Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remain elusive. In this study, we examine the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we find that the directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. The elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further demonstrate that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.

Cancer-specific tissue-resident memory T-cells express ZNF683 in colorectal cancer.

BACKGROUND: Tissue-resident memory T (Trm) cells are associated with cytotoxicity not only in viral infection and autoimmune disease pathologies but also in many cancers. Tumour-infiltrating CD103+ Trm cells predominantly comprise CD8 T cells that express cytotoxic activation and immune checkpoint molecules called exhausted markers. This study aimed to investigate the role of Trm in colorectal cancer (CRC) and characterise the cancer-specific Trm. METHODS: Immunochemical staining with anti-CD8 and anti-CD103 antibodies for resected CRC tissues was used to identify the tumour-infiltrating Trm cells. The Kaplan-Meier estimator was used to evaluate the prognostic significance. Cells immune to CRC were targeted for single-cell RNA-seq analysis to characterise cancer-specific Trm cells in CRC. RESULTS: The number of CD103+/CD8+ tumour-infiltrating lymphocytes (TILs) was a favourable prognostic and predictive factor of the overall survival and recurrence-free survival in patients with CRC. Single-cell RNA-seq analysis of 17,257 CRC-infiltrating immune cells revealed a more increased zinc finger protein 683 (ZNF683) expression in cancer Trm cells than in noncancer Trm cells and in high-infiltrating Trm cells than low-infiltrating Trm in cancer, with an upregulated T-cell receptor (TCR)- and interferon-γ (IFN-γ) signalling-related gene expression in ZNF683+ Trm cells. CONCLUSIONS: The number of CD103+/CD8+ TILs is a prognostic predictive factor in CRC. In addition, we identified the ZNF683 expression as one of the candidate markers of cancer-specific Trm cells. IFN-γ and TCR signalling and ZNF683 expression are involved in Trm cell activation in tumours and are promising targets for cancer immunity regulation.

Non-degradable autophagic vacuoles are indispensable for cell competition.

Cell competition is a process by which unwanted cells are eliminated from tissues. Apical extrusion is one mode whereby normal epithelial cells remove transformed cells, but it remains unclear how this process is mechanically effected. In this study, we show that autophagic and endocytic fluxes are attenuated in RasV12-transformed cells surrounded by normal cells due to lysosomal dysfunction, and that chemical manipulation of lysosomal activity compromises apical extrusion. We further find that RasV12 cells deficient in autophagy initiation machinery are resistant to elimination pressure exerted by normal cells, suggesting that non-degradable autophagic vacuoles are required for cell competition. Moreover, in vivo analysis revealed that autophagy-ablated RasV12 cells are less readily eliminated by cell competition, and remaining transformed cells destroy ductal integrity, leading to chronic pancreatitis. Collectively, our findings illuminate a positive role for autophagy in cell competition and reveal a homeostasis-preserving function of autophagy upon emergence of transformed cells.

N(6)-methyladenosine methylation-regulated polo-like kinase 1 cell cycle homeostasis as a potential target of radiotherapy in pancreatic adenocarcinoma.

In pancreatic cancer, methyltransferase-like 3 (METTL3), a N(6)-methyladenosine (m6A) methyltransferase, has a favorable effect on tumors and is a risk factor for patients' prognosis. However, the details of what genes are regulated by METTL3 remain unknown. Several RNAs are methylated, and what genes are favored in pancreatic cancer remains unclear. By epitranscriptomic analysis, we report that polo-like kinase 1 (PLK1) is an important hub gene defining patient prognosis in pancreatic cancer and that RNA methylation is involved in regulating its cell cycle-specific expression. We found that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) binds to m6A of PLK1 3' untranslated region and is involved in upregulating PLK1 expression and that demethylation of this site activates the ataxia telangiectasia and Rad3-related protein pathway by replicating stress and increasing mitotic catastrophe, resulting in increased radiosensitivity. This suggests that PLK1 methylation is essential for cell cycle maintenance in pancreatic cancer and is a new therapeutic target.

Direct observation of DNA alterations induced by a DNA disruptor.

