Overexpressing Cancer Cells Research Articles

Connective tissue growth factor expression maintains the epithelial phenotype of ovarian cancer in early epithelial to mesenchymal transition.

e17544 Background: Epithelial to Mesenchymal Transition (EMT) is essential to the process of metastasis in ovarian cancer (OC). Early expressions of proteins and pathways during EMT essential for understanding mechanism and potential therapeutic targets in OC. The aim of our study is to evaluate the role and mechanism of Connective Tissue Growth Factor (CTGF) in OC. Methods: R182 and R2615 are well-characterized epithelial OC cell lines. CTGF expression +/- Transforming Growth Factor -b (TGF-b) was determined via Western blot. R182 and R2615 CTGF knock out (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector and confirmed via PCR. Anoikis resistance and invasion assays were performed to characterize phenotypes of R182 and R2615 wild type (WT) and KO cells. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive (ULA) cell plate Promega CellTiter assay which measured cell viability quantified by absorbance at 450 nm at 0, 24, 48, and 72 hrs. For invasion assay, 3000 cells were suspended in 50% reduced growth factor Basement Membrane Extract. Human recombinant CTGF was added at 50 and 100 ng/mL concentrations. Cells were plated in tissue culture plate and placed in Cytation 5/Biospa and imaged at 4-hour interval for up to 6 days. Western blot evaluated expression of mesenchymal markers. Chemo-sensitivity was measured by half maximal inhibitory concentration (IC50) for Cisplatin between the WT and KO cells. RNA sequence analysis was performed for both R182 WT and KO cells and analyzed utilizing iPathway Guide. Results: CTGF is constitutively expressed in R182 and R2615 OC cells line as early as 6 hrs of culture and after 30 min of TGF-b treatment. Loss of CTGF promoted anoikis resistance. At 72 hr, R182 CTGF WT cells displayed 75% viability while R182 KO only have 10% viability. Further, we demonstrated that loss of CTGF increases invasion. Administration of exogeneous CTGF in KO cells suppresses invasion in a dose dependent manner demonstrated. Along with epithelial markers CK-18 and beta-catenin, the KO cells expressed the mesenchymal marker SNAIL. No difference in chemo-sensitivity to Cisplatin was seen between WT and KO in either cell line. RNA sequence analysis identified 1106 out of 14054 differentially expressed genes between the WT and KO cells. PI3KAkt, cell adhesion molecules and ECM receptor interaction were pathway that were most significantly regulated. Conclusions: CTGF expression maintains the epithelial phenotype of OC cells during EMT. Loss of CTGF allows anoikis resistance and invasion which are vital characteristics in the metastatic nature of OC. We suggest that loss of CTGF expression in OC cells could be a biomarker for more aggressive OC.

Selective Colorimetric Detection of Cancer Cells Based on Iron/Copper Nanocatalyst Peroxidase Activity

Nanomaterial based enzyme i.e., nanozymes have the potential to overcome natural enzymes&#x2019; major shortcomings, hence the development of effective artificial enzymes is a burgeoning topic in nanobiotechnology. A novel nanostructured hybrid enzyme was constructed as a mimetic enzyme for <i>i</i> n <i>v</i> itro cancer cell detection and therapeutic treatment in the current study. The iron-copper (FeCu) nanocatalyst was prepared and immobilized with 2-DG, as cancer cell targeting ligand 2-DG can interact with the GLUT receptor overexpressed in several cancer cells. FeCu&#x0040;2-DG showed enhanced peroxidase-like activity and oxidized the substrate 3,<inline-formula> <tex-math notation="LaTeX">$3^\prime $ </tex-math></inline-formula>,5,<inline-formula> <tex-math notation="LaTeX">$5^\prime $ </tex-math></inline-formula>-tetramethylbenzidine (TMB) into blue color product in the presence of hydrogen peroxide (H₂O₂). Peroxidase activity of nanocatalyst can be well utilized as selective and quantitative colorimetric sensor for detection of liver cancer cells (HepG2). Sensor is shown to detect HepG2 cells in linear range of 50&#x2013;1000 cells/mL, with detection limit of 20 cells/mL. Additionally, nanocatalyst can also be used as H₂O₂ and ascorbic acid mediated therapeutic effect in cancer cells. Phloretin (GLUT inhibitor) treated cells showed the negligible interaction with particle in comparison of control non-treated cells. Thus, confirming the selective interaction of synthesized particle with GLUT overexpressed cancer cells. Based on the simple peroxidase function, the developed hybrid might be a promising candidate for clinical cancer diagnosis and therapy.

