Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix.

Small-cell neuroendocrine carcinoma (SCNEC) of the cervix is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. This study established a panel of organoid lines from patients with SCNEC of the cervix and ultimately focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. Tracking single cells indicated the existence of cells with bipotential differentiation toward SCNEC and adenocarcinomas. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for keratin 19 expression, changes in the differentiation of each cell were shown to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

The hepatic niche leads to aggressive natural killer cell leukemia proliferation through the transferrin-transferrin receptor 1 axis.

Aggressive natural killer cell leukemia (ANKL) is a rare lymphoid neoplasm frequently associated with Epstein-Barr virus, with a disastrously poor prognosis. Owing to the lack of samples from patients with ANKL and relevant murine models, comprehensive investigation of its pathogenesis including the tumor microenvironment (TME) has been hindered. Here we established 3 xenograft mice derived from patients with ANKL (PDXs), which enabled extensive analysis of tumor cells and their TME. ANKL cells primarily engrafted and proliferated in the hepatic sinusoid. Hepatic ANKL cells were characterized by an enriched Myc-pathway and proliferated faster than those in other organs. Interactome analyses and in vivo CRISPR-Cas9 analyses revealed transferrin (Tf)-transferrin receptor 1 (TfR1) axis as a potential molecular interaction between the liver and ANKL. ANKL cells were rather vulnerable to iron deprivation. PPMX-T003, a humanized anti-TfR1 monoclonal antibody, showed remarkable therapeutic efficacy in a preclinical setting using ANKL-PDXs. These findings indicate that the liver, a noncanonical hematopoietic organ in adults, serves as a principal niche for ANKL and the inhibition of the Tf-TfR1 axis is a promising therapeutic strategy for ANKL.

Inhibition of transforming growth factor-ß signals suppresses tumor formation by regulation of tumor microenvironment networks.

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-ß (TGF-ß) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-ß receptor containing both TGF-ß type I (TßRI) and type II (TßRII) receptors (TßRI-TßRII-Fc), which trapped all TGF-ß isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TßRI-TßRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TßRI-TßRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TßRI-TßRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1ß (IL-1ß) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1ß and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-ß signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1ß/EREG pathways and that TßRI-TßRII-Fc protein is a promising tool for targeting the TME networks.

Fusion with type 2 macrophages induces melanoma cell heterogeneity that potentiates immunological escape from cytotoxic T lymphocytes.

Evasion from immunity is a major obstacle to the achievement of successful cancer immunotherapy. Hybrids derived from cell-cell fusion are theoretically associated with tumor heterogeneity and progression by conferring novel properties on tumor cells, including drug resistance and metastatic capacity; however, their impact on immune evasion remains unknown. Here, we investigated the potency of tumor-macrophage hybrids in immune evasion. Hybrids were established by co-culture of a melanoma cell line (A375 cells) and type 2 macrophages. The hybrids showed greater migration ability and greater tumorigenicity than the parental melanoma cells. The hybrids showed heterogeneous sensitivity to New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T-cell receptor-transduced T (TCR-T) cells and two out of four hybrid clones showed less sensitivity to TCR-T compared with the parental cells. An in vitro tumor heterogeneity model revealed that the TCR-T cells preferentially killed the parental cells compared with the hybrids and the survival rate of the hybrids was higher than that of the parental cells, indicating that the hybrids evade killing by TCR-T cells efficiently. Analysis of a single-cell RNA sequencing dataset of patients with melanoma revealed that a few macrophages expressed RNA encoding melanoma differentiation antigens including melan A, tyrosinase, and premelanosome protein, which indicated the presence of hybrids in primary melanoma. In addition, the number of potential hybrids was correlated with a poorer response to immune checkpoint blockade. These results provide evidence that melanoma-macrophage fusion has a role in tumor heterogeneity and immune evasion. © 2023 The Pathological Society of Great Britain and Ireland.

Eribulin is an immune potentiator in breast cancer that upregulates human leukocyte antigen class I expression via the induction of NOD-like receptor family CARD domain-containing 5.