DNA alterations, such as base modifications and mutations, are closely related to the activity of transcription factors and the corresponding cell functions; therefore, detection of DNA alterations is important for understanding their relationships. Particularly, DNA alterations caused by exposure to exogenous molecules, such as nucleic acid analogues for cancer therapy and the corresponding changes in cell functions, are of interest in medicine for drug development and diagnosis purposes. However, detection of comprehensive direct evidence for the relationship of DNA modifications/mutations in genes, their effect on transcription factors, and the corresponding cell functions have been limited. In this study, we utilized a single-molecule electrical detection method for the direct observation of DNA alterations on transcription factor binding motifs upon exposure to a nucleic acid analogue, trifluridine (FTD), and evaluated the effects of the DNA alteration on transcriptional activity in cancer cell line cells. We found ~ 10% FTD incorporation at the transcription factor p53 binding regions in cancer cells exposed to FTD for 5 months. Additionally, through single-molecule analysis of p53-enriched DNA, we found that the FTD incorporation at the p53 DNA binding regions led to less binding, likely due to weaken the binding of p53. This work suggests that single-molecule detection of DNA sequence alterations is a useful methodology for understanding DNA sequence alterations.

Clinicopathological significance of MYL9 expression in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, and often involves invasion and distant metastasis from the early tumor stages. Myosin II reportedly plays a key role in regulating tumor progression and metastasis. AIMS: We examined whether myosin regulatory light polypeptide 9 (MYL9) regulates cancer cell proliferation. METHODS AND RESULTS: To investigate the expression pattern and clinical significance of MYL9 in pancreatic ductal adenocarcinoma, we performed immunohistochemical analysis of samples collected from 101 patients with pancreatic ductal adenocarcinoma. The expression of MYL9 was investigated to evaluate its functional role and contribution to proliferation and apoptosis in pancreatic ductal adenocarcinoma cells in vitro. The results showed that MYL9 was predominantly expressed in the cytoplasm and membrane of pancreatic ductal adenocarcinoma cells. Multivariate analysis indicated that MYL9 acted as an independent prognostic factor for overall survival and distant metastasis-free survival. MYL9 expression was strongly associated with malignancy in in vitro analyses, including proliferation and anti-apoptotic activities. CONCLUSIONS: Our findings suggest that MYL9 is an independent prognostic factor of pancreatic ductal adenocarcinoma. MYL9 is a crucial biomarker and potential therapeutic target for pancreatic ductal adenocarcinoma.

Computational healthcare: Present and future perspectives (Review).

Artificial intelligence (AI) has been developed through repeated new discoveries since around 1960. The use of AI is now becoming widespread within society and our daily lives. AI is also being introduced into healthcare, such as medicine and drug development; however, it is currently biased towards specific domains. The present review traces the history of the development of various AI-based applications in healthcare and compares AI-based healthcare with conventional healthcare to show the future prospects for this type of care. Knowledge of the past and present development of AI-based applications would be useful for the future utilization of novel AI approaches in healthcare.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC.

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

Scent test using Caenorhabditis elegans to screen for early-stage pancreatic cancer.

Although early detection and diagnosis are indispensable for improving the prognosis of patients with pancreatic cancer, both have yet to be achieved. Except for pancreatic cancer, other cancers have already been screened through scent tests using animals or microorganisms, including Caenorhabditis elegans. While such a method may greatly improve the prognosis of pancreatic cancer, no studies have investigated the same, mainly given the difficulty of collecting suitable samples from patients with early-stage pancreatic cancer. In this study, we organized a nationwide study group comprising high-volume centers throughout Japan to collect patients with very-early-stage pancreatic cancer (stage 0 or IA). We initially performed an open-label study involving 83 cases (stage 0-IV), with subsequent results showing significant differences after surgical removal in stage 0-IA (×10 dilution: p < 0.001; ×100 dilution: p < 0.001). Thereafter, a blinded study on 28 cases (11 patients with stage 0 or IA disease and 17 healthy volunteers) was conducted by comparing very-early-stage pancreatic cancer patients with healthy volunteers to determine whether C. elegans could detect the scent of cancer for the diagnosis of early-stage pancreatic cancer. Preoperative urine samples had a significantly higher chemotaxis index compared to postoperative samples in patients with pancreatic cancer [×10 dilution: p < 0.001, area under the receiver operating characteristic curve (AUC) = 0.845; ×100 dilution: p < 0.001, AUC = 0.820] and healthy volunteers (×10 dilution: p = 0.034; ×100 dilution: p = 0.088). Moreover, using the changes in preoperative and postoperative chemotaxis index, this method had a higher sensitivity for detecting early pancreatic cancer compared to existing diagnostic markers. The clinical application C. elegans for the early diagnosis of cancer can certainly be expected in the near future.