ELU-FRα-1: A study to evaluate ELU001 in patients with solid tumors that overexpress folate receptor alpha (FRα).

TPS3158 Background: ELU001 is a novel, first-in-class, new molecular entity described as a C’Dot Drug Conjugate (CDC). ELU001 consists of ̃12 folic acid targeting moieties and ̃22 exatecan topoisomerase-1 inhibitor payloads on Cathepsin-B cleavable linkers covalently bound to the surface of each silicon core/polyethylene glycol C’Dot nanoparticle. CDCs are small in size (̃6 nm), have a greater ability to penetrate into and through tumors as compared to ADCs, and are rapidly eliminated by the kidneys. The rapid systemic elimination is expected to lead to less toxicity than is observed with targeting platforms like ADCs that have a longer half-life in circulation. ELU001’s high avidity is believed to promote internalization into FRα overexpressing cancer cells, selectively delivering it’s ̃22 molecules of payload. The first-in-human clinical trial, ELU-FRα-1, is currently recruiting patients that have advanced, recurrent or refractory FRα overexpressing tumors considered to be topoisomerase 1 inhibitor-sensitive based on the literature, and, in the opinion of the Investigator, have no satisfactory therapeutic options available. Methods: This is a Phase 1 / 2 multicenter, open label clinical trial with two parts: Part 1 Dose Escalation and Part 2 Tumor Group Expansion Cohort(s). In Part 1, patients with cancer types with a high likelihood of having FRα overexpressing tumors based on historical data, (specifically, ovarian, endometrial, colorectal, gastric, gastroesophageal junction, triple negative breast, or non-small cell lung cancers, or cholangiocarcinoma), will be enrolled to the study. Patients will receive ELU001 on a weekly dose regimen (QW) (once a week for 3 weeks, 1 week rest) or every other week dose regimen (Q2W, with no rest between cycles). Retrospective analysis of FRα expression will be determined. Part 2 uses a Simon’s Two-Stage design to evaluate 4-6 tumor group expansion cohorts, each consisting of patients with specific tumor types (to be identified based on emerging data) that overexpress FRα (prospectively determined prior to enrollment). The primary objective for Part 1 is to identify the MTD/RP2D and for Part 2 is ORR. Dose Escalation will recruit about 25 patients per dose regimen (QW or Q2W). Dose Expansion will recruit about 15 patients per tumor group expansion cohort. Secondary objectives are anti-tumor activity (DOR, PFS, TFST, PFS2, OS), frequency, severity and tolerability of adverse events, PK, ADA, and FRα expression assessments. The study is actively enrolling in the US – Cohorts 1-2 have been completed without DLT. Enrollment to Cohort 3 began in December 2021. Clinical trial information: NCT05001282 .

A phase 1, first-in-human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in participants with HER2 overexpressing solid tumors.

