Single-Cell Molecular Profiling of Head and Neck Squamous Cell Carcinoma Reveals Five Dysregulated Signaling Pathways Associated With Circulating Tumor Cells.

OBJECTIVES: To determine the dysregulated signaling pathways of head and neck squamous cell carcinoma associated with circulating tumor cells (CTCs) via single-cell molecular characterization. INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) has a significant global burden and is a disease with poor survival. Despite trials exploring new treatment modalities to improve disease control rates, the 5 year survival rate remains low at only 60%. Most cancer malignancies are reported to progress to a fatal phase due to the metastatic activity derived from treatment-resistant cancer cells, regarded as one of the most significant obstacles to develope effective cancer treatment options. However, the molecular profiles of cancer cells have not been thoroughly studied. METHODS: Here, we examined in-situ HNSCC tumors and pairwisely followed up with the downstream circulating tumor cells (CTCs)-based on the surrogate biomarkers to detect metastasis that is established in other cancers - not yet being fully adopted in HNSCC treatment algorithms. RESULTS: Specifically, we revealed metastatic HNSCC patients have complex CTCs that could be defined through gene expression and mutational gene profiling derived from completed single-cell RNASeq (scRNASeq) that served to confirm molecular pathways inherent in these CTCs. To enhance the reliability of our findings, we cross-validated those molecular profiles with results from previously published studies. CONCLUSION: Thus, we identified 5 dysregulated signaling pathways in CTCs to derive HNSCC biomarker panels for screening HNSCC in situ tumors.

ObjectivesInvestigating the dysregulated signaling pathways of head and neck squamous cell carcinoma (HNSCC) linked with circulating tumor cells (CTCs) using single-cell molecular characterization.IntroductionHNSCC poses a significant global health burden with poor survival rates despite advancements in treatment. Metastatic activity from treatment-resistant cancer cells remains a major challenge in developing effective treatments. However, the molecular profiles of cancer cells, particularly CTCs, are not well-understood.MethodsWe analyzed in-situ HNSCC tumors and corresponding CTCs using surrogate biomarkers to detect metastasis, a technique not widely used in HNSCC treatment protocols.ResultsOur study revealed complex CTCs in metastatic HNSCC patients characterized by gene expression and mutational gene profiling via single-cell RNASeq (scRNASeq). These profiles confirmed molecular pathways inherent in CTCs, further validated by previous research.ConclusionThrough our research, we identified five dysregulated signaling pathways in CTCs, suggesting potential biomarker panels for HNSCC screening in situ tumors.

CRIF1 as a potential target to improve the radiosensitivity of osteosarcoma.

Resistance to ionizing radiation (IR), which is a conventional treatment for osteosarcoma that cannot be resected, undermines the efficacy of this therapy. However, the mechanism by which IR induces radioresistance in osteosarcoma is not defined. Here, we report that CR6-interacting factor-1 (CRIF1) is highly expressed in osteosarcoma and undergoes nuclear-cytoplasmic shuttling of cyclin-dependent kinase 2 (CDK2) after IR. Osteosarcoma cells lacking CRIF1 show increased sensitivity to IR, which is associated with delayed DNA damage repair, inactivated G1/S checkpoint, and mitochondrial dysfunction. CRIF1 interacts with the DNA damage checkpoint regulator CDK2, and CRIF1 and CDK2 colocalize in the nucleus after IR. Nuclear localization of CDK2 is associated with phosphorylation changes that promote DNA repair and activation of the G1/S checkpoint. CRIF1 knockdown synergized with IR in an in vivo osteosarcoma model, leading to tumor regression. Based on these findings, we identify CRIF1 as a potential therapeutic target in osteosarcoma that can increase the efficacy of radiotherapy. More broadly, our findings may provide insights into the mechanism for other types of radioresistant cancers and be exploited for therapeutic ends.

FBP1-Altered Carbohydrate Metabolism Reduces Leukemic Viability through Activating P53 and Modulating the Mitochondrial Quality Control System In Vitro.

