Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer ...cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software “Cancer Cell Finder” that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.
Glypican-3 (GPC3), a 65 kD protein consisting of 580 amino acids, is a heparan sulfate proteoglycan bound to the cell membrane by glycosylphosphatidylinositol. This protein is expressed in the liver ...and the kidney of healthy fetuses but is hardly expressed in adults, except in the placenta. Contrarily, GPC3 is specifically expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), yolk sac tumor, and some pediatric cancers. Although the precise function of GPC3 remains unclear, it has been strongly suggested that it is related to the malignant transformation of HCC. We identified GPC3 as a promising target for cancer immunotherapy and have been working on the development of cancer immunotherapeutic agents targeting it through clinical trials. In some trials, it was revealed that the GPC3 peptide vaccines we developed using human leukocyte antigen-A24- and A2-restricted GPC3-derived peptides could induce GPC3-specific cytotoxic T cells in most vaccinated patients and thereby improve their prognosis. To further improve the clinical efficacy of cancer immunotherapy targeting GPC3, we are also developing next-generation therapeutic strategies using T cells engineered to express antigen-specific T-cell receptor or chimeric antigen receptor. In addition, we have successfully monitored the levels of serum full-length GPC3 protein, which is somehow secreted in the blood. The utility of GPC3 as a biomarker for predicting tumor recurrence and treatment efficacy is now being considered. In this review article, we summarize the results of clinical trials carried out by our team and describe the novel agent targeting the cancer-specific shared antigen, GPC3.
Long-read sequencing of full-length cDNAs enables the detection of structures of aberrant splicing isoforms in cancer cells. These isoforms are occasionally translated, presented by HLA molecules, ...and recognized as neoantigens. This study used a long-read sequencer (MinION) to construct a comprehensive catalog of aberrant splicing isoforms in non-small-cell lung cancers, by which novel isoforms and potential neoantigens are identified.
Full-length cDNA sequencing is performed using 22 cell lines, and a total of 2021 novel splicing isoforms are identified. The protein expression of some of these isoforms is then validated by proteome analysis. Ablations of a nonsense-mediated mRNA decay (NMD) factor, UPF1, and a splicing factor, SF3B1, are found to increase the proportion of aberrant transcripts. NetMHC evaluation of the binding affinities to each type of HLA molecule reveals that some of the isoforms potentially generate neoantigen candidates. We also identify aberrant splicing isoforms in seven non-small-cell lung cancer specimens. An enzyme-linked immune absorbent spot assay indicates that approximately half the peptide candidates have the potential to activate T cell responses through their interaction with HLA molecules. Finally, we estimate the number of isoforms in The Cancer Genome Atlas (TCGA) datasets by referring to the constructed catalog and found that disruption of NMD factors is significantly correlated with the number of splicing isoforms found in the TCGA-Lung Adenocarcinoma data collection.
Our results indicate that long-read sequencing of full-length cDNAs is essential for the precise identification of aberrant transcript structures in cancer cells.
Methylglyoxal (MGO) is a reactive α-dicarbonyl compound that causes carbonylation of protein and DNA through the pathways of the Maillard reaction. It is known that MGO is physiologically involved in ...renal dysfunction, vascular disorders, and the acceleration of aging. In this study, we showed for the first time, that a trace amount of MGO was present as an impurity in glycerol preparations used as external medicines and intravenous infusions, when kept unused. The concentration of MGO in the glycerol solutions, diluted to a concentration of 20%, significantly increased after storage for one month when compared to the MGO concentration immediately after opening. Following storage for 6 months at 25°C, MGO concentration increased by about 300 times (approx. 170 µM), and at 40°C, it increased by about 600 times (approx. 350 µM). In the case of intravenous infusion preparations containing 10% glycerol, the MGO concentration increased by 4–15 times (approx. 70 µM) after 2 months of storage at 40°C, and reached over 200 µM after 6 months. Results from the present study showed that glycerol in pharmaceutical preparations is gradually oxidized to form MGO via autoxidation, depending on the temperature and dissolved oxygen content. Thus, we suggest that precautions should be taken when storing glycerol preparations in bottles or plastic containers, with respect to the storage temperature and sealability to prevent MGO formation due to oxidation of glycerol.