TPS2677 Background: Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but have had less success in those with solid tumors. Macrophages are actively recruited and abundantly present in the solid tumor microenvironment (sTME). Tumor associated macrophages are predominantly immunosuppressive and support tumor growth (M2), while a subset of proinflammatory macrophages enhance anti-tumor immunogenicity (M1). M1 macrophage function can be augmented by CAR expression to selectively recognize and phagocytose antigen overexpressing cancer cells. Moreover, CAR macrophages can reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and anti-tumor immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis in many solid tumors (Table). CT-0508 is a cell product comprised of autologous monocyte-derived proinflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden, prolonged survival, and were safe and effective in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. Methods: This Phase 1, FIH study is evaluating safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 in 18 participants (pt) with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2. Pt previously treated with available therapies, including anti-HER2 therapies, as indicated, and subsequent progression are permitted. Filgrastim is used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. CT-0508 is manufactured, prepared, and cryopreserved from mobilized peripheral blood monocytes. Group 1 pt (n=9) receive CT-0508 infusion split over D1, 3, and 5. A Safety Review Committee will review dose limiting toxicities. Group 2 pt (n=9) will receive the full CT-0508 infusion on D1. Pre- and post-treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (RNA scope), CT-0508 persistence in blood and in the tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Radiographic imaging is also being conducted to assess preliminary tumor activity. Clinical trial information: NCT04660929. [Table: see text]

Design, Synthesis and In Vivo Fluorescence Imaging Study of a Cytochrome P450 1B1 Targeted NIR Probe Containing a Chelator Moiety

Cytochrome P450 (CYP) 1B1 has been found to be overexpressed specifically in tumor tissues at an early stage, which makes it a potential cancer biomarker for molecular imaging. Multimodal imaging combines different imaging modalities and offers more comprehensive information. Thus, imaging probes bearing more than one kind of signal fragment have been extensively explored and display great promise. Herein, we developed a near infrared (NIR) probe with a chelator moiety targeting CYP1B1 by conjugating α-naphthoflavone (ANF) derivatives with both an NIR dye and a chelator for potential application in bimodal imaging. Enzymatic inhibitory studies demonstrated inhibitory activity against CYP1B1 and selectivity among CYP1 were successfully retained after chemical modification. Cell-based saturation studies indicated nanomolar range binding affinity between the probe and CYP1B1 overexpressed cancer cells. In vitro competitive binding assays monitored by confocal microscopy revealed that the probe could specifically accumulate in tumor cells. In vivo and ex vivo imaging studies demonstrated that the probe could effectively light-up the tumor tissues as early as 2 hours post-injection. In addition, the fluorescence was significantly blocked by co-injection of CYP1B1 inhibitor, which indicated the probe accumulation in tumor sites was due to specific binding to CYP1B1.

FTX Regulated miR-153-3p/FOXR2 to Promote Cisplatin Resistance in Ovarian Cancer.

Purpose The present study was aimed at exploring the role of FTX in cisplatin (DDP) resistance in ovarian cancer (OC). Methods QPCR was applied to evaluate mRNA expression in OC tissue and cells. CCK-8 assay was conducted to evaluate cell proliferation. Transwell chamber assay was performed to evaluate invasion of SKOV3/DDP cells. The protein expression was evaluated via western blot assay. Flow cytometry was performed to evaluate the apoptosis of SKOV3/DDP cells. Results The expression of FTX in DDP-resistant cells was observably higher in contrast to DDP-sensitive cells and normal ovarian cells. FTX was higher expressed in DDP-resistant tissues by comparison with DDP-sensitive tissues. Knockdown of FTX obviously suppressed the proliferation ability invasion ability of SKOV3/DDP cells. Knockdown of FTX obviously enhanced apoptosis of SKOV3/DDP cells. miR-153-3p was proved to be directly regulated by FTX via the luciferase reporter assays. By comparison with normal cells, miR-153-3p was lower expressed in OC cells. miR-153-3p was lower expressed in SKOV3/DDP cells in contrast to SKOV3 cells. More interestingly, FTX reversed the inhibiting influence of miR-153-3p on cisplatin resistance of OC cells. Moreover, miR-153-3p was proved to directly regulate FOXR2. Knockdown of miR-153-3p attenuated the inhibitory influence of knockdown FOXR2 on cisplatin resistance of OC cells. Conclusion FTX regulated miR-153-3p/FOXR2 to promote cisplatin resistance via inhibiting the apoptosis and promoting the viability and invasion in OC.

