prepared AuNPs conjugated with anti-human epidermal growth factor receptor type 2 (HER2) antibody via polyethylene glycol (PEG) chains (AuNP-PEG-HER2ab). AuNP-PEG-HER2ab specifically bound to the HER2-expressing cancer cells and entered the cells via endocytosis. Whether endocytosis of AuNP-PEG-HER2ab occurred had no effect on radiosensitization efficacy by AuNP-PEG-HER2ab in vitro. The radiosensitization efficacy in vitro depended on dose of AuNP-PEG-HER2ab or dose of X-ray. Moreover, AuNP-PEG-HER2ab administrated into tumor-bearing mice was localized to both the periphery of the tumor tissue and near the nuclei in cancer cells in tumor deep tissue. The localization of AuNP-PEG-HER2ab in tumor tissues was important factors for in vivo powerful radiosensitization efficacy.

CUB Domain-containing Protein 1 (CDCP1) Is Down-regulated by Active Hexose-correlated Compound in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS: Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS: Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p<0.05) compared to untreated cells. CONCLUSION: AHCC down-regulated CDCP1 expression and inhibited the malignant progression of pancreatic cancer cells.

Fabrication of silica-coated gold nanorods and investigation of their property of photothermal conversion.

This study described the preparation of silica-coated Au nanorods (AuNR/SiO2) in a colloidal solution, assessed their property of photothermal conversion, and investigated their ability to kill cancer cells using photothermal conversion. Au-seed nanoparticles were produced by reducing hydrogen tetrachloroaurate (HAuCl4) with sodium borohydride (NaBH4) in aqueous n-hexadecyltrimethylammonium bromide (CTAB) solution. AuNRs were then fabricated by reducing HAuCl4 and silver nitrate (AgNO3) with l-ascorbic acid in the aqueous CTAB solution in the presence of Au-seed nanoparticles. The as-prepared AuNRs were washed by a process composed mainly of centrifugation to remove the CTAB. The washed AuNRs were coated with silica by mixing the AuNR colloidal solution, an aqueous solution of (3-aminopropyl)trimethoxysilane, and tetraethylorthosilicate/ethanol solution with a water/ethanol solution. We found that the addition of AuNR/SiO2 in water, in mice, and in a culture medium with cancer cells, followed by irradiation with a laser, cause an increase in temperature, demonstrating that AuNR/SiO2 have the ability of photothermal conversion. In addition, the cancer cells in the culture medium were found to be killed due to the increase in temperature caused by the photothermal conversion.

Active Hexose-correlated Compound Down-regulates Heat Shock Factor 1, a Transcription Factor for HSP27, in Gemcitabine-resistant Human Pancreatic Cancer Cells.

BACKGROUND: Active hexose-correlated compound (AHCC) is an extract of a basidiomycete mushroom that enhances the therapeutic effects and reduces the side-effects of chemotherapy. Our previous studies demonstrated that heat-shock protein 27 (HSP27) was involved in gemcitabine-resistance of pancreatic cancer cells and it was down-regulated by AHCC-treatment. However, how AHCC down-regulated HSP27 is unknown. In the present study, we focused on two transcription factors reported to induce HSP27, heat shock factor 1 (HSF1) and high-mobility group box 1 (HMGB1) and investigated the effect of AHCC on their expression. MATERIALS AND METHODS: KLM1-R cells were treated with AHCC and the protein expression of HSF1 and HMGB1 were analyzed by western blotting. RESULTS: The protein expression of HSF1 in KLM1-R was down-regulated by AHCC treatment. On the other hand, the protein expression of HMGB1 was not reduced in KLM1-R cells after AHCC treatment. CONCLUSION: The possibility that AHCC down-regulated HSP27 through down-regulation of the HSF1, was herein shown.

High-mobility Group Box 1 and Mitogen-activated Protein Kinase activated Protein Kinase-2 Are Up-regulated in Gemcitabine-resistant Pancreatic Cancer Cells.

BACKGROUND: Results of our previous studies demonstrated that the expression of heat-shock protein 27 (HSP27) was increased and HSP27 was phosphorylated in the GEM-resistant pancreatic cancer cell line, KLM1-R. The expression of HSP27 is regulated mainly by heat-shock factor 1, but other transcription factors or kinases have been reported to activate HSP27. High-mobility group box 1 (HMGB1) is a nuclear transcription factor. It has been reported that HMGB1 regulates HSP27 gene expression. Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) phosphorylates HSP27. In the present study, we investigated the expression of HMGB1 and MAPKAPK2 in KLM1-R cells. MATERIALS AND METHODS: The expression levels of HMGB1 and MAPKAPK2 were compared between KLM1 and KLM1-R cells by western blotting. RESULTS: The protein expression of both HMGB1 and MAPKAPK2 were increased in KLM1-R cells compared to KLM1 cells. CONCLUSION: The increase of both HMGB1 and MAPKAPK2 in KLM1-R cells compared to KLM1 suggest the possibility of the activation of the pathway of HSP27 by HMGB1 and MAPKAPK2 in gemcitabine-resistant KLM1-R cells.

Functional characterization of the mutations in Pepper mild mottle virus overcoming tomato tm-1-mediated resistance.

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm-1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV-899R,F976Y and PMMoV-899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV-899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm-1 protein and enhanced the replication efficiency of PMMoV-899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild-type PMMoV > PMMoV-899R,F976Y > PMMoV-899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side-effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm-1-mediated resistance.

Generation of a panel of monoclonal antibodies against atypical chemokine receptor CCX-CKR by DNA immunization.

INTRODUCTION: Chemokines are regulated by a family of 'atypical' chemokine receptors, D6, DARC and CCX-CKR, each of which efficiently internalizes its cognate chemokine ligands. Development of monoclonal antibodies (MAbs) that would recognize CCX-CKR on the cell surface will be helpful to identify primary CCX-CKR-expressing cell types and analyze the fate of CCX-CKR after ligand binding to the receptor. METHODS: We generated IgG MAbs recognizing the cell-surface CCX-CKR by DNA immunization using a molecular adjuvant, and analyzed the epitope recognized by the MAbs. Then, the reactivities of the MAbs with CCX-CKR-transfected cells, and also hepatocytes and hepatic tumor lines were evaluated. Finally, we also tested the ligand-like activities of the MAbs, namely, induction of internalization of CCX-CKR by the MAbs. RESULTS: A panel of MAbs reacting with CCX-CKR expressed on the cell surface was prepared. The panel was a small one, consisting of only ten MAbs, but was rich in terms of diversity of the Ig isotypes and of the epitopes. Epitope analyses revealed that all the 10 MAbs recognized at least three different, although very close, peptide structures of the N-terminal domain. Three MAbs, namely, 2F11, 13E11 and 14F10, were selected to represent the panel. All of the MAbs were applicable for flow cytometry and immunoflurescent assays and immunoprecipitation. The reactivity of the 2F11 MAb was also confirmed by western blotting. Endogenous expression of CCX-CKR on human hepatocytes and hepatic tumor cell lines was demonstrated using the 13E11 MAb. Interestingly, binding of the 13E11 MAb with B300-19 cells expressing CCX-CKR resulted in induction of CCX-CKR internalization. DISCUSSION: This panel of MAbs may be expected to prove valuable for further study of the functions of this silent chemokine receptor, including those related to the homeostasis of lymphoid cells, and to the growth and metastasis of hepatic cancer.