A Four-Dimensional Organoid System to Visualize Cancer Cell Vascular Invasion.

Vascular invasion of cancer is a critical step in cancer progression, but no drug has been developed to inhibit vascular invasion. To achieve the eradication of cancer metastasis, elucidation of the mechanism for vascular invasion and the development of innovative treatment methods are required. Here, a simple and reproducible vascular invasion model is established using a vascular organoid culture in a fibrin gel with collagen microfibers. Using this model, it was possible to observe and evaluate the cell dynamics and histological positional relationship of invasive cancer cells in four dimensions. Cancer-derived exosomes promoted the vascular invasion of cancer cells and loosened tight junctions in the vascular endothelium. As a new evaluation method, research using this vascular invasion mimic model will be advanced, and applications to the evaluation of the vascular invasion suppression effect of a drug are expected.

Immuno-Surgical Management of Pancreatic Cancer with Analysis of Cancer Exosomes.

Exosomes (EXs), a type of extracellular vesicles secreted from various cells and especially cancer cells, mesenchymal cells, macrophages and other cells in the tumor microenvironment (TME), are involved in biologically malignant behaviors of cancers. Recent studies have revealed that EXs contain microRNAs on their inside and express proteins and glycolipids on their outsides, every component of which plays a role in the transmission of genetic and/or epigenetic information in cell-to-cell communications. It is also known that miRNAs are involved in the signal transduction. Thus, EXs may be useful for monitoring the TME of tumor tissues and the invasion and metastasis, processes that are associated with patient survival. Because several solid tumors secrete immune checkpoint proteins, including programmed cell death-ligand 1, the EX-mediated mechanisms are suggested to be potent targets for monitoring patients. Therefore, a companion therapeutic approach against cancer metastasis to distant organs is proposed when surgical removal of the primary tumor is performed. However, EXs and immune checkpoint mechanisms in pancreatic cancer are not fully understood, we provide an update on the recent advances in this field and evidence that EXs will be useful for maximizing patient benefit in precision medicine.

Hereditary pancreatitis model by blastocyst complementation in mouse.

The application of pluripotent stem cells is expected to contribute to the elucidation of unknown mechanism of human diseases. However, in vitro induction of organ-specific cells, such as pancreas and liver, is still difficult and the reproduction of their disorders in a model has been unfeasible. To study the mechanism of human hereditary pancreatitis (HP), we here performed the blastocyst complementation (BC) method. In the BC method, mouse embryonic stem (ES) cells harboring CRISPR/CAS9-mediated mutations in the Prss1 gene were injected into blastocysts with deficient Pdx1 gene, which is a critical transcription factor in the development of pancreas. The results showed that trypsin was activated extremely in Prss1-mutant mice. This implied that the mouse phenotype mimics that of human HP and that the BC method was useful for the reproduction and study of pancreatic disorders. The present study opens the possibility of investigating uncharacterized human diseases by utilizing the BC method.

State-of-the-Art Technology of Model Organisms for Current Human Medicine.

Since the 1980s, molecular biology has been used to investigate medical field mechanisms that still require the use of crude biological materials in order to achieve their necessary goals. Transcription factor-induced pluripotent stem cells are used in regenerative medicine to screen drugs and to support lost tissues. However, these cells insufficiently reconstruct whole organs and require various intact cells, such as damaged livers and diabetic pancreases. For efficient gene transfer in medical use, virally mediated gene transfers are used, although immunogenic issues are investigated. To obtain efficient detective and diagnostic power in intractable diseases, biological tools such as roundworms and zebrafish have been found to be useful for high-throughput screening (HST) and diagnosis. Taken together, this biological approach will help to fill the gaps between medical needs and novel innovations in the field of medicine.

Convolutional Neural Network Can Recognize Drug Resistance of Single Cancer Cells.