Eribulin inhibits microtubule polymerization and improves the overall survival of patients with recurrent metastatic breast cancer. A subgroup analysis revealed a low neutrophil to lymphocyte ratio (NLR) (<3) to be a prognostic factor of eribulin treatment. We thus hypothesized that eribulin might be related to the immune response for breast cancer cells and we analyzed the effects of eribulin on the immune system. Immunohistochemical staining revealed that human leukocyte antigen (HLA) class I expression was increased in clinical samples after eribulin treatment. In vitro assays revealed that eribulin treatment increased HLA class I expression in breast cancer line cells. RNA-sequencing demonstrated that eribulin treatment increased the expression of the NOD-like family CARD domain-containing 5 (NLRC5), a master regulator of HLA class I expression. Eribulin treatment increased the NY-ESO-1-specific T-cell receptor (TCR) transduced T (TCR-T) cell response for New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) overexpressed breast cancer cells. The eribulin and TCR-T combined therapy model revealed that eribulin and immunotherapy using TCR-T cells has a synergistic effect. In summary, eribulin increases the expression of HLA class 1 via HLA class 1 transactivatior NLRC5 and eribulin combination with immunotherapy can be effective for the treatment of breast cancer.

Cisplatin resistance driver claspin is a target for immunotherapy in urothelial carcinoma.

Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

Lactoferrin and its digestive peptides induce interferon-α production and activate plasmacytoid dendritic cells ex vivo.

Plasmacytoid dendritic cells (pDCs) recognise viral single-stranded RNA (ssRNA) or CpG DNA via Toll-like receptor (TLR)-7 and TLR9, and produce interferon (IFN)-α. Activated pDCs upregulate human leukocyte antigen (HLA)-DR and CD86 expression levels. Ingestion of bovine lactoferrin (LF) activates pDCs, but little is known about its effects. In this study, the effects of LF and its pepsin hydrolysate (LFH) on the production of IFN-α from peripheral blood mononuclear cells (PBMCs) and pDCs were examined. PBMCs were prepared from peripheral blood of healthy adults and incubated with LF, LFH, or lactoferricin (LFcin) in the absence or presence of ssRNA derived from human immunodeficiency virus. The concentration of IFN-α in the supernatant and the expression levels of IFN-α, HLA-DR, and CD86 in pDCs were quantified by enzyme-linked immunosorbent assay and flow cytometry. In the absence of ssRNA, the concentration of IFN-α was negligible and LF had no effect on it. In the presence of ssRNA, IFN-α was detected at a certain level, and LF and LFH significantly increased its concentration. The increase caused by LFH and LFcin were comparable. In addition, LF significantly upregulated the expression levels of IFN-α, HLA-DR, and CD86 in pDCs. LF and its digestive peptides induced IFN-α production and activated pDCs in the presence of ssRNA, suggesting that LF modulates the immune system by promoting pDC activation upon viral recognition.

Allosteric Inhibition of c-Abl to Induce Unfolded Protein Response and Cell Death in Multiple Myeloma.

Endoplasmic reticulum stress activates inositol-requiring enzyme 1α (IRE1α) and protein kinase, R-like endoplasmic reticulum kinase (PERK), the two principal regulators of the unfolded protein response (UPR). In multiple myeloma, adaptive IRE1α signaling is predominantly activated and regulates cell fate along with PERK. Recently, we demonstrated that GNF-2, an allosteric c-Abl inhibitor, rheostatically enhanced IRE1α activity and induced apoptosis through c-Abl conformational changes in pancreatic ß cells. Herein, we analyzed whether the pharmacological modulation of c-Abl conformation resulted in anti-myeloma effects. First, we investigated the effects of GNF-2 on IRE1α activity and cell fate, followed by an investigation of the anti-myeloma effects of asciminib, a new allosteric c-Abl inhibitor. Finally, we performed RNA sequencing to characterize the signaling profiles of asciminib. We observed that both GNF-2 and asciminib decreased cell viability and induced XBP1 mRNA splicing in primary human myeloma cells and myeloma cell lines. RNA sequencing identified the induction of UPR- and apoptosis-related genes by asciminib. Asciminib re-localized c-Abl to the endoplasmic reticulum, and its combination with a specific IRE1α inhibitor, KIRA8, enhanced cell death with the reciprocal induction of CHOP mRNA expression. Together, the allosteric inhibition of c-Abl-activated UPR with anti-myeloma effects; this could be a novel therapeutic target for multiple myeloma.

Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation.

From a mouse triple-negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G-protein-coupled receptor (ADGR) family, G-protein-coupled receptor 56 (GPR56)/adhesion G-protein-coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1-transduced 4T1, TS/A (mouse breast cancer cell line), or MDA-MB-231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1-induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

Immunomodulatory activity of heat-killed Lacticaseibacillus paracaseiMCC1849 based on the activation of plasmacytoid dendritic cells in the peripheral blood of healthy adults.

