An Investigation into the In Vitro Targeted Killing of CD44-Expressing Triple-Negative Breast Cancer Cells Using Recombinant Photoimmunotherapeutics Compared to Auristatin-F-Based Antibody-Drug Conjugates.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors. Therefore, an αCD44(scFv)-SNAP-tag antibody fusion protein was engineered through genetic fusion of a single-chain antibody fragment (scFv) to a SNAPf-tag fusion protein, capable of self-conjugating with benzylguanine-modified light-sensitive near-infrared (NIR) phthalocyanine dye IRDye700DX (BG-IR700) or the small molecule toxin auristatin-F (BG-AURIF). Binding of the αCD44(scFv)-SNAPf-IR700 photoimmunoconjugate to antigen-positive cells was demonstrated by confocal microscopy and flow cytometry. By switching to NIR irradiation, CD44-expressing TNBC was selectively killed through induced phototoxic activities. Likewise, the αCD44(scFv)-SNAPf-AURIF immunoconjugate was able to selectively accumulate within targeted cells and significantly reduced cell viability through antimitotic activities at nano- to micromolar drug concentrations. This study provides an in vitro proof-of-concept for a future strategy to selectively destroy light-accessible superficial CD44-expressing TNBC tumors and their metastatic lesions which are inaccessible to therapeutic light.

Mechanism of DAPK1 for Regulating Cancer Stem Cells in Thyroid Cancer.

Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine (Ser/Thr) protein kinase and is characteristically downregulated in metastatic cancer. Several studies showed that DAPK1 is involved in both the early and late stages of cancer. DAPK1 downregulation is elaborately controlled by epigenetic, transcriptional, posttranscriptional, and posttranslational processes. DAPK1 is known to regulate not only cancer cells but also stromal cells. Recent studies showed that DAPK1 was involved not only in tumor suppression but also in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) formation in colon and thyroid cancers. CSCs are major factors in determining cancer aggressiveness in cancer metastasis and treatment prognosis by influencing EMT. However, the molecular mechanism involved in the regulation of cancer cells by DAPK1 remains unclear. In particular, little is known about the existence of CSCs and how they are regulated in papillary thyroid carcinoma (PTC) among thyroid cancers. In this review, we describe the molecular mechanism of CSC regulation by DAPK1 in PTC progression.

Unveiling Paclitaxel-Induced Mesenchymal Stem Cells: orchestrating Nrf2 Modulation and Apoptosis in CD44+/CD24- Cancer Stem Cells.

Background: Mesenchymal Stem Cells (MSCs) and Cancer Stem Cells (CSC) play pivotal roles in cancer progression and therapeutic responses. This study aimed to explored the effect of MSCs induced by paclitaxel on CSC expressing the CD44+/CD24- phenotype, focusing on Nrf2 modulation and apoptosis induction. Methods: MSCs were characterized for adherence, differentiation potential, and surface markers via standard culture, staining assays, and flow cytometry, respectively. CSCs isolated from MDA-MB-231 using MACS and were characterized based on morphology and CD44+/CD24- expression. Co-culture experiments evaluated the cytotoxic effect of Paclitaxel-induced MSCs on CSC viability using MTT assays. Flow cytometry analysis assessed apoptosis induction via annexin V-PI staining and Nrf2 and Caspase-3 gene expression were measure by qRT-PCR analysis. Results: MSCs exhibited typical adherence and differentiation capabilities, confirming their mesenchymal lineage. CSCs displayed an elongated morphology and expressed CD44+/CD24-, characteristic of stem-like behavior. Paclitaxel induced dose-dependent Nrf2 gene expression in MSCs. Co-culture with Paclitaxel-induced MSCs reduced CSC viability in a dose-dependent manner, with a significant decrease observed at a 5:1 MSCs:CSC ratio. Co-culture decreased the Nrf2 gene expression and increased apoptosis in CSCs, with higher caspase-3 gene expression compared to solitary paclitaxel treatment. Conclusion: Paclitaxel-induced MSCs decreased Nrf2 expression and significantly decreased CSC viability while enhancing apoptosis. This suggests a potential strategy to mitigate paclitaxel resistance in CD44+/CD24- CSCs. Leveraging Paclitaxel-induced MSCs presents a promising avenue for targeting Nrf2 and promoting apoptosis in CSCs, potentially improving the efficacy of chemotherapy and addressing resistance mechanisms in cancer treatment.

