Embryonic Pathways Research Articles

Pan‐Cancer Single‐Cell Transcriptomic Analysis Reveals Divergent Expression of Embryonic Proangiogenesis Gene Modules in Tumorigenesis

ABSTRACTBackgroundAngiogenesis is indispensable for the sustained survival and progression of both embryonic development and tumorigenesis. This intricate process is tightly regulated by a multitude of pro‐angiogenic genes. The presence of gene modules facilitating angiogenesis has been substantiated in both embryonic development and the context of tumor proliferation. However, it remains unresolved whether the pro‐angiogenic gene modules expressed during embryonic development also exist in tumors.MethodsThis study performed a pan‐cancer single‐cell RNA sequencing (scRNA‐seq) analysis on samples from 332 patients across seven cancer types: thyroid carcinoma, lung cancer, breast cancer, hepatocellular carcinoma, colorectal cancer, ovarian carcinoma, and prostate adenocarcinoma. Data processing was carried out using the Seurat R package, with rigorous quality control to filter high‐quality cells and mitigate batch effects across datasets. We used principal component analysis (PCA), shared nearest neighbor graph‐based clustering, and Uniform Manifold Approximation and Projection (UMAP) to visualize cell types and identify distinct cell clusters. Myeloid cell subpopulations were further analyzed for the expression of embryonic pro‐angiogenic gene modules (EPGM) and tumor pro‐angiogenic gene modules (TPGM).ResultsThe analysis identified nine major cell types within the tumor microenvironment, with myeloid cells consistently exhibiting elevated expression of both tumor pro‐angiogenic gene modules (TPGM) and EPGM across all tumor types. In particular, myeloid cells, including macrophages and monocytes, showed high EPGM expression, indicating an active role of embryonic pro‐angiogenesis pathways in tumors. A subset analysis revealed 20 distinct myeloid subtypes with varying EPGM and TPGM expression across different cancers. Treatment and disease stage influenced these gene expressions, with certain subtypes, such as HSPAhi/STAT1+ macrophages in breast cancer, displaying reduced pro‐angiogenic gene activity post‐treatment.ConclusionThis study provides evidence that tumors may exploit EPGM to enhance vascularization and support sustained growth, as evidenced by the elevated EPGM expression in tumor‐associated myeloid cells. The consistent presence of EPGM in TAMs across multiple cancer types suggests a conserved mechanism wherein tumors harness embryonic angiogenic pathways to facilitate their progression. Distinct EPGM expression patterns in specific myeloid cell subsets indicate potential therapeutic targets, particularly in cases where EPGM activation contributes to resistance against anti‐angiogenic therapies. These findings shed new light on the molecular mechanisms underlying tumor angiogenesis and highlight the prognostic relevance of EPGM expression in cancer, underscoring its potential as a biomarker for clinical applications.

4D pathology: translating dynamic epithelial tubulogenesis to prostate cancer pathology.

The Gleason score is the gold standard for grading of prostate cancer (PCa) and is assessed by assigning specific grades to different microscopical growth patterns. Aside from the Gleason grades, individual growth patterns such as cribriform architecture were recently shown to have independent prognostic value for disease outcome. PCa grading is performed on static tissue samples collected at one point in time, whereas in vivo epithelial tumour structures are dynamically invading, branching and expanding into the surrounding stroma. Due to the lack of models that are able to track human PCa microscopical developments over time, our understanding of underlying tissue dynamics is sparse. We postulate that human PCa expansion utilizes embryonic and developmental tubulogenetic pathways. The aim of this study is to provide a comprehensive overview of developmental pathways of normal epithelial tubule formation, elongation, and branching, and relate those to the static microscopical PCa growth patterns observed in daily clinical practise. This study could provide a rationale for the discerned pathological interobserver variability and the clinical outcome differences between PCa growth patterns.

Beyond the Spectrum: Subtype-Specific Molecular Insights into Autism Spectrum Disorder Via Multimodal Data Integration.