Probiotics are widely used in food for their health benefits to the host. Inactivated probiotics also reportedly improve the intestinal environment and immune regulation. Our previous studies showed that heat-killed Lacticaseibacillus paracasei MCC1849 (hk-MCC1849) effectively induced IL-12 production in mouse spleen cells and significantly reduced cold symptoms in clinical trial subjects. To further elucidate the mechanism of host immune regulation by hk-MCC1849, human peripheral blood mononuclear cells (PBMCs) were cocultured with hk-MCC1849. The Toll-like receptor 9 ligands CpG-ODN 2216 and hk-MCC1849 and the heat-killed Lacticaseibacillus rhamnosus ATCC53103 were used as positive and negative controls, respectively. The results showed that, compared with the control, hk-MCC1849 significantly increased the expression of the plasmacytoid dendritic cell (pDC) marker CD86 (p < .0001) and the pDC marker HLA-DR (p < .001) in PBMCs. The expression levels of the IL-12p40, IFNα, IFNα1, IFNγ, and ISG15 genes were significantly increased after coculture with hk-MCC1849 (p < .05, p < .05, p < .05, p < .05, and p < .05, respectively, vs. control). Furthermore, to confirm whether hk-MCC1849 directly interacted with pDCs, DCs were enriched with PBMCs following 24 h of coculture with hk-MCC1849. Phagocytosis of fluorescently labeled hk-MCC1849 by pDCs was observed, and there were significant increases in CD86 (p < .05) and HLA-DR (p < .0001) expression in pDCs. These results suggest that hk-MCC1849 exerts a potential immunomodulatory effect on the host through the activation of peripheral pDCs.

Familial schwannomatosis carrying LZTR1 variant p.R340X with brain tumor: A case report.

Schwannomatosis (SWN) is a rare genetic condition characterized by the risk of developing multiple benign peripheral nerve sheath tumors; however, the risk of developing malignant tumors in patients with SWN remains unclear. This study described the case of a 57-year-old Japanese man diagnosed with SWN whose older brother also had SWN. Whole-exome sequencing identified a heterozygous mutation [c.1018C > T (p.Arg340X)] in the LZTR1 gene, linked to the RAS/MAPK pathway, in the patient and his brother. Moreover, the patient had aphasia and right-sided paralysis because of a brain tumor. RNA sequencing revealed the remarkable upregulation of several genes associated with oxidative stress, such as the reactive oxygen species pathway and oxidative phosphorylation, a downstream effector of the RAS/MAPK pathway, in the the patient and his brother compared with healthy volunteers. The final diagnosis was LZTR1-related familial SWN, and the dysregulated RAS/MAPK pathway in this patient might be associated with brain tumorigenesis.

Astrocyte-induced mGluR1 activates human lung cancer brain metastasis via glutamate-dependent stabilization of EGFR.

There are limited methods to stably analyze the interactions between cancer cells and glial cells in vitro, which hinders our molecular understanding. Here, we develop a simple and stable culture method of mouse glial cells, termed mixed-glial culture on/in soft substrate (MGS), which serves well as a platform to study cancer-glia interactions. Using this method, we find that human lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signaling in the brain microenvironment. Mechanistically, interactions with astrocytes induce mGluR1 in cancer cells through the Wnt-5a/prickle planar cell polarity protein 1 (PRICKLE1)/RE1 silencing transcription factor (REST) axis. Induced mGluR1 directly interacts with and stabilizes the epidermal growth factor receptor (EGFR) in a glutamate-dependent manner, and these cells then become responsive to mGluR1 inhibition. Our results highlight increased dependence on mGluR1 signaling as an adaptive strategy and vulnerability of human lung cancer brain metastasis.

Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis.