The Scr and Csc pathways for sucrose utilization co-exist in E. coli, but only the Scr pathway is widespread in other Enterobacteriaceae.

Most Escherichia coli isolates from humans do not utilize D-sucrose as a substrate for fermentation or growth. Previous work has shown that the Csc pathway allows some E. coli to utilize sucrose for slow growth, and this pathway has been engineered in E. coli W strains to enhance use of sucrose as a feedstock for industrial applications. An alternative sucrose utilization pathway, Scr, was first identified in Klebsiella pneumoniae and has been reported in some E. coli and Salmonella enterica isolates. We show here that the Scr pathway is native to an important subset of E. coli phylogroup B2 lineages that lack the Csc pathway but grow rapidly on sucrose. Laboratory E. coli strains derived from MG1655 (phylogroup A, ST10) are unable to utilize sucrose and lack the scr and csc genes, but a recombinant plasmid-borne scr locus enables rapid growth on and fermentation of sucrose. Genome analyses of Enterobacteriaceae indicate that the scr locus is widespread in other Enterobacteriaceae; including Enterobacter and Klebsiella species, and some Citrobacter and Proteus species. In contrast, the Csc pathway is limited mostly to E. coli, some Shigella species (in which csc loci are rendered non-functional by various mutations), and Citrobacter freundii. The more efficient Scr pathway likely has greater potential than the Csc pathway for bioindustrial applications of E. coli and other Enterobacteriaceae using sucrose as a feedstock.

TSP50 facilitates breast cancer stem cell-like properties maintenance and epithelial-mesenchymal transition via PI3K p110α mediated activation of AKT signaling pathway.

BACKGROUND: Studies have confirmed that epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties are conducive to cancer metastasis. In recent years, testes-specific protease 50 (TSP50) has been identified as a prognostic factor and is involved in tumorigenesis regulation. However, the role and molecular mechanisms of TSP50 in EMT and CSC-like properties maintenance remain unclear. METHODS: The expression and prognostic value of TSP50 in breast cancer were excavated from public databases and explored using bioinformatics analysis. Then the expression of TSP50 and related genes was further validated by quantitative RT-PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). In order to investigate the function of TSP50 in breast cancer, loss- and gain-of-function experiments were conducted, both in vitro and in vivo. Furthermore, immunofluorescence (IF) and immunoprecipitation (IP) assays were performed to explore the potential molecular mechanisms of TSP50. Finally, the correlation between the expression of TSP50 and related genes in breast cancer tissue microarray and clinicopathological characteristics was analyzed by IHC. RESULTS: TSP50 was negatively correlated with the prognosis of patients with breast cancer. TSP50 promoted CSC-like traits and EMT in both breast cancer cells and mouse xenograft tumor tissues. Additionally, inhibition of PI3K/AKT partly reversed TSP50-induced activation of CSC-like properties, EMT and tumorigenesis. Mechanistically, TSP50 and PI3K p85α regulatory subunit could competitively interact with the PI3K p110α catalytic subunit to promote p110α enzymatic activity, thereby activating the PI3K/AKT signaling pathway for CSC-like phenotypes maintenance and EMT promotion. Moreover, IHC analysis of human breast cancer specimens revealed that TSP50 expression was positively correlated with p-AKT and ALDH1 protein levels. Notably, breast cancer clinicopathological characteristics, such as patient survival time, tumor size, Ki67, pathologic stage, N stage, estrogen receptor (ER) and progesterone receptor (PR) levels, correlated well with TSP50/p-AKT/ALDH1 expression status. CONCLUSION: The effects of TSP50 on EMT and CSC-like properties promotion were verified to be dependent on PI3K p110α. Together, our study revealed a novel mechanism by which TSP50 facilitates the progression of breast cancer, which can provide new insights into TSP50-based breast cancer treatment strategies.

Targeting hyaluronan metabolism-related molecules associated with resistant tumor-initiating cells potentiates chemotherapy efficacy in lung cancer.

