Negative Cells Research Articles

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift.

It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. Within 24 h of adding the small concentration of 1X of NSPS (~7 μM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

Preparation, Optimization and In vitro Evaluation of Doxorubicin-loaded into Hyaluronic Acid Coated Niosomes Against Breast Cancer.

Doxorubicin (DOX) is widely used against solid tumors. Niosomes are self-assembled nanocarriers of non-ionic surfactants. DOX loaded into cationic niosomes (DOX-Nio) was prepared via thin film hydration method. DOX-Nio was then decorated with a hyaluronic acid (DOX-HA-Nio) via electrostatic interaction. DOX-Nio and DOX-HA-Nio displayed a particle size of 120.0±1.02 and 182.9±2.3 nm, and charge of + 35.5±0.15 and -15.6±0.25 mV, respectively, with PDI < 0.3. DOX-HA-Nio showed a good stability regarding size and charge over 4 weeks at 4 °C and maintain their integrity after lyophilization. HPLC results showed a 94.1±4.2 % encapsulation efficiency of DOX with good entrapment and slow, prolonged DOX release even after 48 hrs. Cell viability assay showed an IC50 of 14.26 nM for the DOX-HA-Nio against MCF-7 cell line with micromolar IC50 results against CD-44 negative cell lines (NIH/3T3). DOX-HA-Nio was proven to be an effective, targeted nanocarrier for DOX against MCF-7 cell line.

Clinical and Treatment History of Patients with Partial DiGeorge Syndrome and Autoimmune Cytopenia at Multiple Centers.

Patients with partial DiGeorge syndrome (pDGS) can present with immune dysregulation, the most common being autoimmune cytopenia (AIC). There is a lack of consensus on the approach to type, combination, and timing of therapies for AIC in pDGS. Recognition of immune dysregulation early in pDGS clinical course may help individualize treatment and prevent adverse outcomes from chronic immune dysregulation. Objectives of this study were to characterize the natural history, immune phenotype, and biomarkers in pDGS with AIC. Data on clinical presentation, disease severity, immunological phenotype, treatment selection, and response for patients with pDGS with AIC were collected via retrospective chart review. Flow cytometric analysis was done to assess T and B cell subsets, including biomarkers of immune dysregulation. Twenty-nine patients with the diagnosis of pDGS and AIC were identified from 5 international institutions. Nineteen (62%) patients developed Evan's syndrome (ES) during their clinical course and twenty (69%) had antibody deficiency syndrome. These patients demonstrated expansion in T follicular helper cells, CD19hiCD21lo B cells, and double negative cells and reduction in CD4 naïve T cells and regulatory T cells. First-line treatment for 17/29 (59%) included corticosteroids and/or high-dose immunoglobulin replacement therapy. Other overlapping therapies included eltrombopag, rituximab, and T cell immunomodulators. AIC in pDGS is often refractory to conventional AIC treatment paradigms. Biomarkers may have utility for correlation with disease state and potentially even response to therapy. Immunomodulating therapies could be initiated early based on early immune phenotyping and biomarkers before the disease develops or significantly worsens.

Abstract B072: SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms

Abstract As increasing numbers of MRIs and other scans are ordered for unrelated diseases, more and more people are being diagnosed with cystic neoplasms of the pancreas. While many or most of these will likely remain benign, intraductal papillary mucinous neoplasms (IPMNs) have a risk of progression to cancer and IPMN is now recognized as another precursor to pancreatic ductal adenocarcinoma. We are investigating the mechanisms by which IPMNs progress from benign to malignant disease in order to identify markers of progression and targets to block that progression. We found that loss of SMAD4 in conjunction with Krasmutation leads to IPMN that quickly becomes invasive in a mouse model. Examining human IPMN tissues, we found that only one in 46 benign IPMN tumors lacked detectable SMAD4 protein. Among 19 cases of invasive IPMNs, the sites of active invasion were SMAD4 negative in 7 cases, nuclear negative/cytoplasm positive in 3 cases (suggesting no active canonical signaling), and a mix of positive and negative cells in 3 cases, implicating complete or partial loss of SMAD-mediated signaling in 68% of actively invading cells. In order to understand how SMAD signaling blocks progression to invasion, we are examining cell lines from a benign mouse model of IPMN. While these lines do not have endogenous TGF beta signaling, they are capable of responding to exogenous TGF beta, suggesting that TGF beta-related ligands may be supplied by the tumor microenvironment rather than the tumor cells themselves. We are currently determining how different TGF beta family members contribute to regulation of growth and invasion in these cells. In summary, loss of the tumor suppressor SMAD4 allows an otherwise benign disease to progress to invasive cancer. Citation Format: Anna L. Means, Hannah Yoo, Quoc-Huy Trinh, Roshini Thiagarajan, Jinghuan Zi, Frank L. Revetta, Marcus C. Tan, Kathleen E. DelGiorno, Mary K. Washington. SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B072.