LncRNA PART1 Stimulates the Development of Ovarian Cancer by Up-regulating RACGAP1 and RRM2.

Ovarian cancer (OC) is a kind of gynecologic malignancy with a high mortality rate. Long non-coding RNAs (lncRNAs) have been reported to exert regulatory roles in multiple diseases. However, the role of lncRNA prostate androgen-regulated transcript 1 (PART1) has not been investigated in the development of OC. In this study, from RT-qPCR analysis, we discovered that PART1 demonstrated high expression in OC cells. Moreover, data from functional assays manifested that PART1 reduction hindered the proliferative, migratory, and invasive capabilities of OC cells. In vivo uncovered that PART1 knockdown impeded OC tumor growth. Furthermore, from the experimental results of RNA pull down, RIP, and luciferase reporter assays, we discovered that PART1 served as a sponge for microRNA-6884-5p (miR-6884-5p) to modulate the expression of Rac GTPase activating protein 1 (RACGAP1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Finally, rescue assays proved that overexpression of RACGAP1 or RRM2 abrogated the suppressive role of PART1 knockdown on OC cell malignant behaviors. RACGAP1 and RRM2 were also revealed to act as oncogenes in OC cells. In summary, our research verified the PART1/miR-6884-5p/RACGAP1/RRM2 axis in OC cells, which signified that PART1 might act as a novel biomarker in OC.

One-pot preparation of biocompatible folate-functionalized graphitic carbon nitride quantum dots for targeted bioimaging

Graphitic carbon nitride quantum dots (CNQDs) have been widely used in bioimaging due to the unique structure and optical properties, but only a few works demonstrate targeted imaging. Herein, we report a universal one-pot strategy for preparation of targeting agent functionalized CNQDs by taking folate (FA) as an example through hot-fluid annealing of triazine tectons and derived FA. The FA-CNQDs with size of 3 ∼ 8 nm exhibit bright fluorescence, favorable colloidal stability, and well cytocompatibility and in vivo biocompatibility. The remarkable performance of FA-CNQDs in targeted imaging is demonstrated by incubation study with folate receptor overexpressed cancer cells and the cell derived multicellular spheroids. This convenient strategy would open a new avenue for synthesis of various functionalized CNQDs.

Mitochondrial transcription factor B2 overexpression increases M2 macrophage infiltration via cytosolic mitochondrial DNA-stimulated Interleukin-6 secretion in ovarian cancer

ABSTRACT Mitochondrial transcription factor B2 (TFB2M) is a protein modulating both mitochondrial DNA (mtDNA) transcription and compacting. In this study, we explored the expression profile of TFB2M in ovarian cancer, its association with infiltration of tumor-associated macrophages (TAMs), and its influence on macrophage polarization. Serial sections of ovarian cancer tissue arrays were stained to detect TFB2M and CD163 expression. Epithelial ovarian cancer cell line OVISE and CAOV4 were used to assess the influence of TFB2M on IL-6 expression. THP-1 cells were utilized as an in vitro model for macrophage migration and polarization. Results showed that higher TFB2M expression is associated with poor survival in ovarian cancer patients. IHC staining confirmed a moderately positive correlation between TFB2M expression and the infiltration of CD163-positive cells in 68 primary ovarian cancer cases. TFB2M overexpression was associated with increased mtDNA outside the mitochondria and elevated IL-6 expression in ovarian cancer cells. When cytosolic mtDNA was selectively inhibited by DNase I, TFB2M-induced IL-6 upregulation was canceled. TFB2M overexpression could activate the nuclear factor kappa-B (NF-κB) signaling pathway via promoting nucleus entry of p65 and p-p65, which was abrogated by inhibiting cytosolic mtDNA, TLR9, or NF-κB signaling pathway. Conditioned medium from OIVSE cells with TFB2M overexpression could induce macrophage migration and M2 polarization. However, these inducing effects were abrogated by DNase I, TLR9 inhibitor, and anti-IL-6 R pretreatment. In conclusion, this study showed a novel role of TFB2M in the immunosuppressive tumor microenvironment. It promotes M2 macrophage infiltration via a cytosolic mtDNA/TLR9/NF-κB/IL-6 pathway in ovarian cancer.