The success of chemotherapy regimens in patients with non-small cell lung cancer (NSCLC) could be restricted at least in part by cancer stem cells (CSC) niches within the tumor microenvironment (TME). CSC express CD133, CD44, CD47, and SOX2, among other markers and factors. Analysis of public data revealed that high expression of hyaluronan (HA), the main glycosaminoglycan of TME, correlated positively with CSC phenotype and decreased disease-free interval in NSCLC patients. We aimed to cross-validate these findings on human and murine lung cancer cells and observed that CD133 + CSC differentially expressed higher levels of HA, HAS3, ABCC5, SOX2, and CD47 (p < 0.01). We modulated HA expression with 4-methylumbelliferone (4Mu) and detected an increase in sensitivity to paclitaxel (Pa). We evaluated the effect of 4Mu + chemotherapy on survival, HA metabolism, and CSC profile. The combination of 4Mu with Pa reduced the clonogenic and tumor-forming ability of CSC. Pa-induced HAS3, ABCC5, SOX2, and CD47 expression was mitigated by 4Mu. Pa + 4Mu combination significantly reduced in vivo tumor growth, enhancing animal survival and restoring the CSC profile in the TME to basal levels. Our results suggest that HA is involved in lung CSC phenotype and chemosensitivity, and its modulation by 4Mu improves treatment efficacy to inhibit tumor progression.

MUC1-C Dependence for the Progression of Pancreatic Neuroendocrine Tumors Identifies a Druggable Target for the Treatment of This Rare Cancer.

Patients with pancreatic neuroendocrine tumors (pNETs) have limited access to effective targeted agents and invariably succumb to progressive disease. MUC1-C is a druggable oncogenic protein linked to driving pan-cancers. There is no known involvement of MUC1-C in pNET progression. The present work was performed to determine if MUC1-C represents a potential target for advancing pNET treatment. We demonstrate that the MUC1 gene is upregulated in primary pNETs that progress with metastatic disease. In pNET cells, MUC1-C drives E2F- and MYC-signaling pathways necessary for survival. Targeting MUC1-C genetically and pharmacologically also inhibits self-renewal capacity and tumorigenicity. Studies of primary pNET tissues further demonstrate that MUC1-C expression is associated with (i) an advanced NET grade and pathological stage, (ii) metastatic disease, and (iii) decreased disease-free survival. These findings demonstrate that MUC1-C is necessary for pNET progression and is a novel target for treating these rare cancers with anti-MUC1-C agents under clinical development.

Synthesis and characterization of a novel TiO2@chitosan/alginate nanocomposite sponge for highly efficient removal of As(V) ions from aqueous solutions: Adsorption isotherm, kinetics, experiment and adsorption mechanism optimization using Box-Behnken design.

This research uses a novel TiO2@CSC.Alg composite sponge was created by encasing TiO2 nanoparticles in the natural polymers alginate and chitosan, resulting in a nanocomposite that is both ecologically friendly and biocompatible. Using the generated nanocomposite as a new environmentally friendly adsorbent, As(V) heavy metal ions were effectively removed from aqueous media. The following techniques were used to analyse the physicochemical properties of the obtained materials: pHZPC, FTIR, XRD, BET, SEM, and XPS. Utilizing nitrogen adsorption/desorption isotherms, the TiO2@CSC.Alg composite sponge's textural properties were identified. This revealed a BET surface area of 168.42 m2/g and a total pore volume of 1.18 cc/g, indicating its porous nature and potential for high adsorption capacity. Examine the effects of temperature, pH, dose, and beginning concentration on adsorption. The adsorption characteristics were determined based on equilibrium and adsorption kinetics measurements. The adsorption process was both pseudo-second-order (PSOE) and Langmuir isothermally fit. Chemisorption was the adsorption method since the adsorption energy was 25.45 kJ·mol-1. An endothermic and spontaneous adsorption process was indicated by more metal being absorbed as the temperature increased. The optimal conditions for adsorption were optimized via Box-Behnken design software to be pH of 5 in the solution, a dosage of 0.02 g of the TiO2@CSC.Alg composite sponge per 25 mL, and an arsenate (As(V)) solution the adsorption capacity was 202.27 mg/g are ideal for efficient adsorption. These parameters are critical in achieving the maximum adsorption capacity of the composite sponge for arsenate, which could be beneficial for water purification applications. Utilizing Design-Expert software's response surface methodology (RSM) and Box-Behnken design (BBD), the adsorption process was optimized with the fewest planned tests. After six successive cycles of adsorption and desorption, the adsorbent stability was confirmed by the adsorbent reusability test without any noticeable decrease in removal efficacy. Additionally, it displayed good efficiency, the same XRD and XPS data before and after reuse, and no change in chemical composition.