The present study focused on the methylglyoxal (MG) detoxification mechanism in neuroblastoma cells. The involvement of nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated ...protein 1 (Keap1) pathway as a defense response against the formation of MG-modified proteins, which is well-known evidence of carbonyl stress, was also examined.
We found that MG treatment resulted in accumulation of modified proteins bearing the structure of advanced glycation end products (AGEs) derived from MG in SH-SY5Y cells. This accumulation was suppressed by activation of the Nrf2 pathway prior to MG exposure via pre-treatment with an Nrf2 activator, carnosic acid and CDDO-Im, confirming the involvement of the Nrf2 pathway in MG detoxification. Although pre-treatment with the Nrf2 activator did not affect mRNA levels of GLO1, AKR1B1, and AKR7A2, the expressions of GCL and xCT mRNA, involved in GSH synthesis, were induced prior to increase in GSH levels. Furthermore, we demonstrated that a GSH synthesis inhibitor eliminated the MG detoxification effect derived from pretreatment with the Nrf2 activator. These results indicated that increase in GSH levels, induced by pre-treatment with carnosic acid, promoted the formation of the GLO1 substrate, hemithioacetal, thereby accelerating MG metabolism via the glyoxalase system and suppressing its toxicity. It was, therefore, determined that promotion of GSH synthesis via the Nrf2/Keap1pathway is important in the MG detoxification mechanism against neuronal MG-induced carbonyl stress, and Nrf2 activators contribute to reduction in the accumulation and toxic expression of carbonyl proteins.
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•Nrf2 activator prevents methylglyoxal-induced cytotoxicity and protein modifications.•MG treatment resulted in accumulation of modified proteins bearing the structure of CEL and MG-H1.•The promotion of GSH synthesis by activation of Nrf2 is a key response to MG detoxification.
This study performed a comprehensive molecular characterization of microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) gastrointestinal (GI) tumors to elucidate predictors of ...response to PD-1 blockade.
Forty-five patients with MSI-H/dMMR GI tumors, including gastric cancer, colorectal cancer, cholangiocarcinoma, small intestine cancer, pancreatic cancer, and duodenal cancer, receiving PD-1 blockade were analyzed. We conducted the genomic profiling of GI tumors by whole-exome sequencing or targeted next-generation sequencing. The tumor microenvironment was evaluated by transcriptomic analysis and multiplex fluorescence IHC.
Patients with low tumor mutational burdens (TMBs) had lower objective response rates (ORRs; 0% vs. 48.8%) and a significantly shorter progression-free survival (PFS; 2.3 vs. 15.6 months; HR, 6.20;
= 0.002) than those with high TMBs. Among common gene alterations in GI tumors, only
mutations, which were mutually exclusive with a low TMB, were significantly associated with a lower ORRs than wild-type
(21.4 vs. 54.8%; odds, 4.45;
= 0.045). Compared with wild-type
mutations in the phosphatase domain were associated with significantly lower ORRs (12.5 vs. 54.8%;
= 0.049), shorter PFS (2.6 vs. 15.6 months; HR, 5.04;
< 0.001), lower intratumoral CD8
T-cell levels, higher intratumoral CD204
macrophage levels, and PI3K/AKT/mTOR pathway enrichment, whereas
mutations in the C2 domain were not.
Low TMBs and
mutations, especially mutations in the phosphatase domain associated with an immunosuppressive environment, were mutually exclusive and might be negative predictors of PD-1 blockade responses in patients with MSI-H/dMMR GI tumors.