Triphenyltin isoselenocyanate: a novel nuclear retinoid X receptor ligand with antiproliferative and cytotoxic properties in cell lines derived from human breast cancer

Several commercially available triorganotin compounds were previously found to function as agonist ligands for nuclear retinoid X receptor (RXR) molecules. Triphenyltin isoselenocyanate (TPT-NCSe), a novel selenium atom containing a derivative of triorganotin origin, was found to represent a new cognate bioactive ligand for RXRs. TPT-NCSe displayed a concentration- and time-dependent decrease in the cell viability in both human breast carcinoma MCF-7 (estrogen receptor positive) and MDA‑MB‑231 (triple negative) cell lines. Reactive oxygen species levels generated in response to TPT-NCSe were significantly higher in both carcinoma cell lines treated with TPT-NCSe when compared to mock-treated samples. Treatment with 500 nM TPT-NCSe caused a decrease in SOD1 and increased SOD2 mRNA in MCF-7 cells. The levels of SOD2 mRNA were more increased following the treatment with TPT-NCSe along with 1 μM all-trans retinoic acid (AtRA) in MCF-7 cells. An increased superoxide dismutase SOD1 and SOD2 mRNA levels were also detected in combination treatment of 500 nM TPT-NCSe and 1 μM AtRA in TPT-NCSe-treated MDA-MB-231 cells. The data have also shown that TPT-NCSe induces apoptosis via a caspase cascade triggered by the mitochondrial apoptotic pathway. TPT-NCSe modulates the expression levels of apoptosis‑related proteins, Annexin A5, Bcl‑2 and BAX family proteins, and finally, it enhances the expression levels of its cognate nuclear receptor subtypes RXRalpha and RXRbeta.

Inter-Sphincteric Resection of Rectum for Ulcerated S-100 Negative Large Granular Cell Tumour

Non-neural granular cell tumor was first described in 1991 as an unusual primitive, polypoid variant of the conventional GCT (granular cell tumor). To date, this neoplasm remains as a rare entity and the cell of origin is uncertain. While the histological features are similar to the conventional (traditional) GCTs, it represents a distinct entity, as it is negative for S-100 and lacks true nerve sheath differentiation. We report here a case of a 49-year-old male patient with S-100 negative, vimentin positive large benign, ulcerated, bleeding non-neural GCT in the lower rectum. We confirmed the diagnosis histopathologically and immunohistochemically. We treated it by ISRR (Inter-Sphincteric Resection of Rectum) and primary colo-anal anastomosis. We find no other report of non-neural GCT located in the rectum in surgical literature. This is probably the first case of non-neural GCT identified in the rectum. This case report is presented here to document a non-neural GCT located in the rectum for the first time ever reported, and also to highlight the optimum best surgical approach, as we deemed here for this large ulcerated bleeding tumor in the rectum. This rectal non-neural GCT posed here as an unusual differential diagnosis from such more common neoplastic bleeding lesions in the rectum as carcinoma rectum, hemorrhoid, rectal polyp, etc. Here we like to discuss our experience, reviewing in brief the available literature implicating the biological behavior, histopathological and immunohistochemical features, differential diagnosis and therapeutic considerations of neural and non-neural GCTs. KYAMC Journal Volume: 14, No: 03, October 2023: 181-187.