Quantitative optical coherence tomography angiography assessments in the fellow eyes of those with central serous chorioretinopathy.

BACKGROUND: To analyze the vessel density (VD) of the retina and choriocapillaris (CC) layer and the structure of the foveal avascular zone (FAZ) in the fellow eyes of central serous chorioretinopathy (CSC) patients by using optical coherence tomography angiography (OCTA). METHODS: This was a case-control study. Unilateral CSC patients and age-matched healthy subjects were recruited from the Affiliated Eye Hospital of Wenzhou Medical University between July 2016 and July 2021. All eyes were divided into three groups: acute CSC (aCSC), chronic CSC (cCSC), and healthy controls. Both aCSC and cCSC were again divided into two subgroups: the affected eyes and the fellow eyes. In this study, all parameters of VD and FAZ were measured by self-software of OCTA. RESULTS: A total of 231 eyes of 137 subjects were included, with 47 aCSC patients, 47 cCSC patients, and 43 healthy controls. In the fellow eyes of CSC, the retinal VD was significantly lower (all P < 0.05), and the FAZ was significantly larger (all P < 0.05) in the cCSC group than in healthy controls, while no difference was detected in the CC layer. There was no significant difference between the aCSC group and healthy controls in all OCTA parameters. In the affected eyes of CSC, the superficial retinal vessel density (SRVD) was significantly higher (all P < 0.05) in healthy controls than in the aCSC and cCSC groups, while the deep retinal vessel density (DRVD) was significantly lower (all P < 0.05) and the FAZ was larger (all P < 0.05) in the cCSC group than in the aCSC group and healthy controls. A liner regression equation was established: Y (BCVA, best corrected visual acuity) = 3.692-0.036✱X1 (DRVD-Fovea)-0.031✱X2 (FD-300, vessel density around the 300 µm width of the FAZ), R2 = 0.427. CONCLUSION: Based on OCTA measurements, this study revealed that the retinal microvascular network was impaired even in the fellow eyes of those with cCSC, which should arouse attention to the observation of unilateral CSC.

Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis.

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. We found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5G43R + S49T+S54P+S80A+Y88F. Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

CircMALAT1 promotes cancer stem-like properties and chemoresistance via regulating Musashi-2/c-Myc axis in esophageal squamous cell carcinoma.

The primary challenge in treating esophageal squamous cell carcinoma (ESCC) is resistance to chemotherapy. Cancer stem cell (CSC) is the root cause of tumor drug resistance. Therefore, targeting CSCs has been considered promising therapeutic strategy for tumor treatment. Here, we report that circMALAT1 was significantly upregulated in ESCC CSC-like cells and primary tumors from ESCC patients. Clinically, there was a positive correlation between circMALAT1 expression and ESCC stage and lymph node metastasis, as well as poor prognosis for ESCC patients. In vitro and in vivo functional studies revealed that circMALAT1 promoted CSC-like cells expansion, tumor growth, lung metastasis and drug resistance of ESCC. Mechanistically, circMALAT1 directly interacted with CSC-functional protein Musashi RNA Binding Protein 2 (MSI2). CircMALAT1 inhibited MSI2 ubiquitination by preventing it from interacting with ß-transducin repeat containing protein (BTRC) E3 ubiquitin ligase. Also, circMALAT1 knockdown inhibited the expression of MSI2-regulating CSC-markers c-Myc in ESCC. Collectively, circMALAT1 modulated the ubiquitination and degradation of the MSI2 protein signaling with ESCC CSCs and accelerated malignant progression of ESCC. CircMALAT1 has the potential to serve as a biomarker for drug resistance and as a target for therapy in CSCs within ESCC.

Immunotherapeutic Strategies Targeting Breast Cancer Stem Cells.

