Expression Of Sox2 Research Articles

MOLECULAR CLONING AND QUANTITATIVE mRNA EXPRESSION OF sox9 GENE IN GONADAL DEVELOPMENTAL PERIOD OF HYPOATHERINA TSURUGAE

Sox9 is a transcription factor of high mobility group (HMG) box family DNA binding domain. It plays a crucial role in gonadogenesis during embryonic developmental period. 1454 bp of sox9 mRNA transcript of Hypoatherina tsurugae (D. S. Jordan & Starks) was cloned and sequenced. It consists of an open reading frame (ORF) of 1436 bp, that encodes a 479 aa protein, found to be identical to the HMG box of other fish species. A phylogenetic tree was constructed by comparing the mRNA sequence of 50 different fishes across various taxa available in the NCBI database and using as outgroup Acipenser sinensis. The tree shows a high homology of sox9 from H. tsurugae with that from Maelanotaenia boesemani, the two forming a single clade. The expression of sox9 was studied in amhy+ (male) individuals. It begins from baseline at 0 wah (week after hatching) and is expressed in an increasing fashion. In amhy- (female) individuals it is highly expressed at initial stage (0 wah) and the expression reaches its peak at 2 wah then declines, indicating the low expression needed for differentiation of the female sex organs. The histological sections of gonads were studied in different stages of biweekly collected larvae during the sex determination/differentiation period and it showed that differentiation of gonads male/female is decided at 6 wah. In this stage the primary oocytes are clearly recognized and it correlates with the expression of sox9 genes. These finding add to the knowledge for a better understanding of molecular mechanisms of sex determination and differentiation period in fishes.

Loss in Pluripotency Markers in Mesenchymal Stem Cells upon Infection with Chlamydia trachomatis.

The intracellular pathogen Chlamydia trachomatis can inflict substantial damage on the host. Notably, Chlamydia infection is acknowledged for its precise modulation of diverse host signaling pathways to ensure cell survival, a phenomenon intricately connected to genetic regulatory changes in host cells. To monitor shifts in gene regulation within Chlamydia-infected cells, we employed mesenchymal stem cells (MSCs) as a naïve, primary cell model. Utilizing biochemical methods and imaging, our study discloses that acute Chlamydia infection in human MSCs leads to the downregulation of transcription factors Oct4, Sox2, and Nanog, suggesting a loss of pluripotency markers. Conversely, pluripotency markers in MSCs were sustained through treatment with conditioned medium from infected MSCs. Additionally, there is an augmentation in alkaline phosphatase activity, along with elevated Sox9 and CD44 mRNA expression levels observed during acute infection. A comprehensive screening for specific cell markers using touchdown PCR indicates an upregulation of mRNA for the early chondrogenesis gene Sox9 and a decrease in mRNA for the MSC marker vimentin. Real-time PCR quantification further corroborates alterations in gene expression, encompassing increased Sox9 and CD44 mRNA levels, alongside heightened alkaline phosphatase activity. In summary, the infection of MSCs with C. trachomatis induces numerous genetic deregulations, implying a potential trend towards differentiation into chondrocytes. These findings collectively underscore a targeted impact of Chlamydia on the gene regulations of host cells, carrying significant implications for the final fate and differentiation of these cells.

TP8, A Novel Chondroinductive Peptide, Significantly Promoted Neo-Cartilage Repair without Activating Bone Formation.

The repair of large cartilage defects remains highly challenging in the fields of orthopedics and oral and maxillofacial surgery. A chondroinductive agent is promising to activate endogenous mesenchymal stem cells (MSCs) so as to facilitate cartilage regeneration. In this study, we analyzethe crystallographic data of the critical binding domain of transforming growth factor β3 (TGF-β3) with its type II receptor and successfully develop a novel chondroinductive peptide - TGF-β3-derived peptide No. 8 (TP8) that can induce an ectopic cartilage formation without obvious bone formation. TP8 shows a comparable capacity as TGF-β3 in enhancing glycosaminoglycans (GAGs) and proteoglycans (PGs) secretion in the micromass of bone marrow MSCs (BMSCs) and promoting the expression of chondrogenic markers in comparison with the Control group. TP8 induces a significantly higher expression of the SRY-box transcription factor 9 (Sox9) gene than TGF-β3. Moreover, TP8 significantly upregulates the phosphorylation of Smad1/5 but not MAPK/JNK or Smad 2/3. The knockdown of low-density lipoprotein receptor (LDLR) -related protein-1 (Lrp1), a transmembrane endocytosis receptor, nullifies the TP8-induced Sox9 expression. In the critical-size cartilage defects in rabbit medial femoral condyles, TP8 can induce neo-cartilage formation with a significantly thicker deep zone in comparison with the TGF-β3 and Control. These findings suggest a promising application potential of TP8 in cartilage tissue engineering.