Regulation of mitochondrial metabolism by autophagy supports leptin-induced cell migration

Leptin is an adipokine secreted by adipose tissue, which promotes tumor progression by activating canonical signaling pathways such as MAPK/ERK. Recent studies have shown that leptin induces autophagy, and this process is involved in leptin-induced characteristics of malignancy. Autophagy is an intracellular degradation process associated with different hallmarks of cancer, such as cell survival, migration, and metabolic reprogramming. However, its relationship with metabolic reprogramming has not been clearly described. The purpose of this study was to determine the role of leptin-induced autophagy in cancer cell metabolism and its association with cellular proliferation and migration in breast cancer cells. We used ER+/PR+ and triple-negative breast cancer cell lines treated with leptin, autophagy inhibition, or mitochondrial metabolism inhibitors. Our results show that leptin induces autophagy, increases proliferation, mitochondrial ATP production and mitochondrial function in ER+/PR+ cells. Importantly, autophagy was required to maintain metabolic changes and cell proliferation driven by leptin. In triple-negative cells, leptin did not induce autophagy or cell proliferation but increased glycolytic and mitochondrial ATP production, mitochondrial function, and cell migration. In triple negative cells, autophagy was required to support metabolic changes and cell migration, and autophagy inhibition decreased cellular migration similar to mitochondrial inhibitors. In conclusion, leptin-induced autophagy supports mitochondrial metabolism in breast cancer cells as well as glycolysis in triple negative cells. Importantly, leptin-induced mitochondrial metabolism promoted cancer cell migration.

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)β1. We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFβ1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFβ1-induced fibroblast invasion and RHOA pathway activity. Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFβ1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

The Soft Nanodots as Fluorescent Probes for Cell Imaging: Analysis of Cell and Spheroid Penetration Behavior of Single Chain Polymer Dots.

This study describes the formation, size control, and penetration behavior of polymer nanodots (Pdots) consisting of single or few chain polythiophene-based conjugated polyelectrolytes (CPEs) via nanophase separation between good solvent and poor solvent of CPE. Though the chain singularity may be associated with dilution nanophase separation suggests that molecules of a good solvent create a thermodynamically driven solvation layer surrounding the CPEs and thereby separating the single chains even in their poor solvents. This statement is therefore corroborated with emission intensity/lifetime, particle size, and scattering intensity of polyelectrolyte in good and poor solvents. Regarding the augmented features, Pdots are implemented into cell imaging studies to understand the nuclear penetration and to differentiate the invasive characteristics of breast cancer cells. The python based red, green, blue (RGB) color analysis depicts that Pdots have more nuclear penetration ability in triple negative breast cancer cells due to the different nuclear morphology in shape and composition and Pdots have penetrated cell membrane as well as extracellular matrix in spheroid models. The current Pdot protocol and its utilization in cancer cell imaging are holding great promise for gene/drug delivery to target cancer cells by explicitly achieving the very first priority of nuclear intake.

In silico ADMET profiling of Docetaxel and development of camel milk derived liposomes nanocarriers for sustained release of Docetaxel in triple negative breast cancer

This study aimed at encapsulation of commonly administered, highly cytotoxic anticancer drug Docetaxel (DTX) in camel milk fat globule-derived liposomes for delivery in triple negative breast cancer cells. Prior to liposomal encapsulation of drug, in silico analysis of Docetaxel was done to predict off target binding associated toxicities in different organs. For this purpose, the ADMET Predictor (TM) Cloud version 10.4.0.5, 64-bit, was utilized to simulate Docetaxel’s pharmacokinetic and physicochemical parameters. Freshly milked camel milk was bought from local market, from two breeds Brella and Marecha, in suburbs of Islamabad. After extraction of MFGM-derived liposomes from camel milk, docetaxel was loaded into liposomes by thin film hydration method. The physiochemical properties of liposomes were analyzed by SEM, FTIR and Zeta analysis. The results from SEM showed that empty liposomes (Lp-CM-ChT80) had spherical morphology while DTX loaded liposomes (Lp-CM-ChT80-DTX) exhibited rectangular shape, FTIR revealed the presence of characteristic functional groups which confirmed the successful encapsulation of DTX. Zeta analysis showed that Lp-CM-ChT80-DTX had size of 836.6 nm with PDI of 0.088 and zeta potential of − 18.7 mV. The encapsulation efficiency of Lp-CM-ChT80 turned out to be 25% while in vitro release assay showed slow release of DTX from liposomes as compared to pure DTX using dialysis membrane. The in vitro anticancer activity was analyzed by cell morphology analysis and MTT cytotoxicity assay using different concentrations 80 µg/ml, 120 µg/ml and 180 µg/ml of Lp-CM-ChT80-DTX on MDA-MB-231 cells. The results showed cytotoxic effects increased in time and dose dependent manner, marked by rounding, shrinkage and aggregation of cells. MTT cytotoxicity assay showed that empty liposomes Lp-CM-ChT80 did not have cytotoxic effect while Lp-CM-ChT80-DTX showed highest cytotoxic potential of 60.2% at 180 µg/ml. Stability analysis showed that liposomes were stable till 24 h in solution form at 4 °C.