Some toddlers with autism spectrum disorder (ASD) have mild social symptoms and developmental improvement in skills, but for others, symptoms and abilities are moderately or even severely affected. Those with profound autism have the most severe social, language, and cognitive symptoms and are at the greatest risk of having a poor developmental outcome. The little that is known about the underlying biology of this important profound autism subtype, points clearly to embryonic dysregulation of proliferation, differentiation and neurogenesis. Because it is essential to gain foundational knowledge of the molecular biology associated with profound, moderate, and mild autism clinical subtypes, we used well-validated, data-driven patient subtyping methods to integrate clinical and molecular data at 1 to 3 years of age in a cohort of 363 ASD and controls representative of the general pediatric population in San Diego County. Clinical data were diagnostic, language, cognitive and adaptive ability scores. Molecular measures were 50 MSigDB Hallmark gene pathway activity scores derived from RNAseq gene expression. Subtyping identified four ASD, typical and mixed diagnostic clusters. 93% of subjects in one cluster were profound autism and 93% in a different cluster were control toddlers; a third cluster was 76% moderate ability ASD; and the last cluster was a mix of mild ASD and control toddlers. Among the four clusters, the profound autism subtype had the most severe social symptoms, language, cognitive, adaptive, social attention eye tracking, social fMRI activation, and age-related decline in abilities, while mild autism toddlers mixed within typical and delayed clusters had mild social symptoms, and neurotypical language, cognitive and adaptive scores that improved with age compared with profound and moderate autism toddlers in other clusters. In profound autism, 7 subtype-specific dysregulated gene pathways were found; they control embryonic proliferation, differentiation, neurogenesis, and DNA repair. To find subtype-common dysregulated pathways, we compared all ASD vs TD and found 17 ASD subtype-common dysregulated pathways. These common pathways showed a severity gradient with the greatest dysregulation in profound and least in mild. Collectively, results raise the new hypothesis that the continuum of ASD heterogeneity is moderated by subtype-common pathways and the distinctive nature of profound autism is driven by the differentially added profound subtype-specific embryonic pathways.

Abstract We046: Building Mass or Strength: Stage-specific Effects of Insulin and Wnt on Cell Fate Decisions in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose: This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods: We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results: Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions: Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.

Stage specific effects of insulin and Wnt on cell fate decisions in human induced pluripotent stem cell-derived cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation Background Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.Graphical Summary

Circulating microRNAs and cardiomyocyte proliferation in heart failure patients related to 10years survival.

Mechanochemical signalling drives organogenesis and is highly conserved in mammal evolution. Regaining recovery in myocardial jeopardy by inducing principles linking cardiovascular therapy and clinical outcome has been the dream of scientists for decades. Concepts involving embryonic pathways to regenerate adult failing hearts became popular in the early millennium. Since then, abundant data on stem cell research have been published, never reaching widespread application in heart failure therapy. Another conceptual access, using mechanotransduction in cardiac veins to limit myocardial decay, is pressure-controlled intermittent coronary sinus occlusion (PICSO). Recently, we reported acute molecular signs and signals of PICSO activating regulatory miRNA and inducing cell proliferation mimicking cardiac development in adult failing hearts. According to a previously formulated hypothesis, 'embryonic recall', this study aimed to define molecular signals involved in endogenous heart repair during PICSO and study their relation to patient survival. We previously reported a study on the acute molecular effects of PICSO in an observational non-randomized study. Eight out of the thirty-two patients with advanced heart failure undergoing cardiac resynchronization therapy (CRT) were treated with PICSO. Survival was monitored over 10years, and coronary sinus blood samples were collected during intervention before and after 20min and tested for miRNA signalling and proliferation when co-cultured with cardiomyocytes. A numerically lower death rate post-CRT and PICSO as compared with control CRT only, and a non-significant reduction in all-cause mortality risk of 42% was observed (37.5% vs. 54.0%, relative risk=0.58, 95% confidence interval: 0.17-2.05; P=0.402). Four miRNAs involved in cell cycle, proliferation, morphogenesis, embryonic development, and apoptosis significantly increased concomitantly in survivors and PICSO compared with a decrease in non-survivors (hsa-miR Let7b, P<0.01; hsa-miR- 421, P<0.006; hsa-miR 363-3p, P<0.03 and hsa-miR 19b-3p P<0.01). In contrast, three miRNAs involved in proliferation and survival, determining cell fate, and recycling endosomes decreased in survivors and PICSO (hsa miR 101-3p, P<0.03; hsa-miR 25-3p, P<002; hsa-miR 30d-5p P<0.04). In vitro cellular proliferation increased in survivors and lowered in non-survivors showing a pattern distinction, discriminating longevity according to up to 10-year survival in heart failure patients. This study proposes that generating regenerative signals observed during PICSO intervention relate to patient outcomes. Morphogenetic pathways induced by periods of flow reversal in cardiac veins in a domino-like pattern transform embryonic into regenerative signals. Studies supporting the conversion of mechanochemical signals into regenerative molecules during PICSO are warranted to substantiate predictive power on patient longevity, opening new therapeutic avenues in otherwise untreatable heart failure.