Interferon‐γ Induced PD‐L1 Expression in Ovarian Cancer Cells is Regulated by IRF1 Signaling

The immune checkpoint programmed death ligand‐1 (PD‐L1) is highly expressed in cancer cells, including ovarian cancer (OC) cells. High PD‐L1 expression in ovarian cancer correlates with poor prognosis, but the mechanisms that regulate the PD‐L1 expression in OC remain poorly understood. We have recently shown that IFNγ induces the PD‐L1 expression in OC cells, resulting in their increased proliferation and invasion. Here, we show that the IFNγ‐induced PD‐L1 expression in OC cells is associated with increased levels of STAT1, Tyr‐701 pSTAT1 and Ser‐727 pSTAT1 in OC cells. JAK1 and STAT1 suppression significantly decreases the IFNγ‐induced PD‐L1 mRNA and protein levels in OC cells. In addition, IRF1 suppression significantly decreases the IFNγ‐induced gene and protein levels of PD‐L1, indicating that the IFNγ‐induced PD‐L1 expression in OC cells is dependent on IRF1 signaling. Chromatin immunoprecipitation data demonstrate that IFNγ induces occupancy of STAT1, Ser‐727 pSTAT1 and IRF1 at STAT1 and IRF1 binding sites in human PD‐L1 promoter. Together, these results show that the IFNγ‐induced PD‐L1 expression in OC cells is regulated by IRF1/JAK1/STAT1 signaling, and suggest that targeting the IRF1/JAK1/STAT1 pathway may provide leverage to regulate the PD‐L1 levels in ovarian cancer.

Interferon‐γ Induces Bcl3 Expression in Ovarian Cancer Cells by JAK1/STAT1 Signaling

The proto‐oncogene Bcl3 is an atypical member of IkB family, which regulates expression of a number of NFkB target genes, including anti‐apoptotic genes, cytokines, and immune checkpoints. Bcl3 expression is increased in many types of human cancers and is associated with increased tumor growth and malignant potential. Interferon‐γ (IFNγ) is a pleiotropic cytokine that has been used in cancer treatment; in addition, its expression is induced in response to radiation therapy and immune checkpoint blockade during cancer treatment. We have recently shown that IFNγ induces Bcl3 expression in ovarian cancer cells, resulting in their increased proliferation, migration, and invasion. In this study, we have investigated the mechanisms of how IFNγ induces the Bcl3 expression in ovarian cancer cells. Our results demonstrate that the IFNγ‐induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, as well as on p65 NFkB. In addition, we have identified a potential interferon regulatory factor 1 (IRF1) binding site in human Bcl3 promoter and found that the IFNγ‐induced Bcl3 expression in ovarian cancer cells is associated with an increased occupancy of IRF1, Ser‐727 phosphorylated STAT1 and acetylated histone H3 at the IRF1 binding site in Bcl3 promoter. These findings identify Bcl3 as a novel target of IFNγ/JAK1/STAT1 signaling and suggest that targeting the JAK1/STAT1 pathway may suppress the IFNγ‐induced Bcl3 expression in ovarian cancer.

Co-treatment of Low Dose Pacritinib, a Phase III Jak2 Inhibitor, Greatly Increases Apoptosis of P-gp Over-expressing Cancer Cells With Multidrug Resistance.

