PRMT6 methylation of RCC1 regulates mitosis, tumorigenicity, and radiation response of glioblastoma stem cells.

Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.

Reprogramming and multi-lineage transdifferentiation attenuate the tumorigenicity of colorectal cancer cells.

Significant advances have been made in reprogramming various somatic cells into induced pluripotent stem cells (iPSCs) and in multi-lineage differentiation (transdifferentiation) into different tissues. These manipulable transdifferentiating techniques may be applied in cancer therapy. Limited works have been reported that cancer cell malignancy can be switched to benign phenotypes through reprogramming techniques. Here, we reported that two colorectal cancer (CRC) cell lines (DLD1, HT29) could be reprogrammed into iPSCs (D-iPSCs, H-iPSCs). D- and H-iPSCs showed reduced tumorigenesis. Furthermore, we successfully induced D- and H-iPSCs differentiation into terminally differentiated cell types such as cardiomyocyte, neuron, and adipocyte-like cells. Impressively, the differentiated cells exhibited further attenuated tumorigenesis in vitro and in vivo. RNA-Seq further indicated that epigenetic changes occurred after reprogramming and transdifferentiation that caused reduced tumorigenicity. Overall, our study indicated that CRC cells can be reprogrammed and further differentiated into terminally differentiated lineages with attenuation of their malignancy in vitro and in vivo. The current work sheds light on a potential multi-lineage differentiation therapeutic strategy for colorectal cancer.

Glioblastoma stem cell metabolism and immunity.

Despite enormous efforts being invested in the development of novel therapies for brain malignancies, there remains a dire need for effective treatments, particularly for pediatric glioblastomas. Their poor prognosis has been attributed to the fact that conventional therapies target tumoral cells, but not glioblastoma stem cells (GSCs). GSCs are characterized by self-renewal, tumorigenicity, poor differentiation, and resistance to therapy. These characteristics represent the fundamental tools needed to recapitulate the tumor and result in a relapse. The mechanisms by which GSCs alter metabolic cues and escape elimination by immune cells are discussed in this article, along with potential strategies to harness effector immune cells against GSCs. As cellular immunotherapy is making significant advances in a variety of cancers, leveraging this underexplored reservoir may result in significant improvements in the treatment options for brain malignancies.

RBM10 regulates the tumorigenic potential of human cancer cells by modulating PPM1B and YBX1 activities.

RNA binding protein RBM10 participates in various RNA metabolism, and its decreased expression or loss of function by mutation has been identified in many human cancers. However, how its dysregulation contributes to human cancer pathogenesis remains to be determined. Here, we found that RBM10 expression was decreased in breast tumors, and breast cancer patients with low RBM10 expression presented poorer survival rates. RBM10 depletion in breast cancer cells significantly promotes the cellular proliferation and migration. We further demonstrated that RBM10 forms a triple complex with YBX1 and phosphatase 1B (PPM1B), in which PPM1B serves as the phosphatase of YBX1. RBM10 knock-down markedly attenuated association between YBX1 and PPM1B, leading to elevated levels of YBX1 phosphorylation and its nuclear translocation. Furthermore, cancer cells with RBM10 depletion had a significantly accelerated tumor growth in nude mice. Importantly, these enhanced tumorigenic phenotypes can be reversed by overexpression of PPM1B. Our findings provide the mechanistic bases for functional loss of RBM10 in promoting tumorigenicity, and are potentially useful in the development of combined therapeutic strategies for cancer patients with defective RBM10.

A Constitutive Intrinsic Inflammatory Signaling Circuit Composed of miR-196b, Meis2, PPP3CC, and p65 Drives Prostate Cancer Castration Resistance.

Androgen deprivation therapy is the most effective treatment for advanced prostate cancer, but almost all cancer eventually becomes castration resistant, and the underlying mechanisms are largely unknown. Here, we show that an intrinsic constitutively activated feedforward signaling circuit composed of IκBα/NF-κB(p65), miR-196b-3p, Meis2, and PPP3CC is formed during the emergence of castration-resistant prostate cancer (CRPC). This circuit controls the expression of stem cell transcription factors that drives the high tumorigenicity of CRPC cells. Interrupting the circuit by targeting its individual components significantly impairs the tumorigenicity and CRPC development. Notably, constitutive activation of IκBα/NF-κB(p65) in this circuit is not dependent on the activation of traditional IKKß/NF-κB pathways that are important in normal immune responses. Therefore, our studies present deep insight into the bona fide mechanisms underlying castration resistance and provide the foundation for the development of CRPC therapeutic strategies that would be highly efficient while avoiding indiscriminate IKK/NF-κB inhibition in normal cells.