Breast cancer is the most commonly diagnosed cancer in women and is a leading cause of cancer death in women worldwide. Despite the implementation of multiple treatment options, including immunotherapy, breast cancer treatment remains a challenge. In this review, we aim to summarize present challenges in breast cancer immunotherapy and recent advancements in overcoming treatment resistance. We elaborate on the inhibition of signaling cascades, such as the Notch, Hedgehog, Hippo, and WNT signaling pathways, which regulate the self-renewal and differentiation of breast cancer stem cells and, consequently, disease progression and survival. Cancer stem cells represent a rare population of cancer cells, likely originating from non-malignant stem or progenitor cells, with the ability to evade immune surveillance and develop resistance to immunotherapeutic treatments. We also discuss the interactions between breast cancer stem cells and the immune system, including potential agents targeting breast cancer stem cell-associated signaling pathways, and provide an overview of the emerging approaches to breast cancer stem cell-targeted immunotherapy. Finally, we consider the development of breast cancer vaccines and adoptive cellular therapies, which train the immune system to recognize tumor-associated antigens, for eliciting T cell-mediated responses to target breast cancer stem cells.

The roles of cancer stem cell-derived secretory factors in shaping the immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide and has a poor prognosis. Although immune checkpoint inhibitors have entered a new era of HCC treatment, their response rates are modest, which can be attributed to the immunosuppressive tumor microenvironment within HCC tumors. Accumulating evidence has shown that tumor growth is fueled by cancer stem cells (CSCs), which contribute to therapeutic resistance to the above treatments. Given that CSCs can regulate cellular and physical factors within the tumor niche by secreting various soluble factors in a paracrine manner, there have been increasing efforts toward understanding the roles of CSC-derived secretory factors in creating an immunosuppressive tumor microenvironment. In this review, we provide an update on how these secretory factors, including growth factors, cytokines, chemokines, and exosomes, contribute to the immunosuppressive TME, which leads to immune resistance. In addition, we present current therapeutic strategies targeting CSC-derived secretory factors and describe future perspectives. In summary, a better understanding of CSC biology in the TME provides a rational therapeutic basis for combination therapy with ICIs for effective HCC treatment.

Chronic sensory contact with subordinated conspecifics promotes splenic glucocorticoid resistance in experimentally wounded C57BL/6N male mice.

Chronic psychosocial stress induced by the chronic subordinate colony housing (CSC, 19 Days) paradigm promotes functional splenic in vitro glucocorticoid (GC) resistance, but only if associated with significant bite wounding or prior abdominal transmitter implantation. Moreover, sensory contact to social defeat of conspecifics represents a social stressor for the observer individual. As the occurence and severity of bite wounding is not adequately controllable, the present study aimed to develop an animal model, allowing a bite wound-independent, more reliable generation of chronically-stressed mice characterized by functional splenic in vitro GC resistance. Therefore, male C57BL/6N mice received a standardized sterile intraperitoneal (i.p.) incision surgery or SHAM treatment one week prior to 19-days of (i) CSC, (ii) witnessing social defeat during CSC exposure in sensory contact (SENS) or (iii) single-housing for control (SHC), before assessing basal and LPS-induced splenic in vitro cell viability and GC resistance. Our results indicate that individually-housed SENS but not CSC mice develop mild signs of splenic in vitro GC resistance, when undergoing prior i.p.-wounding. Taken together and considering that future studies are warranted, our findings support the hypothesis that the combination of repeated standardized i.p.-wounding with chronic sensory stress exposure represents an adequate tool to induce functional splenic in vitro GC resistance independent of the occurrence of uncontrollable bite wounds required in social stress paradigms to induce a comparable phenotype.

Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence.

Introduction: Cancer stem cells (CSCs), a group of tumor-initiating and tumor-maintaining cells, may be major players in the treatment resistance and recurrence distinctive of chordoma. Characterizing CSCs is crucial to better targeting this subpopulation. Methods: Using flow cytometry, six chordoma cell lines were evaluated for CSC composition. In vitro, cell lines were stained for B7H6, HER2, MICA-B, ULBP1, EGFR, and PD-L1 surface markers. Eighteen resected chordomas were stained using a multispectral immunofluorescence (mIF) antibody panel to identify CSCs in vivo. HALO software was used for quantitative CSC density and spatial analysis. Results: In vitro, chordoma CSCs express more B7H6, MICA-B, and ULBP1, assessed by percent positivity and mean fluorescence intensity (MFI), as compared to non-CSCs in all cell lines. PD- L1 percent positivity is increased by >20% in CSCs compared to non-CSCs in all cell lines except CH22. In vivo, CSCs comprise 1.39% of chordoma cells and most are PD-L1+ (75.18%). A spatial analysis suggests that chordoma CSCs cluster at an average distance of 71.51 mm (SD 73.40 mm) from stroma. Discussion: To our knowledge, this study is the first to identify individual chordoma CSCs and describe their surface phenotypes using in vitro and in vivo methods. PD-L1 is overexpressed on CSCs in chordoma human cell lines and operative tumor samples. Similarly, potential immunotherapeutic targets on CSCs, including B7H6, MICA-B, ULBP1, EGFR, and HER2 are overexpressed across cell lines. Targeting these markers may have a preferential role in combating CSCs, an aggressive subpopulation likely consequential to chordoma's high recurrence rate.