The over-expression of P-glycoprotein (P-gp) is a major mechanism underlying multidrug resistance (MDR). Co-treatment with Janus kinase 2 (Jak2) inhibitors sensitizes P-gp-over-expressing drug-resistant cancer cells. In this study, we evaluated pacritinib, a Jak2 inhibitor currently in phase III clinical trials. Microscopic observation, cell viability assay, colony forming assay, rhodamine uptake tests, annexin V analyses, fluorescence-activated cell sorting (FACS), and western-blot analysis were performed to further investigate the mechanism of action. We found that pacritinib reduced cell viability, induced G2 arrest, and upregulated early apoptosis when administered to P-gp-over-expressing resistant KBV20C cells with vincristine (VIC). Moreover, apoptosis and G2 arrest in VIC-pacritinib-treated cells were involved in the upregulation of pH2AX expression. Pacritinib had an approximately 2-fold higher P-gp-inhibitory activity than the dimethyl sulfoxide (DMSO)-treated control, indicating that VIC-pacritinib sensitization involves the P-gp-inhibitory effects of pacritinib. Similar to VIC, other antimitotic drugs (vinorelbine, vinblastine, and eribulin) could also sensitize against KBV20C cells by co-treatment with pacritinib. Furthermore, comparison of pacritinib with previously characterized Jak2 inhibitors revealed that the VIC-pacritinib combination had sensitization effects similar to those of VIC- CEP-33779 or VIC-NVP-BSK805 combinations at lower doses in KBV20C cells. Generally, Jak2 inhibitor and VIC co-treatment sensitized P-gp-over-expressing resistant cancer cells by inducing early apoptosis. Collectively, pacritinib, induced G2 arrest, reduced cell viability, had high P-gp inhibitory activity, and upregulated the expression of pH2AX when used in combination with VIC. As pacritinib is a Jak2 inhibitor currently in phase III clinical trials, our findings may facilitate the application of this co-treatment in patients with MDR cancer.

A Pan-Cancer Analysis of the Oncogenic Role of WD Repeat Domain 74 in Multiple Tumors.

Although emerging patient-derived samples and cellular-based evidence support the relationship between WDR74 (WD Repeat Domain 74) and carcinogenesis in multiple cancers, no systematic pan-cancer analysis is available. Our preliminary research demonstrated that WDR74 is over-expressed in lung squamous cell carcinoma (LUSC) and related with worse survival. We thus investigated the potential oncogenic roles of WDR74 across 33 tumors based on the database of TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus). WDR74 is highly expressed in most cancers and correlated with poor prognosis in several cancers (all p < 0.05). Mutation analysis demonstrated that WDR74 is frequently mutated in promoter regions of lung cancer. Moreover, we found that CD8+ T-cells and the fibroblast infiltration level increased in WDR74 over-expressed cancer cells. The GO (Gene Ontology) enrichment analysis of the WDR74 pathway revealed its participation in cellular biogenesis of the RNA metabolism and its critical role in cancer initiation and progression through the tumor cell energy metabolism. Our first pan-cancer study inferred a relatively comprehensive understanding of the oncogenic roles of WDR74 across various cancers.

Discovery of a selective and covalent small-molecule inhibitor of BFL-1 protein that induces robust apoptosis in cancer cells

Induction of apoptosis by the FDA-approved drug Venetoclax in cancer cells mainly derives from blocking the interactions between BCL-2 and BH3-only proteins. Anti-apoptotic BFL-1, a homolog of BCL-2, also competitively binds to the BH3-only proteins and is responsible for Venetoclax-induced drug resistance. Compared to BCL-2, small-molecule inhibitors of BFL-1 are relatively underexplored. In order to tackle this issue, in-house compound library was screened and a hit compound was identified and optimized to obtain 12 (ZH97) functioning as a covalent and selective inhibitor of BFL-1. 12 modifies BFL-1 at the C55 residue, blocks BFL-1/BID interaction in vitro, promotes cellular cytochrome c release from mitochondria, and induced apoptosis in BFL-1 overexpressing cancer cells. Mechanistic studies show that 12 inhibited BFL-1/PUMA interaction in cell lysate and is effective in cancer cells that harboring high expression level of BFL-1. In summary, blockade of BFL-1/BH3-only proteins interactions with a covalent small-molecule inhibitor induced apoptosis and elicited antitumor activity. Thus, our study demonstrates an appealing strategy for selective modulation of cellular BFL-1 for cancer therapy.