Acute myeloid leukemia (AML)-the most frequent form of adult blood cancer-is characterized by heterogeneous mechanisms and disease progression. Developing an effective therapeutic strategy that targets metabolic homeostasis and energy production in immature leukemic cells (blasts) is essential for overcoming relapse and improving the prognosis of AML patients with different subtypes. With respect to metabolic regulation, fructose-1,6-bisphosphatase 1 (FBP1) is a gluconeogenic enzyme that is vital to carbohydrate metabolism, since gluconeogenesis is the central pathway for the production of important metabolites and energy necessary to maintain normal cellular activities. Beyond its catalytic activity, FBP1 inhibits aerobic glycolysis-known as the "Warburg effect"-in cancer cells. Importantly, while downregulation of FBP1 is associated with carcinogenesis in major human organs, restoration of FBP1 in cancer cells promotes apoptosis and prevents disease progression in solid tumors. Recently, our large-scale sequencing analyses revealed FBP1 as a novel inducible therapeutic target among 17,757 vitamin-D-responsive genes in MV4-11 or MOLM-14 blasts in vitro, both of which were derived from AML patients with FLT3 mutations. To investigate FBP1's anti-leukemic function in this study, we generated a new AML cell line through lentiviral overexpression of an FBP1 transgene in vitro (named FBP1-MV4-11). Results showed that FBP1-MV4-11 blasts are more prone to apoptosis than MV4-11 blasts. Mechanistically, FBP1-MV4-11 blasts have significantly increased gene and protein expression of P53, as confirmed by the P53 promoter assay in vitro. However, enhanced cell death and reduced proliferation of FBP1-MV4-11 blasts could be reversed by supplementation with post-glycolytic metabolites in vitro. Additionally, FBP1-MV4-11 blasts were found to have impaired mitochondrial homeostasis through reduced cytochrome c oxidase subunit 2 (COX2 or MT-CO2) and upregulated PTEN-induced kinase (PINK1) expressions. In summary, this is the first in vitro evidence that FBP1-altered carbohydrate metabolism and FBP1-activated P53 can initiate leukemic death by activating mitochondrial reprogramming in AML blasts, supporting the clinical potential of FBP1-based therapies for AML-like cancers.

LongGF: computational algorithm and software tool for fast and accurate detection of gene fusions by long-read transcriptome sequencing.

BACKGROUND: Long-read RNA-Seq techniques can generate reads that encompass a large proportion or the entire mRNA/cDNA molecules, so they are expected to address inherited limitations of short-read RNA-Seq techniques that typically generate < 150 bp reads. However, there is a general lack of software tools for gene fusion detection from long-read RNA-seq data, which takes into account the high basecalling error rates and the presence of alignment errors. RESULTS: In this study, we developed a fast computational tool, LongGF, to efficiently detect candidate gene fusions from long-read RNA-seq data, including cDNA sequencing data and direct mRNA sequencing data. We evaluated LongGF on tens of simulated long-read RNA-seq datasets, and demonstrated its superior performance in gene fusion detection. We also tested LongGF on a Nanopore direct mRNA sequencing dataset and a PacBio sequencing dataset generated on a mixture of 10 cancer cell lines, and found that LongGF achieved better performance to detect known gene fusions over existing computational tools. Furthermore, we tested LongGF on a Nanopore cDNA sequencing dataset on acute myeloid leukemia, and pinpointed the exact location of a translocation (previously known in cytogenetic resolution) in base resolution, which was further validated by Sanger sequencing. CONCLUSIONS: In summary, LongGF will greatly facilitate the discovery of candidate gene fusion events from long-read RNA-Seq data, especially in cancer samples. LongGF is implemented in C++ and is available at https://github.com/WGLab/LongGF .

Improving single-cell transcriptome sequencing efficiency with a microfluidic phase-switch device.