Size distribution and mapping analyses of Ag and Au nanoparticles (NPs) have been made using an ICP-MS combined with laser ablation sampling technique (LA-ICP-MS). With the femtosecond laser (Ti:S ...laser) ablation system, the measured size distribution was modified to be smaller, whereas this disintegration could be reduced when the nanosecond laser (ArF Excimer laser) ablation was employed. This suggests that both the size and position of the NPs can be defined by the nanosecond LA-ICP-MS technique. More importantly, based on the peak-height analyses (PHA) of the measured signal intensity profiles, the present form of the analytes, whether particulate or ionic form, could be defined. This is very important to investigate the transport of the NPs within biological samples. To demonstrate the unique feature of the technique, imaging analyses of Ag and Au NPs, together with distribution analysis of the ionic form, were conducted on onion cells, prepared through dosing experiments of the Ag and Au NPs. There were clear differences in both the increasing rates of the numbers of NPs and the concentration range of the ionic form between the Ag and Au. The data obtained here demonstrate clearly that the LA-ICP-MS technique can become a major analytical tool to obtain both the size distribution and position of NPs from tissue samples.
In this study, we developed an analytical method using LC-MS/MS for the simultaneous determination of five bile acids (BAs) that have been recently reported as candidate diagnostic biomarkers for ...Alzheimer’s disease (AD) or AD related factors in the brain. The measurement of BAs in the brains of healthy mice led to the determination of candidate diagnostic markers for AD, such as cholic acid and deoxycholic acid, and other bile acids, such as chenodeoxycholic acid noted for the ameliorating effect on the symptoms of AD. Significant positive correlations were observed between the brain and plasma concentrations of four BAs in healthy young mice. These results indicate that the BA level in the brain may be estimated by the corresponding BA level in the plasma. Thus, our study suggested that the proposed method for the analysis of the five bile acids would aid in the diagnosis of AD or in studies that use AD model mice.
Isotopic analysis, together with size distribution analyses of platinum nanoparticles (Pt NPs) have been made by a multiple collector-ICP-mass spectrometer equipped with a high-time resolution data ...acquisition system (HTR-MC-ICP-MS). With the present system, time-integration efficiency of nearly 100% was achieved from up to four isotopic signals, enabling us to measure both the size and isotopic ratios from a single NP. Based on the 195Pt/194Pt ratios obtained from more than 1000 particles, the precisions of the 195Pt/194Pt ratio measurements on 30 nm, 50 nm, and 70 nm were approximately 40% (2SD), 20% (2SD), and 10% (2SD). These results were comparable to possible variations in the 195Pt/194Pt ratios estimated by counting statistics of the isotopic signals, suggesting that the isotopic ratio measurements could be achieved from transient signal events originating from NPs. The data obtained here demonstrated that the HTR-MC-ICP-MS technique can become a powerful tool to monitor the elemental/isotopic ratios from single NPs.
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•Development of multiple collector inductively coupled plasma mass spectrometer equipped with a high-time resolution data acquisition system•Simultaneous detection of isotopic signals even from transient signal induced by the nanoparticles•Repeatability of isotopic ratio of nanoparticles is controlled by the counting statistics of the signal intensity•Size distribution of samples agreed well with the data shown in product information measured by transmission electron microscope
Immune checkpoint inhibitors have ushered in a new era in cancer therapy, although other therapies or combinations thereof are still needed for many patients for whom these drugs are ineffective. In ...this light, we have identified glypican‐3 an HLA‐24, HLA‐A2 restriction peptide with extreme cancer specificity. In this paper, we summarize results from a number of related clinical trials showing that glypican‐3 peptide vaccines induce specific CTLs in most patients (UMIN Clinical Trials Registry: UMIN000001395, UMIN000005093, UMIN000002614, UMN000003696, and UMIN000006357). We also describe the current state of personalized cancer immunotherapy based on neoantigens, and assess, based on our own research and experience, the potential of such therapy to elicit cancer regression. Finally, we discuss the future direction of cancer immunotherapy.
In this manuscript, we provide a summary of the multiple, related clinical trials we have completed in Japan to test GPC3‐based immunotherapy against hepatocellular carcinoma, ovarian clear‐cell adenocarcinoma, and some pediatric cancers. We also describe the state‐of‐the‐art of personalized cancer immunotherapy, and discuss future directions.
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