Mechanisms of fibrous cap formation in atherosclerosis.

The fibrous cap is formed by smooth muscle cells that accumulate beneath the plaque endothelium. Cap rupture is the main cause of coronary thrombosis, leading to infarction and sudden cardiac death. Therefore, the qualities of the cap are primary determinants of the clinical outcome of coronary and carotid atherosclerosis. In this mini-review, we discuss current knowledge about the formation of the fibrous cap, including cell recruitment, clonal expansion, and central molecular signaling pathways. We also examine the differences between mouse and human fibrous caps and explore the impact of anti-atherosclerotic therapies on the state of the fibrous cap. We propose that the cap should be understood as a neo-media to substitute for the original media that becomes separated from the surface endothelium during atherogenesis and that embryonic pathways involved in the development of the arteria media contribute to cap formation.

Ex vivo mapping of enhancer networks that define the transcriptional program driving melanoma metastasis.

Mortality from vmelanoma is associated with metastatic disease, but the mechanisms leading to spreading of the cancer cells remain obscure. Spatial profiling revealed that melanoma is characterized by a high degree of heterogeneity, which is established by the ability of melanoma cells to switch between different phenotypical stages. This plasticity, likely a heritage from embryonic pathways, accounts for a relevant part of the metastatic potential of these lesions, and requires the rapid and efficient reorganization of the transcriptional landscape of melanoma cells. A large part of the non-coding genome cooperates to control gene expression, specifically through the activity of enhancers (ENHs). In this study, we aimed to identify ex vivo the network of active ENHs and to outline their cooperative interactions in supporting transcriptional adaptation during melanoma metastatic progression. We conducted a genome-wide analysis to map active ENHs distribution in a retrospective cohort of 39 melanoma patients, comparing the profiles obtained in primary (N = 19) and metastatic (N = 20) melanoma lesions. Unsupervised clustering showed that the profile for acetylated histone H3 at lysine 27 (H3K27ac) efficiently segregates lesions into three different clusters corresponding to progressive stages of the disease. We reconstructed the map of super-ENHs (SEs) and cooperative ENHs that associate with metastatic progression in melanoma, which showed that cooperation among regulatory elements is a mandatory requirement for transcriptional plasticity. We also showed that these elements carry out specialized and non-redundant functions, and indicated the existence of a hierarchical organization, with SEs on top as masterminds of the entire transcriptional program and classical ENHs as executors. By providing an innovative vision of how the chromatin landscape of melanoma works during metastatic spreading, our data also point out the need to integrate functional profiling in the analysis of cancer lesions to increase definition and improve interpretation of tumor heterogeneity.