In this paper, we present a novel method to improve the efficiency of single-cell transcriptome sequencing for analyzing valuable cell samples. The microfluidic device we designed integrates multiple single-cell isolation chambers with hydrodynamic traps and achieves a nearly 100% single-cell capture rate and minimal cell loss, making it particularly suitable for samples with limited numbers of cells. Single cells were encapsulated using a novel phase-switch method into picoliter-sized hydrogel droplets and easily recovered for subsequent reactions. Minimizing the reaction volume resulted in a high reverse transcription (RT) efficiency for RNA sequencing (RNA-Seq). With this novel microfluidic platform, we captured dozens of hESCs (H9) simultaneously and obtained live cells in individual picoliter volumes, thus allowing for the convenient construction of a high-quality library for deep single-cell RNA-Seq. Our single-cell RNA-Seq results confirmed that a spectrum of pluripotency existed within an H9 colony. This integrated microfluidic platform can be applied to various cell types for the investigation of rare cellular events, and the phase-switch single-cell processing strategy will improve the efficiency and accessibility of single-cell transcriptome sequencing analysis.

Relapse pathway of glioblastoma revealed by single-cell molecular analysis.

Glioblastoma multiforme (GBM) remains an incurable brain tumor. The highly malignant behavior of GBM may, in part, be attributed to its intraclonal genetic and phenotypic diversity (subclonal evolution). Identifying the molecular pathways driving GBM relapse may provide novel, actionable targets for personalized diagnosis, characterization of prognosis and improvement of precision therapy. We screened single-cell transcriptomes, namely RNA-seq data of primary and relapsed GBM tumors from a patient, to define the molecular profile of relapse. Characterization of hundreds of individual tumor cells identified three mutated genes within single cells, involved in the RAS/GEF GTP-dependent signaling pathway. The identified molecular pathway was further verified by meta-analysis of RNA-seq data from more than 3000 patients. This study showed that single-cell molecular analysis overcomes the inherent heterogeneity of bulk tumors with respect to defining tumor subclonal evolution relevant to GBM relapse.

Crif1 Promotes Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells After Irradiation by Modulating the PKA/CREB Signaling Pathway.

Dysfunction of the hematopoietic microenvironment is the main obstacle encountered during hematopoiesis reconstruction in patients with acute hematopoietic radiation syndrome. Bone marrow mesenchymal stem cells (BM-MSCs) play a crucial supporting role in hematopoiesis by maintaining the balance between adipogenic and osteogenic differentiation. In this study, we found that irradiation decreased the colony-forming efficiency of BM-MSCs and impaired the balance between adipogenic and osteogenic differentiation. Following irradiation, BM-MCSs became strongly predisposed to adipogenesis, as evidenced by increased oil red O staining and elevated mRNA and protein levels of the adipogenic markers and transcription factors PPARγ and AP2. Overexpression of the essential adipogenesis regulator Crif1 in BM-MSCs promoted adipogenesis after irradiation exposure by upregulating adipogenesis-related genes, including C/EBPß, PPARγ, and AP2. We found that Crif1 promoted the phosphorylation of cAMP response element binding protein (CREB) through direct interaction with protein kinase A (PKA)-α. Phosphorylation of CREB was inhibited in Crif1-knockdown BM-MSCs even in the presence of a PKA agonist (db-cAMP) and could be suppressed in Crif1-overexpressing BM-MSCs by a PKAα inhibitor (H-89). These results suggest that Crif1 is an indispensable regulator of PKAα cat that modulates the PKA/CREB signaling pathway to promote adipogenic differentiation of BM-MSCs after irradiation.

Increase developmental plasticity of human keratinocytes with gene suppression.

Recent evidence indicates that p53 suppression increased the efficiency of induced pluripotent stem cell (iPSC) generation. This occurred even with the enforced expression of as few as two canonical transcription factors, Oct4 and Sox2. In this study, primary human keratinocytes were successfully induced into a stage of plasticity by transient inactivation of p53, without enforced expression of any of the transcription factors previously used in iPSC generation. These cells were later redifferentiated into neural lineages. The gene suppression plastic cells were morphologically indistinguishable from human ES cells. Gene suppression plastic cells were alkaline phosphatase-positive, had normal karyotypes, and expressed p53. Together with the accumulating evidence of similarities and overlapping mechanisms between iPSC generation and cancer formation, this finding sheds light on the emerging picture of p53 sitting at the crossroads between two intricate cellular potentials: stem cell vs. cancer cell generation. This finding further supports the crucial role played by p53 in cellular reprogramming and suggests an alternative method to switch the lineage identity of human cells. This reported method offers the potential for directed lineage switching with the goal of generating autologous cell populations for novel clinical applications for neurodegenerative diseases.