Clinicopathological significance of SOX2 and FOXG1 expression patterns in ovarian immature teratomas

Objective: To investigate the relationship between the expression patterns of SOX2 and FOXG1 and the differentiation/development level of neural components in immature teratoma and to determine the clinical significance and potential application of this correlation in a clinical setting. Methods: We conducted a comprehensive whole transcriptome sequencing analysis to identify differentially expressed genes (DEGs) across various subtypes of ovarian germ cell tumors. Additionally, immunohistochemical staining of paraffin-embedded tissue sections was employed to assess the nuclear staining pattern of SOX2 and FOXG1 proteins within the tumor tissues. Results: The transcriptome sequencing data showed that transcription factors SOX2 and FOXG1 exhibited high levels of expression typically in immature teratoma and occupied a pivotal position within the protein-protein interaction network. Immunohistochemical staining revealed the absence of both SOX2 and FOXG1 protein expression in dysgerminoma and yolk sac tumor samples. In immature teratoma, immunohistochemical staining demonstrated diffuse expression of SOX2 and FOXG1 proteins within the inner layer of densely-arranged primitive neuroepithelial tubules. This pattern of expression suggested the presence of stem cell-like properties within these tumor cells. In the sparsely peripheral neurogliocytes, FOXG1 maintained a diffuse nuclear staining pattern resembling that of neuroepithelial cells, while SOX2 exhibited a scattered pattern of positive staining, hinting at a neural lineage differentiation potential. This spatial differential expression pattern of SOX2 and FOXG1 proteins in immature teratoma suggested that primitive neural components within these tumors often recapitulated the trajectory of neural formation and cortical development that was typically observed during embryogenesis. The primitive neural tube acted as the center that constantly moved from inside to outside, with a dynamic shift from the interior to the exterior, paralleled by the sequential differentiation of cell lineages from primitive neuroepithelial stem cells to radial glia, intermediate progenitor cells, and ultimately to precursor glia. Conclusions: This spatial expression pattern of SOX2 and FOXG1 proteins observed in immature teratoma mirrors the lineage differentiation and migration trajectories of primitive neuroepithelial components typically seen in embryonic neurogenesis and cortical development. In daily practice, the combined application of SOX2 and FOXG1 SOX2 and FOXG1 helps identify the primitive neuroepithelial components in immature teratoma, avoid misjudgment of similar morphologies, and thereby assist in the histological grading and clinical decision-making.

Dysregulation of the p53 pathway provides a therapeutic target in aggressive pediatric sarcomas with stem-like traits.

Pediatric sarcomas are bone and soft tissue tumors that often exhibit high metastatic potential and refractory stem-like phenotypes, resulting in poor outcomes. Aggressive sarcomas frequently harbor a disrupted p53 pathway. However, whether pediatric sarcoma stemness is associated with abrogated p53 function and might be attenuated via p53 reactivation remains unclear. We utilized a unique panel of pediatric sarcoma models and tumor tissue cohorts to investigate the correlation between the expression of stemness-related transcription factors, p53 pathway dysregulations, tumorigenicity in vivo, and clinicopathological features. TP53 mutation status was assessed by next-generation sequencing. Major findings were validated via shRNA-mediated silencing and functional assays. The p53 pathway-targeting drugs were used to explore the effects and selectivity of p53 reactivation against sarcoma cells with stem-like traits. We found that highly tumorigenic stem-like sarcoma cells exhibit dysregulated p53, making them vulnerable to drugs that restore wild-type p53 activity. Immunohistochemistry of mouse xenografts and human tumor tissues revealed that p53 dysregulations, together with enhanced expression of the stemness-related transcription factors SOX2 or KLF4, are crucial features in pediatric osteosarcoma, rhabdomyosarcoma, and Ewing's sarcoma development. p53 dysregulation appears to be an important step for sarcoma cells to acquire a fully stem-like phenotype, and p53-positive pediatric sarcomas exhibit a high frequency of early metastasis. Importantly, reactivating p53 signaling via MDM2/MDMX inhibition selectively induces apoptosis in aggressive, stem-like Ewing's sarcoma cells while sparing healthy fibroblasts. Our results indicate that restoring canonical p53 activity provides a promising strategy for developing improved therapies for pediatric sarcomas with unfavorable stem-like traits.