An early midbrain sensorimotor pathway is involved in the timely initiation and direction of swimming in the hatchling Xenopus laevis tadpole.

Vertebrate locomotion is heavily dependent on descending control originating in the midbrain and subsequently influencing central pattern generators in the spinal cord. However, the midbrain neuronal circuitry and its connections with other brainstem and spinal motor circuits has not been fully elucidated. Vertebrates with very simple nervous system, like the hatchling Xenopus laevis tadpole, have been instrumental in unravelling fundamental principles of locomotion and its suspraspinal control. Here, we use behavioral and electrophysiological approaches in combination with lesions of the midbrain to investigate its contribution to the initiation and control of the tadpole swimming in response to trunk skin stimulation. None of the midbrain lesions studied here blocked the tadpole's sustained swim behavior following trunk skin stimulation. However, we identified that distinct midbrain lesions led to significant changes in the latency and trajectory of swimming. These changes could partly be explained by the increase in synchronous muscle contractions on the opposite sides of the tadpole's body and permanent deflection of the tail from its normal position, respectively. We conclude that the tadpole's embryonic trunk skin sensorimotor pathway involves the midbrain, which harbors essential neuronal circuitry to significantly contribute to the appropriate, timely and coordinated selection and execution of locomotion, imperative to the animal's survival.

Phenotypic Spectrum of NFIA Haploinsufficiency: Two Additional Cases and Review of the Literature.

The NFIA (nuclear factor I/A) gene encodes for a transcription factor belonging to the nuclear factor I family and has key roles in various embryonic differentiation pathways. In humans, NFIA is the major contributor to the phenotypic traits of "Chromosome 1p32p31 deletion syndrome". We report on two new cases with deletions involving NFIA without any other pathogenic protein-coding gene alterations. A cohort of 24 patients with NFIA haploinsufficiency as the sole anomaly was selected by reviewing the literature and public databases in order to analyze all clinical features reported and their relative frequencies. This process was useful because it provided an overall picture of the phenotypic outcome of NFIA haploinsufficiency and helped to define a cluster of phenotypic traits that can facilitate clinicians in identifying affected patients. NFIA haploinsufficiency can be suspected by a careful observation of the dysmorphisms (macrocephaly, craniofacial, and first-finger anomalies), and this potential diagnosis is strengthened by the presence of intellectual and developmental disabilities or other neurodevelopmental disorders. Further clues of NFIA haploinsufficiency can be provided by instrumental tests such as MRI and kidney urinary tract ultrasound and confirmed by genetic testing.

Abstract B081: Synergistic paracrine action of Vitamin D and mesenchymal stem cell secretome in targeting pancreatic cancer stem cells

Abstract The knowledge of immunomodulatory and reparative properties of Mesenchymal Stem cells (MSCs) are rapidly advancing and could be best suited as a therapeutic option for inflammatory or adverse immunological conditions such as cancer. In the last decade, Mesenchymal stem cells have attracted significant attention because of their accessibility, tumor-oriented homing capacity and the transplantation feasibility. Until date, MSC-based therapy for pancreatic cancer has not been demonstrated. As per the current understanding of the potentials of MSCs in regenerative medicine, we hypothesized their inhibitory influence on pancreatic cancer cells. For experimentation purposes, we have used human Wharton’s jelly derived MSCs (hWJ-MSCs) and pancreatic cell line models. Our findings show that secretion of soluble biologically active factors from hWJ-MSCs (secretome) had a greater impact on induction of anergy and apoptosis of pancreatic tumor cells. The mechanistic approach directed to influence the tumor microenvironment appear to be two pronged: one, via modulation of tumor glycoproteins (MUC-1 and EpCAM) and second, via GSK3β/Stat3/HIF1-α axis. Treatment with calcitriol, an analogue of Vitamin D known to regulate cell cycle, cell differentiation ca also reduce side effects of chemo-drugs, one of the mainstay of therapy in pancreatic cancer. Yet, the potential mechanism to exert anti-cancer effect remains underexplored. We attempted to elicit the involvement of VitD in one of the crucial stem-cell related embryonic pathway, the Sonic Hedgehog (SHH) pathway. Our data showed enhanced apoptosis of calcitirol treated-pCSCs cells when treated with salinomycin (SHH inhibitor). Further combinatorial treatment with secretome + VitD decreased copy numbers of transcription factors C-myc, SMO, PTCH1, PTCH2, and hypoxia-induced factor (HIF1-α) demonstrating the potential mechanism of action. In conclusion, our experimental evidence postulates a potential mechanism by which VitD-analogue regulate sHH-mTOR axis and in combination with mesenchymal stem cell secretome can affect the proliferation and migratory (oncogenic) capabilities of pancreatic cancer stem cells. So, what next? Do we have the evidence to suggest secretome as a novel cell-free therapeutic candidate!! Citation Format: Sangeeta Choudhury, Neha Chopra, Kriti Jain, Poonam Yadav, Surinder P. Singh, Yashika Charla. Synergistic paracrine action of Vitamin D and mesenchymal stem cell secretome in targeting pancreatic cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B081.