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 µg/mL). We found CSC (10-3 or 10 µg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Knockdown of NeuroD2 leads to seizure-like behavior, brain neuronal hyperactivity and a leaky blood-brain barrier in a Xenopus laevis tadpole model of DEE75.

Developmental and Epileptic Encephalopathies (DEE) are a genetically diverse group of severe, early onset seizure disorders. DEE are normally identified clinically in the first six months of life by the presence of frequent, difficult to control seizures and accompanying stalling or regression of development. DEE75 results from de novo mutations of the NEUROD2 gene that result in loss of activity of the encoded transcription factor, and the seizure phenotype was shown to be recapitulated in Xenopus tropicalis tadpoles. We used CRISPR/Cas9 to make a DEE75 model in Xenopus laevis, to further investigate the developmental etiology. NeuroD2.S CRISPR/Cas9 edited tadpoles were more active, swam faster on average, and had more seizures (C-shaped contractions resembling unprovoked C-start escape responses) than their sibling controls. Live imaging of Ca2+ signaling revealed prolongued, strong signals sweeping through the brain, indicative of neuronal hyperactivity. While the resulting tadpole brain appeared grossly normal, the blood-brain barrier (BBB) was found to be leakier than that of controls. Additionally, the TGFß antagonist Losartan was shown to have a short-term protective effect, reducing neuronal hyperactivity and reducing permeability of the BBB. Treatment of NeuroD2 CRISPant tadpoles with 5 mM Losartan decreased seizure events by more than 4-fold compared to the baseline. Our results support a model of DEE75 resulting from reduced NeuroD2 activity during vertebrate brain development, and indicate that a leaky BBB contributes to epileptogenesis.

Single-cell transcriptomics reveals the intra-tumoral heterogeneity and SQSTM1/P62 and Wnt/ß-catenin mediated epithelial to mesenchymal transition and stemness of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/ß-catenin in TNBC progression. Wnt/ß-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT. Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/ß-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/ß-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.

Double-Layered Hollow Mesoporous Cuprous Oxide Nanoparticles for Double Drug Sequential Therapy of Tumors.

Cancer stem cells (CSCs) are one of the determinants of tumor heterogeneity and are characterized by self-renewal, high tumorigenicity, invasiveness, and resistance to various therapies. To overcome the resistance of traditional tumor therapies resulting from CSCs, a strategy of double drug sequential therapy (DDST) for CSC-enriched tumors is proposed in this study and is realized utilizing the developed double-layered hollow mesoporous cuprous oxide nanoparticles (DL-HMCONs). The high drug-loading contents of camptothecin (CPT) and all-trans retinoic acid (ATRA) demonstrate that the DL-HMCON can be used as a generic drug delivery system. ATRA and CPT can be sequentially loaded in and released from CPT3@ATRA3@DL-HMCON@HA. The DDST mechanisms of CPT3@ATRA3@DL-HMCON@HA for CSC-containing tumors are demonstrated as follows: 1) the first release of ATRA from the outer layer induces differentiation from CSCs with high drug resistance to non-CSCs with low drug resistance; 2) the second release of CPT from the inner layer causes apoptosis of non-CSCs; and 3) the third release of Cu+ from DL-HMCON itself triggers the Fenton-like reaction and glutathione depletion, resulting in ferroptosis of non-CSCs. This CPT3@ATRA3@DL-HMCON@HA is verified to possess high DDST efficacy for CSC-enriched tumors with high biosafety.