Aging negatively affects postoperative recovery of biomechanical strength through decreased recruitment of Scx+/Sox9+ enthesis-related progenitors after rotator cuff repair in rats

BackgroundAlthough older adult patients have a higher retear rate after rotator cuff (RC) repair, the influence of aging on the healing process remains unclear. During mouse enthesis development, a multipotent progenitor co-expressing scleraxis (Scx) and SRY-box 9 (Sox9) contributes to enthesis formation. Scx+/Sox9+ cells may act as enthesis progenitors even during postnatal healing, and their number decreases with maturation. However, the pathophysiology of decreased RC healing ability due to aging remains unclear. We aimed to evaluate the effects of aging on tendon-to-bone healing after surgical RC repair in rats through biomechanical and histological analyses of Scx+ and Sox9+ progenitors in a ScxGFP transgenic rat model. MethodsThis was a controlled laboratory study. Adult Sprague-Dawley rats (n = 111) underwent unilateral surgery for supraspinatus tendon repair immediately after transection. The rats were divided into aged (95 weeks old) and control (12 weeks old) groups. The effects of aging were assessed using biomechanical tests at 6 weeks postoperatively and histologically at 2 and 6 weeks postoperatively. ScxGFP transgenic rats were used for the immunohistochemical assessment of Scx- and Sox9-expressing progenitors during the repair process. Sox9, Scx, and tenomodulin expression was assessed using real-time reverse transcription polymerase chain reaction. ResultsIn the biomechanical test at 6 weeks postoperatively, the aged group had lower ultimate load to failure (Control: 20.4 ± 6.1 N, Aged: 14.9 ± 6.6 N, P = 0.007), stiffness (Control: 16.1 ± 6.2 N/mm, Aged: 12.6 ± 5.5 N/mm, P = 0.023), and ultimate stress to failure (Control: 6.0 ± 3.4 N/mm2, Aged: 3.4 ± 2.5 N/mm2, P < 0.001) than the control group. The total histological score of the aged group was lower than that of the control group at 6 weeks postoperatively (Control: 8.8 ± 0.8, Aged: 5.8 ± 0.4, P = 0.029). Immunohistochemistry tests showed that the percentages of Sox9+ (Control: 6.6 ± 1.1, Aged: 2.0 ± 1.0, P = 0.029) and Scx+/Sox9+ (Control: 3.6 ± 0.8, Aged: 1.1 ± 0.6, P = 0.029) cells at the reparative site were lower in the aged group than in the control group at 2 weeks postoperatively. ConclusionIn a rat RC tendon-to-bone healing model, the decreased recruitment of Scx+/Sox9+ enthesis-related progenitor cells in the early phase may be associated with delayed reparative tissue remodeling in aging animals. These findings encourage the development of therapeutic strategies that biologically promote healing and reduce postoperative retears in older adult patients.

Astrocytic Regulation of Cocaine Locomotor Sensitization in EcoHIV Infected Mice

Cocaine use disorder (CUD) is highly comorbid with HIV infection and worsens HIV outcomes. Preclinical research on the outcomes of HIV infection may yield crucial information on neurobehavioral changes resulting from chronic drug exposure in people living with HIV (PLWH). Repeated exposure to cocaine alters behavioral responses to cocaine. This includes development of cocaine locomotor sensitization - or increased locomotor responses to the same doses of cocaine - which depends on nucleus accumbens (NAc) neural plasticity. NAc astrocytes are key regulators of neural activity and plasticity, and their function can be impaired by cocaine exposure and HIV infection, thus implicating them as potential regulators of HIV-induced changes in behavioral response to cocaine. To characterize the effects of HIV infection on cocaine locomotor sensitization, we employed the EcoHIV mouse model in male and female mice to assess changes in locomotor responses after repeated cocaine (10mg/kg) exposure and challenge. EcoHIV infection potentiated expression of cocaine sensitization. We also identified EcoHIV-induced increases in expression of the astrocytic nuclear marker Sox9 selectively in the NAc core. To investigate whether modulation of NAc astrocytes could reverse EcoHIV-induced deficits, we employed a chemogenetic approach. We found that chemogenetic activation of NAc astrocyte Gq signaling attenuated EcoHIV-enhanced cocaine sensitization. We propose that HIV infection contributes to cocaine behavioral sensitization and induces adaptations in NAc astrocytes, while promoting NAc astrocytic Gq-signaling can recover EcoHIV-induced behavioral changes. These findings identify potential cellular substrates of disordered cocaine-driven behavior in the context of HIV infection and point toward strategies to reduce cocaine-related behavior in PLWH.