BACKGROUND: Acute liver failure (ALF) is a life-threatening disorder that progresses from self-limiting acute liver injury (ALI). Microcirculatory disturbance characterized by sinusoidal hypercoagulation and subsequent massive hypoxic hepatocyte damage have been proposed to be the mechanism by which ALI deteriorates to ALF; however, the precise molecular pathway of the sinusoidal hypercoagulation remains unknown. Here, we analyzed ALI patients and mice models to uncover the pathogenesis of ALI with microcirculatory disturbance. METHODS: We conducted a single-center retrospective study for ALI and blood samples and liver tissues were analyzed to evaluate the microcirculatory disturbance in ALI patients (n = 120). Single-cell RNA sequencing analysis (scRNA-seq) was applied to the liver from the concanavalin A (Con A)­induced mouse model of ALI. Interferon-gamma (IFNγ) and tumor necrosis factor-alpha knockout mice, and primary human liver sinusoidal endothelial cells (LSECs) were used to assess the mechanism of microcirculatory disturbance. RESULTS: The serum IFNγ concentrations were significantly higher in ALI patients with microcirculatory disturbance than in patients without microcirculatory disturbance, and the IFNγ was upregulated in the Con A mouse model which presented microcirculatory disturbance. Hepatic IFNγ expression was increased as early as 1 hour after Con A treatment prior to sinusoidal hypercoagulation and hypoxic liver damage. scRNA-seq revealed that IFNγ was upregulated in innate lymphoid cells and stimulated hepatic vascular endothelial cells at the early stage of liver injury. In IFNγ knockout mice treated with Con A, the sinusoidal hypercoagulation and liver damage were remarkably attenuated, concomitant with the complete inhibition of CD40 and tissue factor (TF) upregulation in vascular endothelial cells. By ligand-receptor analysis, CD40-CD40 ligand interaction was identified in vascular endothelial cells. In human LSECs, IFNγ upregulated CD40 expression and TF was further induced by increased CD40-CD40 ligand interaction. Consistent with these findings, hepatic CD40 expression was significantly elevated in human ALI patients with microcirculatory disturbance. CONCLUSION: We identified the critical role of the IFNγ-CD40 axis as the molecular mechanism of microcirculatory disturbance in ALI. This finding may provide novel insights into the pathogenesis of ALI and potentially contribute to the emergence of new therapeutic strategies for ALI patients.

Single-cell RNA sequencing to detect age-associated genes that identify senescent cells in the liver of aged mice.

Senescent cells are predicted to occur and increase in animal tissues with aging. However, senescent cells in the tissues of aged animals remain to be identified. We refer to the marker genes to identify senescent cells in tissues as "age-associated genes". In this study, we searched for age-associated genes to identify senescent cells in the livers of aged animals. We performed single-cell RNA sequencing (scRNA-seq) to screen candidates for age-associated genes using young and aged rat primary hepatocytes. To remove animal species specificity, gene expression analyses in mouse livers were performed, confirming age-associated increases in the mRNA expression levels of Glipr1, Clec12a, and Phlda3. Moreover, the mRNA expression levels of Glipr1 and Phlda3 were increased by stress-induced premature senescence using doxorubicin in primary hepatocytes and livers of young mice. Transcriptome data of aged rat hepatocytes suggested that Glipr1, Clec12a, and Phlda3 were expressed in almost identical cells. Fluorescence in situ hybridization (FISH) confirmed the presence of cells with abundant Glipr1, Clec12a, and Phlda3 mRNA in 27-month-old mouse primary hepatocytes, which are considered to be senescent cells. This study is the first to identify Glipr1, Clec12a, and Phlda3 as age-associated genes in the mouse liver.

Application of spatial transcriptomics analysis using the Visium system for the mouse nasal cavity after intranasal vaccination.

Intranasal vaccines that elicit mucosal immunity are deemed effective against respiratory tract infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but their ability to induce humoral immunity characterized by immunoglobulin A (IgA) and IgG production is low. It has been reported that vaccination with a mixture of a viscous base carboxyvinyl polymer (CVP) and viral antigens induced robust systemic and mucosal immune responses. In this study, we analyzed the behavior of immunocompetent cells in the nasal cavity over time by spatial transcriptome profiling induced immediately after antigen vaccination using CVP. We established a method for performing spatial transcriptomics using the Visium system in the mouse nasal cavity and analyzed gene expression profiles within the nasal cavity after intranasal vaccination. Glycoprotein 2 (Gp2)-, SRY-box transcription factor 8 (Sox8)-, or Spi-B transcription factor (Spib)-expressing cells were increased in the nasal passage (NP) region at 3-6 hr after SARS-CoV-2 spike protein and CVP (S-CVP) vaccination. The results suggested that microfold (M) cells are activated within a short period of time (3-6 hr). Subsequent cluster analysis of cells in the nasal cavity showed an increase in Cluster 9 at 3-6 hr after intranasal vaccination with the S-CVP. We found that Il6 in Cluster 9 had the highest log2 fold values within the NP at 3-6 hr. A search for gene expression patterns similar to that of Il6 revealed that the log2 fold values of Edn2, Ccl20, and Hk2 also increased in the nasal cavity after 3-6 hr. The results showed that the early response of immune cells occurred immediately after intranasal vaccination. In this study, we identified changes in gene expression that contribute to the activation of M cells and immunocompetent cells after intranasal vaccination of mice with antigen-CVP using a time-series analysis of spatial transcriptomics data. The results facilitated the identification of the cell types that are activated during the initial induction of nasal mucosal immunity.

The Potential Immunomodulatory Effect of Bifidobacterium longum subsp. longum BB536 on Healthy Adults through Plasmacytoid Dendritic Cell Activation in the Peripheral Blood.