Profiling Analysis of N6-Methyladenosine mRNA Methylation Reveals Differential m6A Patterns during the Embryonic Skeletal Muscle Development of Ducks.

Simple SummaryRecent studies show that N6-methyladenosine (m6A) modification, the most common RNA chemical modification, influences the modification, processing, transport, and translation of RNA. N6-methyladenosine is an epigenetic modification that influences skeletal myogenesis and skeletal muscle development. However, the N6-methyladenosine modification profile and its function during poultry muscle development is unclear, and there is only one report about m6A modification in ducks, which focuses on duck hepatitis A virus infection. Here, we compared the m6A modification profiles between E13 (embryonic day 13) and E19 (embryonic day 19) in duck breast muscle differentiation using MeRIP-seq, and evaluated the expression profile of the methyl transferase METTL14 and its cofactors during breast muscle development. This is the first study of N6-methyladenosine modification patterns in duck muscle tissue. The current study not only elucidates the regulation mechanisms of duck skeletal muscle development, but also lays the groundwork for studying the role of RNA modification in poultry muscle development.N6-Methyladenosine is a reversible epigenetic modification that influences muscle development. However, the m6A modification profile during poultry skeletal muscle development is poorly understood. Here, we utilized m6A-specific methylated RNA immunoprecipitation sequencing to identify m6A sites during two stages of breast muscle development in ducks: embryonic days 13 (E13) and E19. MeRIP-seq detected 19,024 and 18,081 m6A peaks in the E13 and E19 groups, respectively. Similarly to m6A distribution in mammalian transcripts, our results revealed GGACU as the main m6A motif in duck breast muscle; they also revealed that m6A peaks are mainly enriched near the stop codons. In addition, motif sequence analysis and gene expression analysis demonstrated that m6A modification in duck embryo skeletal muscles may be mediated by the methyltransferase-like 14. GO and KEGG analysis showed that m6A peaks containing genes at E19 were mainly enriched in muscle-differentiation- and muscle-growth-related pathways, whereas m6A peaks containing genes in E13 were mainly enriched in embryonic development and cell proliferation pathways. Combined analysis of MeRIP-seq and RNA-seq showed that the mRNA expression may be affected by m6A modification. Moreover, qRT-PCR analysis of the expression of METTL14 and its cofactors (WTAP, ZC3H13, RBM15 and VIRMA) during duck embryonic skeletal muscle development in breast and leg muscle samples revealed a significant downward trend as the developmental age progressed. Our results demonstrated that m6A mRNA methylation modifications control muscle development in ducks. This is the first study of m6A modification patterns in duck muscle tissue development, and it lays the foundation for the study of the effects of RNA modification on poultry skeletal muscle development.