Alpha-lipoic acid targets KLF7 expression to inhibit cervical cancer progression.

It is unclear what part KLF7 plays in cervical cancer. In this study, immunohistochemical and bioinformatics analyses reveal that KLF7 expression is lower in normal cervical tissues than in cervical cancer tissues ( P≤0.05), and the high level of KLF7 transcripts in cervical cancer tissues is negatively correlated with patients' overall and disease-free survival ( P<0.05). In addition, KLF7 overexpression facilitates the proliferation, migration, and invasion of cervical cells, reduces PFKL expression, and increases the expressions of KLF4, Nanog, OCT4, CD44, SOX2, and ACADL ( P<0.05). Additionally, knocking out the Exon 2 of KLF7 in HeLa cells results in a decrease in the total expression of KLF7 but an increase in the nuclear expression of KLF7, an increase in the capacity for proliferation, migration, invasion, and oncogenicity ( P<0.05), and an increase in the density and ridge density of mitochondria. Consistent with these findings, RNA-seq analysis shows that knocking out the Exon 2 of KLF7 facilitates the expression of gene sets associated with cancer compared with that in wild-type HeLa cells ( P<0.05). Moreover, the administration of alpha-lipoic acid (ALA) leads to a reduction in KLF7 expression in cells and tumor tissues, a suppression of the proliferation, migration, and invasion of HeLa and SiHa cells ( P<0.05), and an increase in the carcinogenic potential of HeLa cells ( P<0.05), while KLF7 overexpression shows the opposite effect on the expressions of ACADL and PFKL in HeLa and SiHa cells. In conclusion, KLF7 promotes the development of cervical cancer, and ALA can downregulate KLF7 expression and play a positive role in cervical cancer treatment.

Association of SOX10 Immunohistochemical Expression with Triple Negative Breast cancer

Abstract Background: The term triple-negative breast cancer (TNBC) refers to a particular class of aggressive, poorly differentiated neoplasms that show the absence of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor2 (HER2) antibodies. The aim of this study is to assess the immunohistochemical expression of SOX10 in TNBC, its correlation with tumor grade and other clinicopathologic characteristics. Materials and methods: A Cross sectional study which included thirty paraffin blocks of primary (TNBC) , that were collected from Karbala teaching centre of histopathology in the period (from Jan. 2023 to Dec. 2023). For each of the collected block, two sections were taken. One stained by hematoxylin and eosin and the other was stained by immunohistochemical method for SOX10. Results: SOX10 reported positive in 43% (13) positive cases versus 57% (17) negative cases, this positivity was significantly correlated with pathological grade (p value =0.012) and the pathological stage (p value =0.038). A particular statistical significance was found in association with the lymph node group and distant metastasis. (p value 0.039 and 0.037) respectively. No significant statistical difference between SOX10 expression and the patient’s age, tumor size, presence of Ductal carcinoma in situ (DCIS) and lymphovascular invasion. Conclusion: The study suggested that SOX10 expression is a prognostic biomarker for TNBC. It also highlighted the clinical significance of targeting SOX10 as a promising potential therapeutic target gene for TNBC therapy. Keywords: SOX10; triple negative breast cancer; immunohistochemistry.

Duhuo Jisheng Decoction in reduction of inflammatory response via Transforming growth factor-β/Smad signaling pathway for repairing rabbit articular cartilage Injury: A Randomized Controlled Trial