The interaction between the gut microbiota and the host can influence the host's immune system. Bifidobacterium, a commensal genus of gut bacteria, seems to have positive effects on host health. Our previous clinical research showed that B. longum subsp. longum BB536 enhanced innate and adaptive immune responses in elderly individuals with a lower grade of immunity, but the immunomodulatory mechanism is still unclear. In this study, dendritic cell (DC) surface markers in peripheral blood mononuclear cells isolated from healthy individuals were evaluated through coculture with heat-killed BB536. DC markers, innate immune activity and cytokine levels in plasma were also evaluated by a randomized, double-blind, placebo-controlled, parallel-group study (UMIN000045564) with 4 weeks of continuous live BB536 intake. BB536 significantly increased the expression of CD86 and HLA-DR on plasmacytoid DCs (pDCs) in vitro. Compared to placebo (n = 48), a significant increase in the expression of CD86 on peripheral pDCs was detected at week 4 of live BB536 intake (n = 49; 1 × 1010 CFU/day). Furthermore, coculture with hk-BB536 significantly increased the IFNγ expression level and demonstrated trends of increased IFNα1 and IFNß expression. These findings suggest that consumption of BB536 has potential immunomodulatory effects on healthy individuals through the activation of peripheral pDCs.

Phospholipase A2 Group IIA Is Associated with Inflammatory Hepatocellular Adenoma.

Although benign hepatocellular adenomas (HCA) are very rare, recent observations have shown their occurrence in patients with diabetes mellitus. Consequently, most of these cases are treated by resection due to concerns regarding their potential progression to hepatocarcinoma (HCC). This decision is largely driven by the limited number of studies on HCC subtyping and the lack of molecular and biological insights into the carcinogenic potential of benign tumors. This study aimed to comprehensively investigate the subtype classification of HCA and to compare and analyze gene expression profiling between HCA and HCC tissues. One fresh inflammatory HCA (I-HCA), three non-B non-C HCCs, two hepatitis B virus-HCCs, and one normal liver tissue sample were subjected to single-cell RNA sequencing (scRNA-seq). Comparative analysis of scRNA-seq among different tissues showed that phospholipase A2 group IIA (PLA2G2A) mRNA was specifically expressed in I-HCA, following RNA-seq analysis in formalin-fixed paraffin-embedded tissues from other HCAs. Immunohistochemistry using the PLA2G2A antibody in these tissues indicated that the positive reaction was mainly observed in hepatocytes of I-HCAs and stromal cells surrounding the tumor tissue in HCC were also stained. According to a clinical database, PLA2G2A expression in HCC does not correlate with poor prognosis. This finding may potentially help develop a new definition for I-HCA, resulting in a significant clinical contribution, but it requires validation with other fresh HCA samples.

SOX10 Inhibits T Cell Recognition by Inducing Expression of the Immune Checkpoint Molecule PD-L1 in A375 Melanoma Cells.

BACKGROUND/AIM: Malignant melanoma is a fatal skin cancer and is among the most immunogenic malignancies expressing melanoma-differentiation antigens and neoantigens. SRY-related HMG-box 10 (SOX10) is a transcription factor and a neural-crest differentiation marker that is used as a diagnostic marker for melanoma whilst playing a role in melanoma initiation through activation of the SOX10-MITF axis. SOX10 was shown to play a role in melanoma initiation by inducing expression of immune checkpoint molecules (e.g., HVEM and CEACAM1). In this study, we aimed to investigate the relationship between SOX10 and the expression an immune checkpoint molecule, programmed death-1 ligand 1 (PD-L1). MATERIALS AND METHODS: SOX10 overexpression and knockdown was performed using SOX10 gene transfection and SOX10 siRNA transfection into A375 melanoma cells. PD-L1 expression was assessed by flow cytometry and western blotting. T cell response was evaluated using NY-ESO-1 specific TCR-transduced T (TCR-T) cells by IFNγ ELISPOT assay. RESULTS: SOX10 overexpression increased the expression of PD-L1, whereas SOX10 knockdown, using siRNA, decreased its expression. IFNγ ELISPOT assay revealed that overexpression of SOX10 decreased the susceptibility of cells to NY-ESO-1-specific TCR-T cells. CONCLUSION: SOX10 has a role in the intrinsic immune suppressive mechanisms of melanoma through expression of PD-L1.

Molecular insights of a CBP/ß-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder.

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/ß-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724. Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly. PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/ß-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.

B cell receptor repertoire analysis from autopsy samples of COVID-19 patients.

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.