Linc-NSC affects cell differentiation, apoptosis and proliferation in mouse neural stem cells and embryonic stem cells in vitro and in vivo.

BACKGROUND: Stem cell therapy is a promising therapeutic strategy. In a previous study, we evaluated tumorigenicity by the stereotactic transplantation of neural stem cells (NSCs) and embryonic stem cells (ESCs) from experimental mice. Twenty-eight days later, there was no evidence of tumor formation or long-term engraftment in the NSCs transplantation group. In contrast, the transplantation of ESCs caused tumor formation; this was due to their high proliferative capacity. Based on transcriptome sequencing, we found that a long intergenic non-coding RNA (named linc-NSC) with unknown structure and function was expressed at 1100-fold higher levels in NSCs than in ESCs. This finding suggested that linc-NSC is negatively correlated with stem cell pluripotency and tumor development, but positively correlated with neurogenesis. In the present study, we investigated the specific role of linc-NSC in NSCs/ESCs in tumor formation and neurogenesis. METHODS: Whole transcriptome profiling by RNA sequencing and bioinformatics was used to predict lncRNAs that are widely associated with enhanced tumorigenicity. The expression of linc-NSC was assessed by quantitative real-time PCR. We also performed a number of in vitro methods, including cell proliferation assays, differentiation assays, immunofluorescence assays, flow cytometry, along with in vivo survival and immunofluorescence assays to investigate the impacts of linc-NSC on tumor formation and neurogenesis in NSCs and ESCs. RESULTS: Following the knockdown of linc-NSC in NSCs, NSCs cultured in vitro and those transplanted into the cortex of mice showed stronger survival ability (P < 0.0001), enhanced proliferation(P < 0.001), and reduced apoptosis (P < 0.05); the opposite results were observed when linc-NSC was overexpressed in ESCs. Furthermore, the overexpression of linc-NSC in ECSs induced enhanced apoptosis (P < 0.001) and differentiation (P < 0.01), inhibited tumorigenesis (P < 0.05) in vivo, and led to a reduction in tumor weight (P < 0.0001). CONCLUSIONS: Our analyses demonstrated that linc-NSC, a promising gene-edited target, may promote the differentiation of mouse NSCs and inhibit tumorigenesis in mouse ESCs. The knockdown of linc-NSC inhibited the apoptosis in NSCs both in vitro and in vivo, and prevented tumor formation, revealing a new dimension into the effect of lncRNA on low survival NSCs and providing a prospective gene manipulation target prior to transplantation. In parallel, the overexpression of linc-NSC induced apoptosis in ESCs both in vitro and in vivo and attenuated the tumorigenicity of ESCs in vivo, but did not completely prevent tumor formation.

A PRC2-Kdm5b axis sustains tumorigenicity of acute myeloid leukemia.

Acute myeloid leukemias (AMLs) with the NUP98-NSD1 or mixed lineage leukemia (MLL) rearrangement (MLL-r) share transcriptomic profiles associated with stemness-related gene signatures and display poor prognosis. The molecular underpinnings of AML aggressiveness and stemness remain far from clear. Studies with EZH2 enzymatic inhibitors show that polycomb repressive complex 2 (PRC2) is crucial for tumorigenicity in NUP98-NSD1+ AML, whereas transcriptomic analysis reveal that Kdm5b, a lysine demethylase gene carrying "bivalent" chromatin domains, is directly repressed by PRC2. While ectopic expression of Kdm5b suppressed AML growth, its depletion not only promoted tumorigenicity but also attenuated anti-AML effects of PRC2 inhibitors, demonstrating a PRC2-|Kdm5b axis for AML oncogenesis. Integrated RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and Cleavage Under Targets & Release Using Nuclease (CUT&RUN) profiling also showed that Kdm5b directly binds and represses AML stemness genes. The anti-AML effect of Kdm5b relies on its chromatin association and/or scaffold functions rather than its demethylase activity. Collectively, this study describes a molecular axis that involves histone modifiers (PRC2-|Kdm5b) for sustaining AML oncogenesis.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

Diagnostic potential of soluble ST2 and D-dimer for Stanford Type B aortic dissection and intramural aortic hematoma.