ObjectiveThis study aims to investigate the mechanism underlying the effect of Duhuo Jisheng Decoction on the repair of rabbit articular cartilage injury through a reduction in the inflammatory response mediated by the Transforming growth factor (TGF)-β/Smad signaling pathway. MethodsA rabbit articular cartilage injury model was constructed using a ring bone extraction drill. Twenty-four Japanese white rabbits were randomly divided into six groups, namely Sham operation, model, low-dose Duhuo Jisheng Decoction, medium-dose Duhuo Jisheng Decoction, high-dose Duhuo Jisheng Decoction, and positive control groups. The treatment lasted 12 weeks. Gross observation, International Cartilage Repair Society score, Wakitani score, and Micro-computed tomography analysis were used to evaluate the structural repair of cartilage injury. Histology and immunohistochemistry were used to observe the proteoglycan, P-TβRII, P-Smad2, and type II collagen expression levels. Enzyme-linked immunosorbent assay was used to analyze the concentrations of Matrix Metalloproteinase-13 and Syndecan-4 in the joint fluid; and RT-PCR and Western Blot were used to observe the mRNA and protein expressions of ALK5, Sox-9, P-Smad3, and TGF-β1 at the injury repair site. ResultsThe repair effect of cartilage injury, as seen through gross observation and quantitative scoring, was better in all the Duhuo Jisheng Decoction treatment groups than in the model group. The medium dose group of Duhuo Jisheng Decoction had the best repair effect. We observed remarkable structural restoration of cartilage injury in the medium-dose Duhuo Jisheng Decoction group, with the subchondral bone presenting a distinct hierarchy, and parameters such as bone volume fraction and trabecular separation/spacing being significantly augmented. We found high expression levels of proteoglycans, P-TβRII, P-Smad2, and type II collagen. The concentrations of Matrix Metalloproteinase-13 and Syndecan-4 in the joint fluid were significantly lower following treatment. The low gene expression levels of ALK5, Sox-9, P-Smad3, and TGF-β1 in the injury site of the model group could be reversed in the medium-dose Duhuo Jisheng Decoction group. ConclusionDuhuo Jisheng Decoction can repair rabbit cartilage injury and reverse the levels of inflammatory factors in the joint fluid. The mechanism underlying its therapeutic effect is related to the activation of the TGF-β/Smad signaling pathway. This study provides a reliable basis for using Duhuo Jisheng Decoction to treat cartilage injury following knee osteoarthritis.

The role of the SOX2 gene in cervical cancer: focus on ferroptosis and construction of a predictive model

BackgroundThe intricate interplay between stemness markers and cell death pathways significantly influences the pathophysiology of cervical cancer. SOX2, a pivotal regulator of stem cell pluripotency, has recently been implicated in the modulation of ferroptosis, a specialized form of iron-dependent cell death, in cancer dynamics. This study delineates the role of SOX2 in the ferroptotic landscape of cervical carcinoma.ObjectiveTo delineate the association between SOX2 expression and ferroptosis in cervical cancer and develop a robust, SOX2-centric model for predicting prognosis and enhancing personalized treatment.MethodsA multidimensional approach integrating advanced bioinformatics, comprehensive molecular profiling, and state-of-the-art machine learning algorithms was employed to assess SOX2 expression patterns and their correlation with ferroptosis marker expression patterns in cervical cancer tissues. A prognostic model incorporating the expression levels of SOX2 and ferroptosis indicators was meticulously constructed.ResultsThis investigation revealed a profound and intricate correlation between SOX2 expression and ferroptotic processes in cervical cancer, substantiated by robust molecular evidence. The developed predictive model based on SOX2 expression exhibited superior prognostic accuracy and may guide therapeutic decision-making.ConclusionThis study underscores the critical role of SOX2 in orchestrating the ferroptosis pathway in cervical cancer and presents a novel prognostic framework. The SOX2-centric predictive model represents a significant advancement in prognosis evaluation, offering a gateway to personalized treatment for gynaecologic cancers.

Short-range Fgf signalling patterns hindbrain progenitors to induce the neurogenesis-to-oligodendrogenesis switch.

In the vertebrate nervous system, neurogenesis generally precedes gliogenesis. The mechanisms driving the switch in cell type production and generation of the correct proportion of cell types remain unclear. Here, we show that Fgf20 signalling patterns progenitors to induce the switch from neurogenesis to oligodendrogenesis in the zebrafish hindbrain. Fgf20 emanating from earlier-born neurons signals at a short range to downregulate proneural gene expression in the segment centre with high spatial precision along both anterior-posterior (AP) and dorsal-ventral (DV) axes. This signal induces oligodendrocytes in the segment centre by upregulating olig2 and sox10 expression in pre-patterned competent progenitors. We show that the magnitude of proneural gene downregulation and the quantity of OPCs specified is dependent on the extent of Fgf20 signalling. Overexpression of fgf20a induces precocious specification and differentiation of oligodendrocytes among olig2+ progenitors, resulting in an increase in oligodendrocytes at the expense of neurogenesis. Thus, Fgf20 signalling defines the proportion of each cell type produced. Taken together, Fgf20 signalling from earlier-born neurons patterns hindbrain segments spatially and temporally to induce the neurogenesis-to-oligodendrogenesis switch.