Potential methylation-regulated genes and pathways in hepatocellular neoplasm, not otherwise specified.

Background and AimsThe molecular basis of hepatocellular neoplasm, not otherwise specified (HCN-NOS) is unknown. We aimed to identify gene expression patterns, potential methylation-regulated genes and pathways that characterize the tumor, and its possible relationship to hepatoblastoma and hepatocellular carcinoma (HCC).Approach & ResultsParallel genome-wide profiling of gene expression (RNAseq) and DNA methylation (EPIC850) was performed on 4 pairs of pre-treatment HCN-NOS tumors and adjacent non-tumor controls. 2530 significantly differentially expressed genes (DEGs) were identified between tumors and controls. Many of these DEGs were associated with hepatoblastoma and/or HCC. Analysis Match in Ingenuity Pathway Analysis determined that the gene expression profile of HCN-NOS was unique but significantly similar to that of both hepatoblastoma and HCC. A total of 27,195 CpG sites (CpGs) were significantly differentially methylated (DM) between tumors and controls, with a global hypomethylation pattern and predominant CpG island hypermethylation in promotor regions. Aberrant DNA methylation predominated in Developmental Process and Molecular Function Regulator pathways. Embryonic stem cell pathways were significantly enriched. In total, 1055 aberrantly methylated (at CpGs) and differentially expressed genes were identified, including 25 upstream regulators and sixty-one potential CpG island methylation-regulated genes. Eight methylation-regulated genes (TCF3, MYBL2, SRC, HMGA2, PPARGC1A, SLC22A1, COL2A1 and MYCN) had highly consistent gene expression patterns and prognostic value in patients with HCC, based on comparison to publicly available datasets.ConclusionsHCN-NOS has a unique, stem-cell like gene expression and DNA methylation profile related to both hepatoblastoma and HCC but distinct therefrom. Further, 8 methylation-regulated genes associated with prognosis in HCC were identified.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-β and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.

How maternal age and environmental cues influence embryonic developmental pathways and diapause dynamics in a North American annual killifish.

Temporary pools are variable environments with seasonal drought/flood phases. Annual killifish have adapted to life in temporary pools by producing embryos that undergo diapause to traverse the dry phase. To fill existing knowledge gaps about embryo diapause regulation and evolution in annual killifishes, we test the effect of maternal age, incubation temperature, and incubation medium on diapause induction and length in Millerichthys robustus, the only North American fish species that has evolved an annual life history. All embryos at extreme temperatures follow a defined developmental pathway: skipping diapause at 30°C, and entering diapause at 18°C, both regardless of maternal age, and incubation medium. However, maternal age, and incubation medium influenced whether diapause is entered, and time arrested in diapause for embryos incubated at 25°C. At 25°C, five-week-old, and 52-week-old females produced more embryos that entered diapause than 26-week-old females. Also, embryos incubated in aqueous medium skipped diapause more frequently at this intermediate temperature. Millerichthys developmental dynamics associated with maternal age under intermediate range of temperatures are likely adapted to the particular patterns of flood/drought in North American temporary pools. Millerichthys also exhibits developmental patterns largely comparable with other annual fishes, probably due to common seasonal patterns in temporary pools.

Embryonic Programs in Cancer and Metastasis-Insights From the Mammary Gland.

Cancer is characterized as a reversion of a differentiated cell to a primitive cell state that recapitulates, in many aspects, features of embryonic cells. This review explores the current knowledge of developmental mechanisms that are essential for embryonic mouse mammary gland development, with a particular focus on genes and signaling pathway components that are essential for the induction, morphogenesis, and lineage specification of the mammary gland. The roles of these same genes and signaling pathways in mammary gland or breast tumorigenesis and metastasis are then summarized. Strikingly, key embryonic developmental pathways are often reactivated or dysregulated during tumorigenesis and metastasis in processes such as aberrant proliferation, epithelial-to-mesenchymal transition (EMT), and stem cell potency which affects cellular lineage hierarchy. These observations are in line with findings from recent studies using lineage tracing as well as bulk- and single-cell transcriptomics that have uncovered features of embryonic cells in cancer and metastasis through the identification of cell types, cell states and characterisation of their dynamic changes. Given the many overlapping features and similarities of the molecular signatures of normal development and cancer, embryonic molecular signatures could be useful prognostic markers for cancer. In this way, the study of embryonic development will continue to complement the understanding of the mechanisms of cancer and aid in the discovery of novel therapeutic targets and strategies.