OBJECTIVE: Type B aortic dissection (TBAD) and intramural aortic hematoma (IMH) are common manifestations of Acute Aortic Syndrome (AAS), exhibiting overlapping clinical features. The timely and accurate diagnosis and differentiation between TBAD and IMH are critical for appropriate management. Tumorigenicity 2 (sST2) and D-dimer have been shown to elevate levels in both TBAD and IMH, making them valuable as "rule-out" markers. Hence, we aimed to assess the diagnostic utility of sST2 and D-dimer in distinguishing TBAD from IMH. METHODS: In this retrospective study, we analyzed serum levels of sST2 and D-dimer in 182 AAS patients, comprising 90 TBAD cases, 92 IMH cases, and 90 non-AAS cases. Serial measurements were taken at 1 h, 6 h, 12 h, 24 h, and 72 h post-admission. Comparative analyses were conducted between TBAD and non-AAS cases, IMH and non-AAS cases, and TBAD and IMH cases. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic accuracy of sST2 and D-dimer in identifying TBAD or IMH cases. RESULTS: Both TBAD and IMH patients displayed elevated levels of sST2 and D-dimer compared to non-AAS cases. Notably, sST2 levels were significantly higher in TBAD patients than in IMH patients, whereas D-dimer levels exhibited moderate differences. TBAD patients tended to exhibit elevated levels of either sST2 or D-dimer, with a modest correlation between the two (Pearson correlation coefficient = 0.3614). In contrast, IMH patients showed elevations in both markers, with a positive correlation between them (Pearson correlation coefficient = 0.6814). The ROC analysis revealed that both sST2 (AUC, 0.657; 95 % CI, 0.552-0.753; cutoff value, 27.54 ng/ml) and D-dimer (AUC, 0.695; 95 % CI, 0.591-0.787, cutoff value, 1.215 ng/ml) demonstrated favorable diagnostic performance for TBAD. sST2 exhibited a sensitivity of 80.92 % and a specificity of 75.00 %, while D-dimer showed a sensitivity of 80.92 % and a specificity of 75.00 %. For the diagnosis of IMH, the combined assessment of sST2 and D-dimer (AUC, 0.674; 95 % CI, 0.599-0.768; sensitivity, 69.20 %; specificity, 80.00 %) proved effective. CONCLUSIONS: Our results indicate that both sST2 and D-dimer show diagnostic potential for TBAD. Elevated levels of either serve as an indicator of TBAD onset. However, concurrent elevation of both markers seems to be indicative of IMH. The combination of increased sST2 and D-dimer levels demonstrates strong diagnostic performance in identifying IMH cases.

Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products.

BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

GFP Transfection Alters Protein Expression Patterns in Prostate Cancer Cells: A Proteomic Study.

PURPOSE: Green Fluorescent Protein is widely used as a cellular marker tool, but its potential influence on cells has been questioned. Although the potential off-target effects of GFP on tumor cells have been studied to some extent, the findings at the molecular level are insufficient to explain the effect of GFP expression on the tumorigenic capacity of cancer cells. Here, we aimed to investigate the effect of GFP expression on the tumorigenicity of PC3 prostate cancer cells. METHODS: Using GFP-expressing and wild-type PC-3 cells, xenograft models were generated in athymic BALB/C mice. To identify differentially expressed proteins, the change in cells proteome was investigated by label-free quantification with nano-high performance liquid chromatography to tandem mass spectrometry (nHPLC-MS/MS). Proteins that showed significantly altered expression levels were evaluated using the bioinformatics tools. RESULTS: Unlike the wild-type PC-3 cells, GFP-expressing cells failed to develop tumor. Comparative proteome analysis of GFP-expressing cells with WT PC-3 cells revealed a total of 216 differentially regulated proteins, of which 98 were upregulated and 117 were downregulated. CONCLUSION: Upon GFP expression, differential changes in several pathways including the immune system, translational machinery, energy metabolism, elements of cytoskeletal and VEGF signaling pathway were observed. Therefore, care should be taken into account to prevent reporting deceitful mechanisms generated from studies utilizing GFP.

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors including transforming growth factor beta (TGF-ß), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-ß, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Serum Soluble Suppression of Tumorigenicity-2 Levels Predict Cardiovascular Events in Patients Undergoing Incident Peritoneal Dialysis: A Prospective Cohort Study.