Rhaponticin suppresses the stemness phenotype of gastric cancer stem-like cells CD133+/CD166 + by inhibiting programmed death-ligand 1

BackgroundGastric cancer stem cells (GCSCs) are key contributors to tumorigenesis, recurrence and metastasis, complicating gastric cancer (GC) treatment. Rhaponticin (RA), a potential novel anticancer drug, has unexplored effects on GCSCs.MethodsGCSCs were isolated using CD133 and CD166 markers with magnetic bead separation method and then evaluated their response to the IC50 concentrations of RA (16.90 µg/mL for BGC-823 and 22.18 µg/mL for SGC-7901), and effects on cell proliferation, migration, invasion, and stemness were measured. We analyzed the GCSC-related microarray dataset GSE111556 and explored RA’s role in restoring programmed cell death ligand 1 (PD-L1) function in CD133+/CD166 + cells post-PD-L1 knockdown. RA’s impact on tumour growth and immune microenvironment was assessed in a xenograft mouse model.ResultsThe CD133+/CD166 + subpopulation exhibited stem-like characteristics, with the highest proportion in BGC-823 (38.85%) and SGC-7901 (43.81%) cells. These cells formed tumour spheres and had increased expression of stemness markers Sox2 and Oct-4 (compared to the parental cell line, P < 0.001). RA treatment showed no toxicity to normal GES-1 cells but reduced the viability of CD133+/CD166 + cells in a dose-dependent manner, with IC50 values of 16.90 µg/ml for BGC-823 and 22.18 µg/ml for SGC-7901. RA also decreased the proportion of CD133+/CD166 + cells and their stem-like properties (P < 0.001). Analysis of the GEO database identified PD-L1 as a key target gene of RA, with high expression in GC tissues. Knocking down PD-L1 in CD133+/CD166 + cells and introducing RA did not significantly change PD-L1 expression (P>0.05), suggesting RA’s effect may be PD-L1 dependent. In a xenograft mouse model, the tumour size in the RA treatment group was approximately one-sixth that of the CD133+/CD166 + group (P < 0.001). Post-RA treatment, there was an elevation in the expression levels of CD4 and CD8, alongside a reduction in PD-L1 expression (P < 0.001).ConclusionsRA suppresses GCSC stem - like phenotype by inhibiting PD - L1 and enhancing T cell tumour infiltration in the studied models. These findings suggest that RA may have potential for further exploration as a candidate for GC treatment, but extensive preclinical and clinical studies are required to determine its true therapeutic value.

Abstract C026: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

Abstract Epithelial-to-mesenchymal transition (EMT) plays important roles during development, injury repair, cancer invasion and metastasis. The heterogeneity of EMT programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. Through in vivo selection, we established dormant metastasis models from human and mouse early-stage LUAD cell lines. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers, whereas cells derived from overt metastases express high level of the late-stage progenitor marker Sox9. Using a TGF-β signaling reporter, we showed that in the primitive progenitors, TGF-β induces growth inhibition accompanied by a classic EMT response that subsequently transitions into an atypical mesenchymal state of round morphology as the disseminated cells establish dormancy. Prolonged treatment with TGF-β in vitro recapitulates this transition in these primitive progenitors, but not in the late-stage progenitors. Given this dramatic morphological change, we focused on actin-cytoskeleton regulators and found that TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy. Citation Format: Zhenghan Wang, Yassmin Elbanna, Inês Godet, Lila Peters, George Lampe, Yanyan Chen, Joao Xavier, Morgan Huse, Joan Massagué. TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C026.

Abstract A032: Uncovering the impact of current therapies on LUAD dormant metastasis