Contactless Single Cell Extraction from Monolayer Cell Culture Using A MEMS Acoustic Droplet Ejector.

OBJECTIVE: To achieve a contactless and damage-less extraction of a single (or a few) human retinal pigment epithelial (RPE) cell(s) from a cell monolayer with acoustic droplet ejection. METHODS: An acoustic droplet ejector based on a self-focusing acoustic transducer (SFAT) is designed, microfabricated, and placed on a precision movable stage that aligns it to the targeted cell(s) in a Petri dish. The device delivers 20.1 MHz focused ultrasound (FUS) with a focal diameter of 100 µm, which ejects droplets capable of extracting and transferring only the targeted cell(s) from a monolayer. RESULTS: The extraction and collection of 1-10 cells are successfully demonstrated. The number of ejected cells can be controlled by the FUS pulse width. As confirmed by fluorescence-based cell viability assays and re-culture experiments, the ejected cell(s) and remaining monolayer cells are intact without damage after cell ejection. Furthermore, real-time reverse transcription polymerase chain reaction (RT-PCR) tests for both housekeeping genes (GAPDH and ß -actin) and RPE-specific genes (MITF, PEDF, and PMEL17) show no significant difference between the acoustically ejected cells and those collected manually with a micropipette. CONCLUSION: The proposed technology realizes a contactless, damage-free extraction of cells with high spatial resolution and precise control of the number of cells ejected, with a simple setup. SIGNIFICANCE: This powerful technology not only enables efficient, high-precision cell extraction for quality check applications but also opens new avenues for other advanced biotechnologies such as bioprinting.

DeepDecon accurately estimates cancer cell fractions in bulk RNA-seq data.

Understanding the cellular composition of a disease-related tissue is important in disease diagnosis, prognosis, and downstream treatment. Recent advances in single-cell RNA-sequencing (scRNA-seq) technique have allowed the measurement of gene expression profiles for individual cells. However, scRNA-seq is still too expensive to be used for large-scale population studies, and bulk RNA-seq is still widely used in such situations. An essential challenge is to deconvolve cellular composition for bulk RNA-seq data based on scRNA-seq data. Here, we present DeepDecon, a deep neural network model that leverages single-cell gene expression information to accurately predict the fraction of cancer cells in bulk tissues. It provides a refining strategy in which the cancer cell fraction is iteratively estimated by a set of trained models. When applied to simulated and real cancer data, DeepDecon exhibits superior performance compared to existing decomposition methods in terms of accuracy.

Mesenchymal Stem Cells for Prophylaxis of Chronic Graft-vs-Host Disease After Haploidentical Hematopoietic Stem Cell Transplant: An Open-Label Randomized Clinical Trial.

Importance: Chronic graft-vs-host disease (GVHD) limits the long-term benefit of haploidentical hematopoietic stem cell transplant (HSCT). This clinical trial evaluated repeated infusions of umbilical cord mesenchymal stem cells (MSCs) during the early stage (45 days and 100 days) after haplo-HSCT to prevent chronic GVHD. Objective: To determine whether repeated infusions of MSCs during the early stage after haplo-HSCT decreases the incidence of severe chronic GVHD. Design, Setting, and Participants: This open-label, multicenter, parallel randomized clinical trial was conducted from April 2016 to January 2022. Eligibility criteria included a diagnosis of acute leukemia and having a haploidentical, suitable related donor for HSCT. The median (range) follow-up time was 39.0 (1.5-67.0) months. Interventions: The enrolled patients with a haploidentical relative for HSCT received the modified busulfan/cyclophosphamide + antithymocyte globulin modified regimen and standard GVHD prophylaxis. Patients were randomly chosen to receive MSCs (the MSC group) (1 × 106 cells/kg, every 2 weeks, starting from 45 days after transplant, 4 times total) or regular prophylaxis (control group). Main Outcome and Measure: The cumulative incidence of severe chronic GVHD. Results: Of 158 patients, 58 (36.7%) were female individuals; the median (range) age for the MSC and control groups was 28 (18-60) years and 28 (18-56) years, respectively. A total of 158 patients were screened, and 148 patients were randomly assigned to the MSC group (n = 74) or control group (n = 74) 1 day before MSCs infusion. The estimated 2-year cumulative incidence of severe chronic GVHD was 5.4% (95% CI, 1.8%-14.0%) in the MSC group and 17.4% (95% CI, 10.1%-28.5%) in the control group (hazard ratio [HR], 0.29; 95% CI, 0.10-0.88; P = .03). There was no difference between the MSC and control groups in the cumulative incidence of leukemia relapse (HR, 1.17; 95% CI, 0.55-2.47; P = .68). The cumulative incidence of stage II to IV acute GVHD in the MSC group was significantly lower than that in the control group (HR, 0.25; 95% CI, 0.09-0.67; P = .01). The MSC group had better GVHD-free and relapse-free survival rates than the control group (HR, 0.62; 95% CI, 0.39-0.98; P = .04). Conclusions and Relevance: The results of this randomized clinical trial show that early repeated infusions of MSCs decreased the incidence and severity of chronic GVHD, and the incidence and severity of acute GVHD manifested as a better GVHD-free and relapse-free survival rate for patients after haplo-HSCT. Trial Registration: Chinese Clinical Trial Registry: ChiCTR-IIR-16007806.