Abstract A007: Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair

Abstract Ser/Thr kinase BUB1 is known to regulate chromosomal segregation during mitosis. However, its exact role in DNA damage repair in response to radiotherapy remained elusive. Here, we demonstrate that inhibition of BUB1 kinase activity (BAY1816032) or genomic depletion (CRISPR) lead to radiation sensitization in triple negative breast cancer and lung cancer cell lines and in TNBC tumor xenograft models. Mechanistic studies demonstrated reduced DNA-DSB repair (γ-H2AX foci assay), and effect on the NHEJ pathway by the bioluminescent DNA repair reporter (BLRR). Gene expression analysis show that BUB1 expression correlates tightly with key members of the NHEJ machinery including H2AX, ATM, 53BP1, DNAPKcs, Ku70, Ku80, Lig4 among others. Cycloheximide-chase experiments demonstrate that BUB1 ablation (pharmacological or genomic) stabilizes DNAPKcs after irradiation. The half-lives (t1/2) of total-DNAPK, phospho-DNAPK could not be estimated in BUB1 ablated cells post radiotherapy. Conversely, we observed BUB1 stabilization following radiotherapy alone while pre-treatment with BUB1i or DNAPKi caused robust degradation of BUB1 (significantly shorter t1/2). Nuclear and chromatin fractionation assays revealed an increase in the phospho- and total- DNAPK, ATM, and KAP1 in nuclear fractions and in chromatin enriched fractions in BUB1 inhibited or depleted cells. There was no significant change in the levels of Ku70 and Ku80 in the nuclear fractions or in the chromatin enriched fractions. This data indicates that BUB1 may be involved in the activation and recruitment of key NHEJ proteins (ATM, DNAPKcs) to DSBs. Further experiments are underway to determine how BUB1 stabilizes DNAPKcs and regulate chromatin loading of key NHEJ factors following radiation. Additionally, we demonstrate that BUB1 mediate apoptotic cell death of cancer cells post-radiation. Our findings suggest that BUB1 may serve as a novel molecular target for radiosensitization in various solid cancers including TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Stephen Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A007.

Sodium Butyrate as Histone Deacetylase Inhibitor Can Alter miR-101, ZEB1, ZEB2, and E-cadherin Expression in MDA-MB-468 Cells as Triple Negative Breast Cancer Cells

Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype in women worldwide. The various alterations in the expression of different microRNAs (miRNAs) have been reported as crucial in the development of metastasis in breast tumors. Objectives: This study investigated the effect of sodium butyrate (NaB) on cell survival, cell metastasis and expression of miR-101, ZEB1, ZEB2 and E- cadherin in MDA-MB-468 cells as a TNBC cell line. Methods: Cell viability was evaluated using the (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay), and the metastasis potential of MDA-MB-468 cells was investigated using the scratch and transwell assay. The expression of genes involved in the metastasis process was measured using real-time polymerase chain reaction (PCR). Results: The MTT assay showed that NaB attenuated MDA-MB-468 cell survival dose-dependently with an IC50 value of 3.1 mM after 72 h treatment. The scratch and transwell assays also showed the anti-metastatic potential of NaB. The expression of miR-101, E-cadherin, ZEB1, and ZEB2 was significantly difference in MDA-MB-468 cells treated with 3.1 mM NaB after 72 hours (P &lt; 0.05). E-cadherin and miR-101 were up-regulated, while the expression of ZEB1 and ZEB2 was significantly down-regulated compared to the untreated cells. This suggests that NaB increased cell attachment and prevented metastasis. In addition, NaB (IC50 value) restored the expression of miR-101, as a tumor suppressor, in MDA-MB-468 cells confirming its anti-cancer potency. Conclusions: Sodium butyrate can be used as a drug to suppress invasion and cell migration in TNBC cells. However, further studies are needed to demonstrate the putative anti-metastatic mechanism of NaB in preclinical and clinical settings.