INTRODUCTION: Biomarkers are urgently required to identify peritoneal dialysis (PD) patients at risk of cardiovascular (CV) events. This study aimed to investigate the predictive value of soluble suppression of tumorigenicity-2 (sST2) for CV events in patients undergoing incident PD. METHODS: In this prospective cohort study, incident PD patients were enrolled. Blood samples to measure sST2 levels were obtained before PD catheter implantation. The patients underwent a standard peritoneal equilibration test (PET) after initiation of PD for 4-6 weeks. The sST2 levels in both serum and dialysate were determined using enzyme-linked immunosorbent assay. CV events were recorded during the follow-up period. RESULTS: A total of 137 patients were enrolled. During the follow-up period of 17.3 months, 49 (35.76%) patients experienced CV events. When patients were dichotomized based on the median values and the calculated cutoff values of sST2, the higher sST2 group had 2.980- and 3.048-fold increased risks of CV events, respectively, when compared with the lower sST2 group. Moreover, the prognostic value of sST2 remained significant as a continuous variable (per 1 standard deviation increase, hazard ratio [HR] = 1.037, 95% confidence interval [CI] 1.010-1.066, p = 0.008). N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were found to indicate a higher risk only when dichotomized based on the calculated cutoff values. Furthermore, serum sST2 and NT-proBNP levels simultaneously above the calculated cutoff values were associated with a higher risk of CV events (HR = 3.398, 95% CI 1.813-6.367, p < 0.001). CONCLUSION: Baseline serum sST2 level is an independent predictor of the risk of CV events in patients receiving incident PD, and in combination with NT-proBNP level, it can provide a more accurate predictive value.

Serum interleukin-33 and soluble suppression of tumorigenicity 2 in pediatric leukemia with febrile neutropenia.

The purpose of this study was to evaluate the association between interleukin-33 (IL-33) and its receptor Soluble Suppression of Tumorigenicity-2 (sST2) levels and bacterial infections during febrile neutropenia (FN) in pediatric patients with acute lymphoblastic leukemia (ALL). In this prospective, case-control study, participants were divided into 3 groups: ALL patients with FN (Group A), ALL patients without neutropenia and fever (Group B), and healthy children without infection and chronic disease (Group C). There were 30 cases in each group. Blood samples for IL-33 and sST2 have been drawn from patients in Group A before the initiation of treatment and on days 1 and 5 of treatment, and from patients in Groups B and C at initiation. At admission, mean IL-33 level (39.02 ± 26.40 ng/L) in Group B and mean sST2 level (185.3 ± 371.49 ng/ml) in Group A were significantly higher than the other groups (p = 0.038, p < 0.001, respectively). No difference was observed in the mean IL-33 and sST2 levels in the 5-day follow-up of patients in Group A (p = 0.82, p = 0.86, respectively). IL-33 and sST2 levels were not associated with fever duration, neutropenia duration or length of hospitalization. While C-reactive protein (CRP) was significantly higher in patients with positive blood culture (p = 0.021), IL-33 (p = 0.49) and sST2 (p = 0.21) levels were not associated with culture positivity.  Conclusion: IL-33 and sST2 levels were not found valuable as diagnostic and prognostic markers to predict bacterial sepsis in patients with FN. What is Known: • Neutropenic patients are at high risk of serious bacterial and viral infections, but the admission symptom is often only fever. • Febrile neutropenia has a high mortality rate if not treated effectively. What is New: • Febrile neutropenia is not only caused by bacterial infections. Therefore, new biomarkers should be identified to prevent overuse of antibiotics. • Specific biomarkers are needed to diagnose bacterial sepsis in the early phase of febrile neutropenia.

Assessment of Genetic Stability in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Using Droplet Digital PCR.

Unintended genetic modifications that occur during the differentiation and proliferation of human induced pluripotent stem cells (hiPSCs) can lead to tumorigenicity. This is a crucial concern in the development of stem cell-based therapies to ensure the safety and efficacy of the final product. Moreover, conventional genetic stability testing methods are limited by low sensitivity, which is an issue that remains unsolved. In this study, we assessed the genetic stability of hiPSCs and hiPSC-derived cardiomyocytes using various testing methods, including karyotyping, CytoScanHD chip analysis, whole-exome sequencing, and targeted sequencing. Two specific genetic mutations in KMT2C and BCOR were selected from the 17 gene variants identified by whole-exome and targeted sequencing methods, which were validated using droplet digital PCR. The applicability of this approach to stem cell-based therapeutic products was further demonstrated with associated validation according to the International Council for Harmonisation (ICH) guidelines, including specificity, precision, robustness, and limit of detection. Our droplet digital PCR results showed high sensitivity and accuracy for quantitatively detecting gene mutations, whereas conventional qPCR could not avoid false positives. In conclusion, droplet digital PCR is a highly sensitive and precise method for assessing the expression of mutations with tumorigenic potential for the development of stem cell-based therapeutics.