It is known that single or isolated tumor cells enter cancer patients' circulatory systems. These circulating tumor cells (CTCs) are thought to be an effective tool for diagnosing cancer malignancy. However, handling CTC samples and evaluating CTC sequence analysis results are challenging. Recently, the convolutional neural network (CNN) model, a type of deep learning model, has been increasingly adopted for medical image analyses. However, it is controversial whether cell characteristics can be identified at the single-cell level by using machine learning methods. This study intends to verify whether an AI system could classify the sensitivity of anticancer drugs, based on cell morphology during culture. We constructed a CNN based on the VGG16 model that could predict the efficiency of antitumor drugs at the single-cell level. The machine learning revealed that our model could identify the effects of antitumor drugs with ~0.80 accuracies. Our results show that, in the future, realizing precision medicine to identify effective antitumor drugs for individual patients may be possible by extracting CTCs from blood and performing classification by using an AI system.

COVID-19 Drug Discovery Using Intensive Approaches.

Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

Theoretical analyses and experimental validation of the effects caused by the fluorinated substituent modification of DNA.

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH-[Formula: see text], lone pair (LP)-[Formula: see text], and [Formula: see text]-[Formula: see text] interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone.

One-carbon metabolism for cancer diagnostic and therapeutic approaches.

Altered metabolism is critical for the rapid and unregulated proliferation of cancer cells; hence the requirement for an abundant source of nucleotides. One characteristic of this metabolic reprogramming is in one-carbon (1C) metabolism, which is particularly noteworthy for its role in DNA synthesis. Various forms of methylation are also noteworthy as they relate to cancer cell survival and proliferation. In recent years, 1C metabolism has received substantial attention for its role in cancer malignancy via these functions. Therefore, therapeutic inhibitors targeting 1C metabolism have been utilized as anticancer drugs. This review outlines the importance of 1C metabolism and its clinical application in cancer. Understanding 1C metabolism could aid the development of novel cancer diagnostic and therapeutic methods.

CD44/CD133-Positive Colorectal Cancer Stem Cells are Sensitive to Trifluridine Exposure.

Cancer stem cells (CSCs) are involved in metastatic colorectal cancer recurrence, but no effective therapy targeting these cells is currently available. Because trifluridine (FTD)/tipiracil therapy is used for refractory colorectal cancer, we sought to determine whether FTD is effective against CSC-like cells. CD44+CD133+ high-expressing and other populations of human DLD-1 colon cancer cells were separately isolated through fluorescence-activated cell sorting. The sphere-forming activity of each population and the anti-sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells were then measured. CD44+CD133+ DLD-1 cells formed substantially more spheres than other cells. Moreover, treating CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) inhibited sphere formation, and this was superior to the effect of subtoxic concentrations (1 µM) of 5-FU. The associated inhibition rates for FTD and 5-FU were 58.2% and 26.1%, respectively. Further, CD44+CD133+ DLD-1 cells expressed higher levels of thymidine kinase 1, which is responsible for FTD phosphorylation, than DLD-1 cells, and FTD was incorporated into the DNA of CD44+CD133+ DLD-1 cells. Thus, our data show that FTD treatment is effective against CSC-like cells and might be applied as CSC-targeting chemotherapy for tumor subtypes with high CD44 and CD133 expression.

Application of C. elegans cancer screening test for the detection of pancreatic tumor in genetically engineered mice.

Pancreatic ductal adenocarcinoma (PDAC) exhibits a very early onset of metastasis. Thus, early detection and treatment are pivotal to successful eradication of pancreatic cancers. Economical and non-invasive cancer screening systems is indispensable for this purpose. Previously our group developed a novel method to detect various kinds of human cancer using nematode Caenorhabditis elegans (C. elegans) that respond to cancer odor in urine; however, whether this method is useful for non-human species remains to be understood. In this study, we examined its effectiveness in the detection of murine pancreatic tumor spontaneously generated in genetically-engineered mice. We generated pancreas-specific Kras G12D and/or c-Met deletion mutant mice and measured the probability of spontaneous tumor generation in these mice. The chemotactic indexes of C. elegans to the urine samples of these mutant mice were measured. As previously described, oncogenic KrasG12D was necessary to induce pancreatic intraepithelial neoplasia in this mouse model, while c-Met mutation did not show further effect. The chemotactic analysis indicated that C. elegans avoids urine of healthy recipient mice, while they tended to be attracted to urine of mice with KrasG12D . Our study demonstrated that C. elegans can recognize the odor of pancreatic cancer in urine of KrasG12D model mouse, suggesting the similarity of cancer odor between species. Our result facilitates further studies on mechanism of cancer detection by C. elegans.