Abstract 3164: Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells

Abstract Multiple myeloma (MM) is the second most common hematological malignancy characterized by a malignant expansion of abnormal antibody-producing cells. Despite available treatments, relapsed disease is a common occurrence with patients receiving survival predictions of less than one year. Relapsed MM is often refractory to multiple lines of therapy and occurs due to the uncontrolled regeneration of a population of malignant cells displaying progenitor-cell-like phenotypes. Selectively targeting this cancer stem cell (CSC) population provides an effective way to limit incidence of relapsed disease, improving the prognosis for high-risk patients. In MM, the defining characteristics of CSCs remain poorly understood, though signaling pathways promoting ‘stemness’ that are often deregulated in CSCs, such as interleukin-6 (IL-6) signaling, as well as canonical and non-canonical Wnt signaling are also aberrantly activated in MM. In this context, the central objective of this project was to explore the functionality of two proteins which have been shown to be downstream targets of IL-6/JAK2/STAT3 signaling and potentiate Wnt signaling, the embryonic G-protein coupled receptor LGR4 and the fetal-oncogene tyrosine kinase like orphan receptor-1 (ROR1), as CSC biomarker candidates, and explore the feasibility of CSC targeting therapeutics for MM. Receptor expression was assessed by quantitative PCR and flow cytometry in primary plasma cell dyscrasia patient samples across the spectrum of disease, as well as in human myeloma cell lines (HMCLs). Whole transcriptome sequencing analyses were carried out in HMCLs induced to overexpress LGR4, which enriched genes associated with ion transport and embryonic development. Cell adhesion and extracellular matrix genes were downregulated, along with genes associated with metabolic and mitochondrial pathways. Results of ROR1 gene expression and flow cytometry studies suggest that a subset of MM patient samples harbor a low-frequency ROR1-positive cell population, and expression is enriched in an in vitro model of acquired drug resistance. Overexpression of interferon regulatory factor-4 (IRF4) - a myeloma cell-enriched transcription factor that we recently showed promotes myeloma regeneration - also contributed to increased human ROR1 protein levels. HMCLs with high endogenous ROR1 expression were sensitive to in vitro killing by anti-ROR1 CAR T-cell treatment, which employs an scFv generated from the fully humanized ROR1-specific monoclonal antibody cirmtuzumab, at effector:target cell ratios (ET) as low as 0.33:1. Together, these results suggest a central role for Wnt signaling in MM stem cell phenotypes and activation of embryonic transcriptional pathways, while also paving the way towards development of clinical therapies that target a CSC population in MM. Citation Format: Lara C. Avsharian, Christopher S. Oh, Ashni A. Vora, Jayde Fernandez, Caitlin Shin, Navyaa Sinha, Charles Prussak, Leslie Crews. Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3164.

Targeting cancer signaling pathways by natural products: Exploring promising anti-cancer agents.