Pathogenesis of chemically induced nasal cavity tumors in rodents: contribution to adverse outcome pathway.

The pathogenesis of nasal cavity tumors induced in rodents has been critically reviewed. Chemical substances that induce nasal cavity tumors in rats, mice, and hamsters were searched in the National Toxicology Program (NTP), International Agency for Research on Cancer (IARC), and Japan Bioassay Research Center (JBRC) databases, in addition to PubMed. Detailed data such as animal species, administration routes, and histopathological types were extracted for induced nasal cavity tumors. Data on non-neoplastic lesions were also extracted. The relationship between the tumor type and non-neoplastic lesions at equivalent sites was analyzed to evaluate tumor pathogenesis. Genotoxicity data were also analyzed. Squamous cell carcinoma was the most frequent lesion, regardless of the dosing route, and its precursor lesions were squamous metaplasia and/or respiratory epithelial hyperplasia, similar to squamous cell papilloma. The precursor lesions of adenocarcinoma, the second most frequent tumor type, were mainly olfactory epithelial hyperplasia, whereas those of adenoma were respiratory epithelial lesions. These pathways were consistent among species. Our results suggest that the responsible lesions may be commonly linked with chemically-induced cytotoxicity in each tumor type, irrespective of genotoxicity, and that the pathways may largely overlap between genotoxic and non-genotoxic carcinogens. These findings may support the documentation of adverse outcome pathways (AOPs), such as cytotoxicity, leading to nasal cavity tumors and the integrated approaches to testing and assessment (IATA) for non-genotoxic carcinogens.

Neural stemness unifies cell tumorigenicity and pluripotent differentiation potential.

Neural stemness is suggested to be the ground state of tumorigenicity and pluripotent differentiation potential. However, the relationship between these cell properties is unclear. Here, by disrupting the neural regulatory network in neural stem and cancer cells and by serial transplantation of cancer cells, we show that tumorigenicity and pluripotent differentiation potential are coupled cell properties unified by neural stemness. We show that loss of neural stemness via inhibition of SETDB1, an oncoprotein with enriched expression in embryonic neural cells during vertebrate embryogenesis, led to neuronal differentiation with reduced tumorigenicity and pluripotent differentiation potential in neural stem and cancer cells, whereas enhancement of neural stemness by SETDB1 overexpression caused the opposite effects. SETDB1 maintains a regulatory network comprising proteins involved in developmental programs and basic cellular functional machineries, including epigenetic modifications (EZH2), ribosome biogenesis (RPS3), translation initiation (EIF4G), and spliceosome assembly (SF3B1); all of these proteins are enriched in embryonic neural cells and play active roles in cancers. In addition, SETDB1 represses the transcription of genes promoting differentiation and cell cycle and growth arrest. Serial transplantation of cancer cells showed that neural stemness, tumorigenicity, and pluripotent differentiation potential were simultaneously enhanced; these effects were accompanied by increased expression of proteins involved in developmental programs and basic machineries, including SETDB1 and the abovementioned proteins, as well as by increased alternative splicing events. These results indicate that basic machineries work together to define a highly proliferative state with pluripotent differentiation potential and also suggest that neural stemness unifies tumorigenicity and differentiation potential.

Performance Evaluation of AFIAS ST2 and Ichroma ST2 Assays in Comparison with Presage ST2 Assay.