Investigating evolutionary perspective of carcinogenesis with single-cell transcriptome analysis.

We developed phase-switch microfluidic devices for molecular profiling of a large number of single cells. Whole genome microarrays and RNA-sequencing are commonly used to determine the expression levels of genes in cell lysates (a physical mix of millions of cells) for inferring gene functions. However, cellular heterogeneity becomes an inherent noise in the measurement of gene expression. The unique molecular characteristics of individual cells, as well as the temporal and quantitative information of gene expression in cells, are lost when averaged among all cells in cell lysates. Our single-cell technology overcomes this limitation and enables us to obtain a large number of single-cell transcriptomes from a population of cells. A collection of single-cell molecular profiles allows us to study carcinogenesis from an evolutionary perspective by treating cancer as a diverse population of cells with abnormal molecular characteristics. Because a cancer cell population contains cells at various stages of development toward drug resistance, clustering similar single-cell molecular profiles could reveal how drug-resistant sub-clones evolve during cancer treatment. Here, we discuss how single-cell transcriptome analysis technology could enable the study of carcinogenesis from an evolutionary perspective and the development of drug-resistance in leukemia. The single-cell transcriptome analysis reported here could have a direct and significant impact on current cancer treatments and future personalized cancer therapies.

BCR-ABL1-driven exosome-miR130b-3p-mediated gap-junction Cx43 MSC intercellular communications imply therapies of leukemic subclonal evolution.