Do patients with cN0 oral squamous cell carcinoma benefit from elective neck dissection? A large-scale population-based study

BackgroundThe neck management of clinical-nodal negative (cN0) oral squamous cell carcinoma (OSCC) remains controversial. Elective neck dissection (END) and observation are the main strategies, but it is still not clear who could benefit the most from END. The purpose of this study was to clarify the potential clinical factors that affect the therapeutic value of END and to explore the actual characteristics associated with benefit from END.MethodsPatients with cN0 OSCC were identified in the SEER database from 2000 to 2019. 5-year Overall survival (OS) and disease-specific survival (DSS) were analyzed using the Kaplan‒Meier method, and the hazard ratios (HRs) for survival were estimated using the Cox regression model. Multiple subgroup analyses of DSS and OS among different factors, comparing END and No END, were performed.ResultsA total of 17,019 patients with cN0 OSCC were included. The basic survival analysis and Cox regression model showed that END increased the probability of 5-year DSS and OS and was an independent prognostic factor. However, among patients who underwent only primary tumor surgery, no significant differences were found between the END and No END groups in 5-year DSS (P = 0. 585) and OS (P = 0.465). Further subgroup analysis showed that primary sites and T stage, but not other factors, might influence the benefit of END. Significant differences were found for T1 (P < 0.001 for OS) and T2 (P = 0.001 for DSS and < 0.001 for OS) tongue squamous cell carcinoma (TSCC) but not for other primary tumor sites.ConclusionThis large-scale retrospective population-based cohort study suggests that not all patients with cN0 OSCC could benefit from END. Patients with cN0 TSCC are recommended to undergo END, especially with early-stage tumors.

Nanointegrative Glycoengineering-Activated Necroptosis of Triple Negative Breast Cancer Stem Cells Enables Self-Amplifiable Immunotherapy for Systemic Tumor Rejection.

Triple-negative breast cancer stem cells (TCSCs) are considered as the origin of recurrence and relapse. It is difficult to kill not only for its resistance, but also the lacking of targetable molecules on membrane. Here, it is confirmed that ST6 β-galactoside alpha-2,6-sialyltransferase 1 (ST6Gal-1) is highly expressed in TCSCs that may be the key enzyme involved in glycoengineering via sialic acid (SA) metabolism. SA co-localizes with a microdomain on cell membrane termed as lipid rafts that enrich CSCs marker and necroptosis proteins mixed lineage kinase domain-like protein (MLKL), suggesting that TCSCs may be sensitive to necroptosis. Thus, the triacetylated N-azidoacetyl-d-mannosamine (Ac3ManNAz) is synthesized as the glycoengineering substrate and applied to introduce artificial azido receptors, dibenzocyclooctyne (DBCO)-modified liposome is used to deliver Compound 6i (C6), a receptor-interacting serine/threonine protein kinase 1(RIPL1)-RIP3K-mixed lineage kinase domain-like protein(MLKL) activator, to induce necroptosis. The pro-necroptosis effect is aggravated by nitric oxide (NO), which is released from NO-depot of cholesterol-NO integrated in DBCO-PEG-liposome@NO/C6 (DLip@NO/C6). Together with the immunogenicity of necroptosis that releases high mobility group box 1(HMGB1) of damage-associated molecular patterns, TCSCs are significantly killed in vitro and in vivo. The results suggest a promising strategy to improve the therapeutic effect on the non-targetable TCSCs with high expression of ST6Gal-1 via combination of glycoengineering and necroptosis induction.

2-methoxyestradiol inhibits the malignant behavior of triple negative breast cancer cells by altering their miRNome.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with no effective targeted treatment currently available. Estrogen and its metabolites influence the growth of mammary cancer. Previously, we demonstrated the anti-cancer effects of 2-methoxyestradiol (2ME2) on mammary carcinogenesis. In the present study, we investigated the effects of 2ME2 on TNBC cells. TNBC (MDA-MB-231 and MDA-MB-468) and non-tumorigenic breast (MCF10A) cell lines were used to determine the effects of 2ME2 on cell proliferation (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS assay), cell cycle (flow cytometric assay), migration (transwell migration assay), invasion (matrigel invasion assay), apoptosis (annexin V/propidium iodide assay), colony formation (soft agar assay), and miRNome (human miRNA profiling array). The miRNome data were analyzed using the c-BioPortal and Xena platforms. Moreover, Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and reactome pathway analyses were performed. We found that 2ME2 effectively inhibited cell proliferation and induced apoptosis. Furthermore, 2ME2 treatment arrested TNBC cells in the S-phase of the cell cycle. Treatment with 2ME2 also significantly decreased the aggressiveness of TNBC cells by inhibiting their migration and invasion. In addition, 2ME2 altered the miRNA expression in these cells. In silico analysis of the miRNome profile of 2ME2-treated MDA-MB-468 cells revealed that miRNAs altered the target genes involved in many different cancer hallmarks. 2ME2 inhibits triple negative breast cancer by impacting major cellular processes like proliferation, apoptosis, metastasis, etc. It further modifies gene expression by altering the miRNome of triple negative breast cancer cells. Overall, our findings suggest 2ME2 as a potent anti-cancer drug for the treatment of TNBC.

Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

Nano iron doped calcium phosphates act as pH-Sensitive and biodegradable peroxidase-like nanozymes for cancer chemodynamic therapy and MRI contrast

All types of cancer are a threat to human health and among the current therapies, the highly invasive chemotherapy is still the standard in several cases. Currently, more specific and less invasive therapies are being developed and chemodynamic therapy arose as a viable alternative. This strategy takes advantage of the tumour biochemical characteristics such as the acidic pH (∼6.0) and the high concentration of H2O2 (∼50 μM–∼1 mM). In these specific conditions, nanoparticles with peroxidase-like catalytic properties can convert H2O2 into highly toxic reactive oxygen species that induce oxidative stress in cancer cells. One of the remarkable advantages of chemodynamic therapy concerns the peroxidase-like activity, which only occurs in specific pH environments, as found in the tumour microenvironment. At the same time, the produced reactive oxygen species are short-lived, meaning that they will not travel outside the tumoral region, thus not affecting healthy surrounding tissues.This work reports on the production and characterization of iron doped calcium phosphate nanoparticles, as a fully biodegradable alternative to existing chemodynamic agents. By incorporating iron in different oxidation states into the lattice through calcium substitution, these nanoparticles exhibit high catalytic conversion of H2O2 into reactive oxygen species, more specifically into the hydroxyl radical, independently of the iron oxidation state. Outstanding results concern the significant toxicity, only obtained at pH 6.0 and in the presence of H2O2, while remaining non-toxic in physiological conditions (pH7.4) when tested against triple negative breast cancer cells, thus proving the specificity of the treatment in tumoral conditions. Moreover, the presented magnetic properties allowed them to produce significant contrast in magnetic resonance imaging, with different results depending on the iron oxidation state.

A pooled analysis of clinical outcome in driver-gene negative non-small cell lung cancer patients with MET overexpression treated with gumarontinib.

MET overexpression represents the most MET aberration in advanced non-small-cell lung cancer (NSCLC). However, except MET exon 14 (METex14) skipping mutation was recognized as a clinical biomarker, the role of MET overexpression as a predictive factor to MET inhibitor is not clear. The purpose of the pooled analysis is to explore the safety and efficiency of gumarontinib, a highly selective oral MET inhibitor, in drive-gene negative NSCLC patients with MET overexpression. NSCLC patients with MET overexpression [immunohistochemistry (IHC) ⩾3+ as determined by central laboratory] not carrying epidermal growth factor receptor mutation, METex14 skipping mutation or other known drive gene alternations who received Gumarontinib 300 mg QD from two single arm studies were selected and pooled for the analysis. The efficacy [objective response rate (ORR), disease control rate (DCR), duration of response, progression-free survival (PFS) and overall survival (OS)] and safety [treatment emergent adverse event (TEAE), treatment related AE (TRAE) and serious AE (SAE) were assessed. A total of 32 patients with MET overexpression were included in the analysis, including 12 treatment naïve patients who refused or were unsuitable for chemotherapy, and 20 pre-treated patients who received ⩾1 lines of prior systemic anti-tumour therapies. Overall, the ORR was 37.5% [95% confidence interval (CI): 21.1-56.3%], the DCR was 81.3% (95% CI: 63.6-92.8%), median PFS (mPFS) and median OS (mOS) were 6.9 month (95% CI: 3.6-9.7) and 17.0 month (95% CI: 10.3-not evaluable), respectively. The most common AEs were oedema (59.4%), hypoalbuminaemia (40.6%), alanine aminotransferase increased (31.3%). Gumarontinib showed promising antitumour activity in driver-gene negative locally advanced or metastatic NSCLC patients with MET overexpression, which warranted a further clinical trial. ClinicalTrials.gov identifier: NCT03457532; NCT04270591.