Background: Elevated soluble suppression of tumorigenicity 2 (sST2) levels may predict mortality in heart failure (HF) patients. The AFIAS ST2 assay (AFIAS ST2, Boditech Med Inc., Chuncheon, Korea) and ichroma ST2 assay (ichroma ST2, Boditech Med Inc.) are newly developed point-of-care (POC) assays for measuring sST2 level. We evaluated the performance of these assays, in terms of cut-off validation and prognosis, and compared them with that of the Presage ST2 assay (Presage ST2, Critical Diagnostics, San Diego, CA, USA). Methods: We validated the US FDA-claimed sST2 clinical cut-off of 35 ng/mL using 420 serum samples (298 samples from the universal sample bank of the American Association for Clinical Chemistry and 122 samples from reference individuals from Konkuk University Medical Center). We compared AFIAS ST2 and ichroma ST2 with Presage ST2, using 206 samples from patients with HF. We assessed prognosis using the three assays in 252 samples from the Barcelona ambulatory HF cohort subsets. Results: The upper reference limits of AFIAS ST2 and ichroma ST2 were within the clinical cut-off of Presage ST2. The results of AFIAS ST2 and ichroma ST2 were highly correlated with those of Presage ST2 (r = 0.82 and 0.81, respectively). Based on this cut-off, all three assays predicted cardiovascular death. Conclusions: The new POC assays, AFIAS ST2 and ichroma ST2, would be useful in clinical practice for managing HF patients, with performances equivalent to that of Presage ST2.

Identification of marker genes to monitor residual iPSCs in iPSC-derived products.

BACKGROUND: Engineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC-derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As a consequence, highly sensitive and specific methods for detecting residual undifferentiated iPSCs are indispensable for safety evaluations of iPSC-based therapies. The present study provides an approach for identifying potential marker genes for iPSC impurities in iPSC-derived cells using RNA sequencing data from iPSCs and various differentiated cell types. METHODS: Identifying iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, the authors focused on identifying markers for iPSC impurities in iPSC-derived cardiomyocytes (iCMs) and validated the selected genes by reverse transcription quantitative polymerase chain reaction. The sensitivity of the candidate genes was determined by spiking different amounts of iPSCs into iCMs and their performance was compared with the previously suggested marker lin-28 homolog A (LIN28A). RESULTS: Embryonic stem cell-related gene (ESRG), long intergenic non-protein coding RNA 678 (LINC00678), CaM kinase-like vesicle-associated (CAMKV), indoleamine 2,3-dioxygenase 1 (IDO1), chondromodulin (CNMD), LINE1-type transposase domain containing 1 (L1DT1), LIN28A, lymphocyte-specific protein tyrosine kinase (LCK), vertebrae development-associated (VRTN) and zinc finger and SCAN domain containing 10 (ZSCAN10) detected contaminant iPSCs among iCMs with a limit of detection that ranged from 0.001% to 0.1% depending on the gene and iCM batch used. CONCLUSIONS: Using the example of iCMs, the authors provide a strategy for identifying a set of highly specific and sensitive markers that can be used for quality assessment of iPSC-derived products.

The impact and outcomes of cancer-macrophage fusion.

BACKGROUND: Cancer's hallmark feature is its ability to evolve, leading to metastasis and recurrence. Although genetic mutations and epigenetic changes have been implicated, they don't fully explain the leukocytic traits that many cancers develop. Cell fusion between cancer and somatic cells, particularly macrophages, has been suggested as an alternative pathway for cancer cells to obtain new traits by acquiring exogenous genetic material. METHODS: This study aims to investigate the potential biological outcomes of tumor-myeloid cell fusion by generating tumor-macrophage hybrid cells. Two clones with markedly different tumorigenicity were selected, and RNA-seq was used to compare their RNA expressions with that of the control cells. Based on the results that the hybrid cells showed differential activation in several upstream regulator pathways that impact their biological behaviors, the hybrid cells' abilities to recruit stromal cells and establish angiogenesis as well as their cell cycle distributions were investigated through in vitro and in vivo studies. RESULTS: Although both hybrid clones demonstrated p53 activation and reduced growth rates, they exhibited distinct cell cycle distributions and ability to grow in vivo. Notably, while one clone was highly tumorigenic, the other showed little tumorigenicity. Despite these differences, both hybrid clones were potent environmental modifiers, exhibiting significant abilities to recruit stromal and immune cells and establish angiogenesis. CONCLUSIONS: The study revealed that tumor-somatic cell fusion is a potent environmental modifier that can modulate tumor survival and evolution, despite its relatively low occurrence. These findings suggest that tumor-somatic cell fusion could be a promising target for developing new cancer therapies. Furthermore, this study provides an experimental animal platform to investigate cancer-myeloid fusion and highlights the potential role of tumor-somatic cell fusion in modulating the tumor environment.