Rationale: In the bone marrow microenvironment (BMME), mesenchymal stem/stromal cells (MSCs) control the self-renewal of both healthy and cancerous hematopoietic stem/progenitor cells (HSPCs). We previously showed that in vivo leukemia-derived MSCs change neighbor MSCs into leukemia-permissive states and boost leukemia cell proliferation, survival, and chemotherapy resistance. But the mechanisms behind how the state changes are still not fully understood. Methods: Here, we took a reverse engineering approach to determine BCR-ABL1+ leukemia cells activated transcriptional factor C/EBPß, resulting in miR130a/b-3p production. Then, we back-tracked from clinical specimen transcriptome sequencing to cell co-culture, molecular and cellular assays, flow cytometry, single-cell transcriptome, and transcriptional regulation to determine the molecular mechanisms of BCR-ABL1-driven exosome-miR130b-3p-mediated gap-junction Cx43 MSC intercellular communications. Results: BCR-ABL1-driven exosome-miR130a/b-3p mediated gap-junction Cx43 (a.k.a., GJA1) BMSC intercellular communications for subclonal evolution in leukemic microenvironment by targeting BMSCs-expressed HLAs, thereby potentially maintaining BMSCs with self-renewal properties and reduced BMSC immunogenicity. The Cx43low and miR-130a/bhigh subclonal MSCs subsets of differentiation state could be reversed to Cx43high and miR-130a/blow subclones of the higher stemness state in Cx43-overexpressed subclonal MSCs. Both miR-130a and miR-130b might only inhibit Cx43 translation or degrade Cx43 proteins and did not affect Cx43 mRNA stability. The subclonal evolution was further confirmed by single-cell transcriptome profiling of MSCs, which suggested that Cx43 regulated their stemness and played normal roles in immunomodulation antigen processing. Thus, upregulated miR-130a/b promoted osteogenesis and adipogenesis from BMSCs, thereby decreasing cancer progression. Our clinical data validated that the expression of many genes in human major histocompatibility was negatively associated with the stemness of MSCs, and several immune checkpoint proteins contributing to immune escape in tumors were overexpressed after either miR-130a or miR-130b overexpression, such as CD274, LAG3, PDCD1, and TNFRSF4. Not only did immune response-related cytokine-cytokine receptor interactions and PI3K-AKT pathways, including EGR3, TNFRSF1B, but also NDRG2 leukemic-associated inflammatory factors, such as IFNB1, CXCL1, CXCL10, and CCL7 manifest upon miR-130a/b overexpression. Either BCR siRNAs or ABL1 siRNAs assay showed significantly decreased miR-130a and miR-130b expression, and chromatin immunoprecipitation sequencing confirmed that the regulation of miR-130a and miR-130b expression is BCR-ABL1-dependent. BCR-ABL1 induces miR-130a/b expression through the upregulation of transcriptional factor C/EBPß. C/EBPß could bind directly to the promoter region of miR-130b-3p, not miR-130a-3p. BCR-ABL1-driven exosome-miR130a-3p could interact with Cx43, and further impact GJIC in TME. Conclusion: Our findings shed light on how leukemia BCR-ABL1-driven exosome-miR130b-3p could interact with gap-junction Cx43, and further impact GJIC in TME, implications for leukemic therapies of subclonal evolution.

Amplifying STING activation by bioinspired nanomedicine for targeted chemo- and immunotherapy of acute myeloid leukemia.

Chemotherapy resistance and the tumor immune microenvironment are dual reasons for the poor therapeutic efficacy of treating acute myeloid leukemia (AML), causing suboptimal clinical outcomes and high relapse rates. Activation of the stimulator of interferon genes (STING) pathway based on innate immunity can effectively improve antitumor immunity. However, traditional STING agonists are limited due to their easy degradation and difficult membrane transport. Here, a bioinspired nanomedicine synergizing chemo- and immunotherapy was developed by activating the STING pathway for targeted and systemic AML cell damage. We show that a leukemia cell membrane (LCM)-camouflaged hollow MnO2 nanocarrier (HM) with encapsulated doxorubicin (DOX) (denoted LHMD) could bind specifically to AML cells with a homologous targeting effect. Then, MnO2 was decomposed into Mn2+ in response to endosomal acid and glutathione (GSH), which improved the magnetic resonance imaging (MRI) signal for AML detection and activated the STING pathway. In mouse models, LHMD was confirmed to eradicate established AML and prevent the engraftment of AML cells. The percentages of T-helper 1 (Th1) and T-helper 17 (Th17) cells and the concentrations of type I interferon (IFN-Ⅰ) and proinflammatory cytokines increased, while the percentage of T-helper 2 (Th2) cells decreased, reflecting the anti-AML immune response induced by Mn2+ after treatment with LHMD. This nanotechnology-based therapeutic regimen may represent a generalizable strategy for generating an anti-leukemia immune response. STATEMENT OF SIGNIFICANCE: Relapse and chemotherapy refractoriness are main causes for the dismal prognosis of AML, making it urgent to develop more effective anti-AML therapies. This study proposes an innovative strategy to combat this issue by designing a biomimetic BM-targeted nanomedicine based on a MnO2 nano-carrier to rationally deliver chemotherapeutic agents and to trigger Mn2+ mediated STING pathway activation for potent immune- and chemotherapy against AML cells. Hence, the nanomedicine design addresses the challenges associated with AML therapy and proposes a promising strategy to improve the therapeutic efficacy against AML.