Abstract Despite recent therapeutic advances, Lung Adenocarcinoma (LUAD) remains a leading cause of mortality worldwide, with most deaths attributed to metastasis. Metastasis is mediated by disseminated metastasis initiating cells (MICs) that can persist in a dormant state in distant sites for months to years and ultimately drive metastatic relapse, which is clinically incurable. Adjuvant therapy seeks to prevent cancer relapse, however, efforts to improve adjuvant treatments are hindered by an insufficient understanding of the molecular mechanisms supporting the long-term viability of dormant MICs. We have established that primitive LUAD cells characterized by the spontaneous expression of the pluripotency transcription factor SOX2 can enter an immune-evasive dormant state with downregulation of immune response mediators (STING, NK ligands, MHC-I) to evade immune surveillance during dormancy. Now, we hypothesize that dormant and immune evasive progenitor MICs may also escape therapies targeting growth pathways. We have generated latency competent cells (LCCs) that can remain in a latent state for months by isolating progenitor cells from the Kras LSL-G12D/+ ;Trp53 flox/flox mouse model and screening their metastatic potential and progenitor state in the B6 background. LCCs entering a slow-cycling state in response to dormancy signals remain sensitive to targeted KRAS inhibitors but contain a resistant subpopulation that survives. To specifically characterize the progenitor state in these LCC populations, we have developed and validated two strategies to fluorescently mark SOX2 expression and activity, respectively. As speculated, the resistant population is mostly composed of SOX2+ progenitor LCCs after treatment with KRAS inhibitor under dormancy signaling. Furthermore, induced-dormancy also drives an accumulation of cells in the SOX2+ progenitor state in these LCC-rich populations. Ongoing studies are focused on delineating the emergence of the SOX2+ progenitor state in LCCs during dormancy and the biological mechanisms driving their persistence and resistance to targeted therapies. We aim to define (1) the dependence of dormant LCCs on oncogenic signaling, the (2) effect of targeted therapy on MICs, and (3) strategies to overcome potential resistance mechanisms in dormant MICs. Our ultimate goal is to delineate strategies to clear latent MICs and prevent LUAD recurrence. Citation Format: Inês Godet, Zhenghan Wang, Joan Massagué. Uncovering the impact of current therapies on LUAD dormant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A032.

Rotating culture regulates the formation of HepaRG-derived liver organoids via YAP translocation

BackgroundLiver organoid serves as an alternative model for liver pathophysiology in carbohydrate or lipid metabolism and xenobiotic metabolism transformation. Biomechanical cues including spaceflight mission can affect liver organoid construction and their related functions, but their underlying mechanisms are not fully understood yet. Here, a rotating cell culture device, namely Rotating Flat Chamber (RFC), was specifically designed for adhering cells or cell aggregated to elucidate the effects of altered gravity vector on HepaRG-derived liver organoids construction.ResultsThe organoids so formed under RFC presented the fast growth rate and large projection area. Meanwhile, the expressions of two pluripotency markers of SOX9 and CD44 were enhanced. This finding was positively correlated with the increased YAP expression and nuclear translocation as well as the elevated α4β6-integrin expression. Inhibition of YAP expression and nuclear translocation decreased the expression of SOX9 and CD44 under RFC, thereby attenuating the pluripotency of HepaRG-derived liver organoids.ConclusionsIn conclusion, we proposed a novel liver organoid construction method using rotating culture, by which the pluripotency of liver organoids so constructed is mediated by α4β6-integrin and YAP translocation. This work furthered the understanding in how the gravity vector orientation affects the construction of liver organoids and the related mechanotransductive pathways.

Abstract 4136598: Osteochondrogenic transdifferentiation of vascular smooth muscle cells and microenvironmental dynamics in medial arterial calcification at single-cell resolution

Background: Medial arterial calcification (MAC) significantly contributes to cardiovascular mortality, yet effective treatments remain limited due to unclear molecular mechanisms. This study aimed to develop a mouse model of MAC using O-ring-induced transverse aortic constriction (OTAC) and to elucidate key genes, pathways, and cellular interactions involved in MAC formation through lineage tracing and single-cell RNA sequencing (scRNA-seq). Methods: An OTAC mouse model was established to replicate MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were conducted at various time points. Histological and immunohistochemical assessments were performed to monitor changes in vascular smooth muscle cells (VSMCs). Lineage tracing was utilized to verify the origin of osteochondrogenic VSMCs. Furthermore, scRNA-seq was employed to capture dynamic changes in VSMCs and other cell types contributing to MAC development. Results: The OTAC model successfully induced osteochondrogenic transdifferentiation of VSMCs, leading to MAC. Immunohistochemical analysis demonstrated time-dependent decreases in α-SMA expression and increases in SOX9 and RUNX2 expression. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic VSMCs originated from contractile VSMCs. scRNA-seq identified osteochondrogenic VSMCs and revealed dynamic changes in surrounding cells, including macrophages and fibroblasts, which were implicated in calcification-related and inflammatory processes. Conclusion: The OTAC method effectively mimicked human MAC pathology, offering comprehensive insights into the microenvironmental dynamics of MAC progression at the single-cell level. This model serves as a robust platform for future research on therapeutic interventions.