Cancer is one of the leading causes of death and significantly burdens the healthcare system. Due to its prevalence, there is undoubtedly an unmet need to discover novel anticancer drugs. The use of natural products as anticancer agents is an acceptable therapeutic approach due to accessibility, applicability, and reduced cytotoxicity. Natural products have been an incomparable source of anticancer drugs in the modern era of drug discovery. Along with their derivatives and analogs, natural products play a major role in cancer treatment by modulating the cancer microenvironment and different signaling pathways. These compounds are effective against several signaling pathways, mainly cell death pathways (apoptosis and autophagy) and embryonic developmental pathways (Notch pathway, Wnt pathway, and Hedgehog pathway). The historical record of natural products is strong, but there is a need to investigate the current role of natural products in the discovery and development of cancer drugs and determine the possibility of natural products being an important source of future therapeutic agents. Many target-specific anticancer drugs failed to provide successful results, which accounts for a need to investigate natural products with multi-target characteristics to achieve better outcomes. The potential of natural products to be promising novel compounds for cancer treatment makes them an important area of research. This review explores the significance of natural products in inhibiting the various signaling pathways that serve as drivers of carcinogenesis and thus pave the way for developing and discovering anticancer drugs.

Maternal dietary methionine restriction alters the expression of energy metabolism genes in the duckling liver

BackgroundIn mammals, the nutritional status experienced during embryonic development shapes key metabolic pathways and influences the health and phenotype of the future individual, a phenomenon known as nutritional programming. In farmed birds as well, the quantity and quality of feed offered to the dam can impact the phenotype of the offspring. We have previously reported that a 38% reduction in the intake of the methyl donor methionine in the diet of 30 female ducks during the growing and laying periods - from 10 to 51 weeks of age - reduced the body weight of their 180 mule ducklings compared to that of 190 ducklings from 30 control females. The maternal dietary methionine restriction also altered the hepatic energy metabolism studied in 30 of their ducklings. Thus, their plasma glucose and triglyceride concentrations were higher while their plasma free fatty acid level was lower than those measured in the plasma of 30 ducklings from the control group. The objective of this new study was to better understand how maternal dietary methionine restriction affected the livers of their newly hatched male and female ducklings by investigating the hepatic expression levels of 100 genes primarily targeting energy metabolism, amino acid transport, oxidative stress, apoptotic activity and susceptibility to liver injury.ResultsSixteen of the genes studied were differentially expressed between the ducklings from the two groups. Maternal dietary methionine restriction affected the mRNA levels of genes involved in different pathways related to energy metabolism such as glycolysis, lipogenesis or electron transport. Moreover, the mRNA levels of the nuclear receptors PPARGC1B, PPARG and RXRA were also affected.ConclusionsOur results show that the 38% reduction in methionine intake in the diet of female ducks during the growing and egg-laying periods impacted the liver transcriptome of their offspring, which may explain the previously observed differences in their liver energy metabolism. These changes in mRNA levels, together with the observed phenotypic data, suggest an early modulation in the establishment of metabolic pathways.

Molecular mechanisms of developmental pathways in neurological disorders: a pharmacological and therapeutic review.

Developmental signalling pathways such as Wnt/β-catenin, Notch and Sonic hedgehog play a central role in nearly all the stages of neuronal development. The term 'embryonic' might appear to be a misnomer to several people because these pathways are functional during the early stages of embryonic development and adulthood, albeit to a certain degree. Therefore, any aberration in these pathways or their associated components may contribute towards a detrimental outcome in the form of neurological disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and stroke. In the last decade, researchers have extensively studied these pathways to decipher disease-related interactions, which can be used as therapeutic targets to improve outcomes in patients with neurological abnormalities. However, a lot remains to be understood in this domain. Nevertheless, there is strong evidence supporting the fact that embryonic signalling is indeed a crucial mechanism as is manifested by its role in driving memory loss, motor impairments and many other processes after brain trauma. In this review, we explore the key roles of three embryonic pathways in modulating a range of homeostatic processes such as maintaining blood-brain barrier integrity, mitochondrial dynamics and neuroinflammation. In addition, we extensively investigated the effect of these pathways in driving the pathophysiology of a range of disorders such as Alzheimer's, Parkinson's and diabetic neuropathy. The concluding section of the review is dedicated to neurotherapeutics, wherein we identify and list a range of biological molecules and compounds that have shown enormous potential in improving prognosis in patients with these disorders.