Vitamin D activates FBP1 to block the Warburg effect and modulate blast metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) has the lowest survival rate among the leukemias. Targeting intracellular metabolism and energy production in leukemic cells can be a promising therapeutic strategy for AML. Recently, we presented the successful use of vitamin D (1,25VD3) gene therapy to treat AML mouse models in vivo. In this study, recognizing the importance of 1,25VD3 as one of only 2 molecules (along with glucose) photosynthesized for energy during the beginning stage of life on this planet, we explored the functional role of 1,25VD3 in AML metabolism.Transcriptome database (RNA-seq) of four different AML cell lines revealed 17,757 genes responding to 1,25VD3-treatment. Moreover, we discovered that fructose-bisphosphatase 1 (FBP1) noticeably stands out as the only gene (out of 17,757 genes) with a 250-fold increase in gene expression, which is known to encode the key rate-limiting gluconeogenic enzyme fructose-1,6-bisphosphatase. The significant increased expression of FBP1 gene and proteins induced by 1,25VD3 was confirmed by qPCR, western blot, flow cytometry, immunocytochemistry and functional lactate assay. Additionally, 1,25VD3 was found to regulate different AML metabolic processes including gluconeogenesis, glycolysis, TCA, de novo nucleotide synthesis, etc. In summary, we provided the first evidence that 1,25 VD3-induced FBP1 overexpression might be a novel therapeutic target to block the "Warburg Effect" to reduce energy production in AML blasts.

Molecular Characterization of Differentiated-Resistance MSC Subclones by Single-Cell Transcriptomes.

Background: The mechanism of tumorigenicity potentially evolved in mesenchymal stem cells (MSCs) remains elusive, resulting in inconsistent clinical application efficacy. We hypothesized that subclones in MSCs contribute to their tumorgenicity, and we approached MSC-subclones at the single-cell level. Methods: MSCs were cultured in an osteogenic differentiation medium and harvested on days 12, 19, and 25 for cell differentiation analysis using Alizarin Red and followed with the single-cell transcriptome. Results: Single-cell RNA-seq analysis reveals a discrete cluster of MSCs during osteogenesis, including differentiation-resistant MSCs (DR-MSCs), differentiated osteoblasts (DO), and precursor osteoblasts (PO). The DR-MSCs population resembled cancer initiation cells and were subjected to further analysis of the yes associated protein 1 (YAP1) network. Verteporfin was also used for YAP1 inhibition in cancer cell lines to confirm the role of YAP1 in MSC--involved tumorigenicity. Clinical data from various cancer types were analyzed to reveal relationships among YAP1, OCT4, and CDH6 in MSC--involved tumorigenicity. The expression of cadherin 6 (CDH6), octamer-binding transcription factor 4 (OCT4), and YAP1 expression was significantly upregulated in DR-MSCs compared to PO and DO. YAP1 inhibition by Verteporfin accelerated the differentiation of MSCs and suppressed the expression of YAP1, CDH6, and OCT4. A survey of 56 clinical cohorts revealed a high degree of co-expression among CDH6, YAP1, and OCT4 in various solid tumors. YAP1 inhibition also down-regulated HeLa cell viability and gradually inhibited YAP1 nuclear localization while reducing the transcription of CDH6 and OCT4. Conclusions: We used single-cell sequencing to analyze undifferentiated MSCs and to discover a carcinogenic pathway in single-cell MSCs of differentiated resistance subclones.

The Machine-Learning-Mediated Interface of Microbiome and Genetic Risk Stratification in Neuroblastoma Reveals Molecular Pathways Related to Patient Survival.

Currently, most neuroblastoma patients are treated according to the Children's Oncology Group (COG) risk group assignment; however, neuroblastoma's heterogeneity renders only a few predictors for treatment response, resulting in excessive treatment. Here, we sought to couple COG risk classification with tumor intracellular microbiome, which is part of the molecular signature of a tumor. We determine that an intra-tumor microbial gene abundance score, namely M-score, separates the high COG-risk patients into two subpopulations (Mhigh and Mlow) with higher accuracy in risk stratification than the current COG risk assessment, thus sparing a subset of high COG-risk patients from being subjected to traditional high-risk therapies. Mechanistically, the classification power of M-scores implies the effect of CREB over-activation, which may influence the critical genes involved in cellular proliferation, anti-apoptosis, and angiogenesis, affecting tumor cell proliferation survival and metastasis. Thus, intracellular microbiota abundance in neuroblastoma regulates intracellular signals to affect patients' survival.