Phytosesquiterpene lactones deregulate mitochondrial activity and phenotypes associated with triple-negative breast cancer metastasis

Triple-negative breast cancer (TNBC) recurrence and metastasis are the major causes of failure in TNBC therapy. The difficulties in treating TNBCs may be because of increased cancer cell plasticity that involves the fine-tuning of cellular redox homeostasis, mitochondrial bioenergetics, metabolic characteristics, and the development of cancer stem cells (CSCs). To investigate the effects and the underlying mechanisms of the phytosesquiterpene lactone deoxyelephantopin (DET) and its semi-synthesized derivative (DETD-35) in suppressing different phenotypic TNBC cell populations that contribute to tumor metastasis. A timelapse microfluidic-based system was established to analyze the effects of DETD-35 and DET on cell migration behavior in an oxygen gradient. Seahorse real-time cell metabolic analyzer and gas chromatography/quadrupole-time-of-flight mass spectrometry (GC/Q-TOF MS) were utilized to analyze the effects of the compounds on mitochondrial bioenergetics in TNBC cells. A miRNA knockout technique and miRNA sponges were employed to evaluate the miR-4284 involvement in the anti-TNBC cell effect of either compound. DETD-35 and DET attenuated TNBC cell migration toward hypoxic regions under a 2-19 % oxygen gradient in a timelapse microfluidic-based system. DETD-35 and DET also suppressed CSC-like phenotypes, including the expression of Sox2, Oct4, and CD44 in TNBC cells under hypoxic conditions. DETD-35 and DET affected mitochondrial basal respiration, ATP production, proton leak, and primary metabolism, including glycolysis, the TCA cycle, and amino acid metabolism in the lung-metastatic TNBC cells. Furthermore, the expression of mitophagy markers PARKIN, BNIP3, PINK1, LC3-II, and apoptotic markers Bax, cleaved caspase 7, and cleaved PARP in hypoxic and lung-metastatic TNBC cells was also regulated by treatment with either compound. In miR-4284 knockout cells or miR-4284 inhibitor co-treated TNBC cells, DET- and DETD-35-induced over-expression of mitophagic and apoptotic markers was partially reversed, indicating miR-4284 involved with the compounds caused programmed cell death. This study demonstrated the novel activities of DETD-35 and DET in suppressing CSC-like phenotypes and metastatic TNBC cells through the de-regulation of mitochondrial bioenergetics.

Impact of endogenous viral elements on glioma clinical phenotypes by inducing OCT4 in the host.

Endogenous viral elements (EVEs) are viral sequences integrated within the host genome that can influence gene regulation and tumor development. While EVEs have been implicated in cancer, their role in regulating key transcription factors in glioblastoma (GBM) remains underexplored. This study investigates the relationship between EVEs and the activation of OCT4, a critical transcription factor in GBM progression. We utilized CancerHERVdb and HervD Atlas databases to identify potential interactions between EVEs and key genes involved in GBM. Data from 273 GBM patient samples in the TCGA database were analyzed to examine the correlation between OCT4 expression and mutations in glioma-related genes. Furthermore, glioblastoma stem cells (GSCs) were assessed for the expression levels of OCT4 and SOX2, and Pearson correlation analysis was performed. Our analysis revealed that OCT4 is a pivotal gene activated by EVEs in GBM. OCT4 expression was significantly correlated with mutations in key glioma-associated genes. Higher OCT4 levels were associated with poorer patient prognosis, higher tumor grades, and older age. Additionally, GSCs exhibited elevated expression of both OCT4 and SOX2, with a positive correlation observed between these two genes in GBM patients. This study highlights the potential role of EVEs in driving GBM progression through the activation of OCT4. The findings emphasize the importance of OCT4 in GBM malignancy and suggest that targeting EVE-mediated pathways may provide new therapeutic approaches for GBM treatment.

Morphogen-driven differentiation is precluded by physical confinement in human iPSCs spheroids.

Cell lineage specification is tightly associated with profound morphological changes in the developing human embryo, particularly during gastrulation. The interplay between mechanical forces and biochemical signals is poorly understood. Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model based on spheroids formed from human induced pluripotent stem cells (hiPSCs). First, we compare self-renewing versus differentiating media conditions in free-floating cultures and observe the emergence of tri-germ layers. In these unconfined conditions, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Our study combines high-content imaging, synthetic hydrogels, and hiPSCs-derived models of early development to define the drivers that cause changes in the shape and the emergence of germ layers.