Targeting TKI-Activated NFKB2-MIF/CXCLs-CXCR2 Signaling Pathways in FLT3 Mutated Acute Myeloid Leukemia Reduced Blast Viability.

Disease relapse is a common cause of treatment failure in FMS-like tyrosine kinase 3 (FLT3) mutated acute myeloid leukemia (AML). In this study, to identify therapeutic targets responsible for the survival and proliferation of leukemic cells (blasts) with FLT3 mutations after gilteritinib (GILT, a 2nd generation tyrosine kinase inhibitor (TKI)) treatment, we performed proteomic screening of cytokine release and in vitro/ex vivo studies to investigate their associated signaling pathways and transcriptional regulation. Here, we report that macrophage migration inhibition factor (MIF) was significantly increased in the supernatant of GILT-treated blasts when compared to untreated controls. Additionally, the GILT-treated blasts that survived were found to exhibit higher expressions of the CXCR2 gene and protein, a common receptor for MIF and pro-inflammatory cytokines. The supplementation of exogenous MIF to GILT-treated blasts revealed a group of CD44High+ cells that might be responsible for the relapse. Furthermore, we identified the highly activated non-classical NFKB2 pathway after GILT-treatment. The siRNA transient knockdown of NFKB2 significantly reduced the gene expressions of MIF, CXCR2, and CXCL5. Finally, treatments of AML patient samples ex vivo demonstrated that the combination of a pharmaceutical inhibitor of the NFKB family and GILT can effectively suppress primary blasts' secretion of tumor-promoting cytokines, such as CXCL1/5/8. In summary, we provide the first evidence that targeting treatment-activated compensatory pathways, such as the NFKB2-MIF/CXCLs-CXCR2 axis could be a novel therapeutic strategy to overcome TKI-resistance and effectively treat AML patients with FLT3 mutations.

Targeting the Tumor Microenvironment in Acute Myeloid Leukemia: The Future of Immunotherapy and Natural Products.

The tumor microenvironment (TME) plays an essential role in the development, proliferation, and survival of leukemic blasts in acute myeloid leukemia (AML). Within the bone marrow and peripheral blood, various phenotypically and functionally altered cells in the TME provide critical signals to suppress the anti-tumor immune response, allowing tumor cells to evade elimination. Thus, unraveling the complex interplay between AML and its microenvironment may have important clinical implications and are essential to directing the development of novel targeted therapies. This review summarizes recent advancements in our understanding of the AML TME and its ramifications on current immunotherapeutic strategies. We further review the role of natural products in modulating the TME to enhance response to immunotherapy.

Neurotrophin Pathway Receptors NGFR and TrkA Control Perineural Invasion, Metastasis, and Pain in Oral Cancer.

Oral squamous cell carcinoma (OSCC) patients suffer from poor survival due to metastasis or locoregional recurrence, processes that are both facilitated by perineural invasion (PNI). OSCC has higher rates of PNI than other cancer subtypes, with PNI present in 80% of tumors. Despite the impact of PNI on oral cancer prognosis and pain, little is known about the genes that drive PNI, which in turn drive pain, invasion, and metastasis. In this study, clinical data, preclinical, and in vitro models are leveraged to elucidate the role of neurotrophins in OSCC metastasis, PNI, and pain. The expression data in OSCC patients with metastasis, PNI, or pain demonstrate dysregulation of neurotrophin genes. TrkA and nerve growth factor receptor (NGFR) are focused, two receptors that are activated by NGF, a neurotrophin expressed at high levels in OSCC. It is demonstrated that targeted knockdown of these two receptors inhibits proliferation and invasion in an in vitro and preclinical model of OSCC, and metastasis, PNI, and pain. It is further determined that TrkA knockdown alone inhibits thermal hyperalgesia, whereas NGFR knockdown alone inhibits mechanical allodynia. Collectively the results highlight the ability of OSCC to co-opt different components of the neurotrophin pathway in metastasis, PNI, and pain.