Carboxymethylated pachyman induces ferroptosis in ovarian cancer by suppressing NRF1/HO-1 signaling.

Carboxymethylated pachyman (CMP) is characterized by immune regulatory, antitumor and antioxidant activities. However, whether CMP contributes to the treatment of ovarian cancer has yet to be explored. The role of CMP in ovarian cancer cell death was analyzed using CCK-8 and flow cytometry assays. The data showed that CMP induced ovarian cancer cell death in a dose-dependent manner. Furthermore, CMP-induced cell death could be largely reversed by preincubation with ferrostatin-1 (Fer-1) but not 3-methyladenine or necrostatin-1. Reverse transcription-quantitative PCR analysis indicated that CMP significantly increased prostaglandin-endoperoxide synthase 2 (PTGS2) and Chac glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) mRNA levels, but preincubation with Fer-1 obviously reduced PTGS2 and CHAC1 mRNA levels in SKOV3 and Hey cells. The intracellular levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and Fe2+ were then quantified The data showed that 100 and 200 µg/ml CMP enhanced the production of SOD, MDA and Fe2+ but decreased GSH levels in SKOV3 and HEY cells. These data indicated that CMP could induce ferroptosis in ovarian cancer cells. More importantly, in vitro and in vivo studies indicated that CMP significantly suppressed nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), cystine/glutamate antiporter system X(c)(−) (xCT) and glutathione peroxidase 4 (GPX4) expression in ovarian cancer cells and tumors. In conclusion, the present study showed novel data that CMP could induce ferroptotic death in ovarian cancer cells by suppressing Nrf2/HO-1/xCT/GPX4. All these findings indicate that CMP may have great potential in anti-ovarian cancer cell therapy by inducing ferroptosis.

KLF8 is activated by TGF-β1 via Smad2 and contributes to ovarian cancer progression.

Krüppel-like factor 8 (KLF8) is a transcription factor expressed abnormally in various cancer types and promotes oncogenic transformation. However, the role of KLF8 in ovarian cancer (OC) progression remains unclear. This study reports that transforming growth factor-β1(TGF-β1)/Smad2/KLF8 axis regulates epithelial-mesenchymal transition (EMT) and contributes to OC progression. We analyzed the KLF8 expression in OC cells and tissues, wherein a significant overexpression of KLF8 was observed. Increased KLF8 expressions were correlated with higher cell proliferation, EMT, migration, and invasion and conferred poor clinical outcomes in OC patients. Overexpressed KLF8 increases F-actin polymerization and induces cytoskeleton remodeling of OC cells. Furthermore, a dissection of the molecular mechanism defined that TGF-β1 triggers KLF8 through the Smad2 pathway and regulates EMT. Pharmacological and genetic inhibition of Smad2 followed by TGF-β1 treatment failed to activate KLF8 expression and induction of EMT. Using promoter-luciferase reporter assays, we defined that upon TGF-β1 activation, phosphorylated Smad2 binds and promotes the KLF8 promoter activity, and knockdown of Smad2 inhibits KLF8 promoter activation. Together, these results demonstrate that TGF-β1 activates KLF8 expression by the Smad2 pathway, and KLF8 contributes to OC progression and may serve as a potential therapeutic strategy for treating OC patients.

Inhibition of cancer cells using target-specific 2A3 antibody-conjugated gold nanoclusters

Background: Metal nanoclusters (NCs) with outstanding structural and optical properties have been intensively validated for applications in nanomedicine and nanotechnology. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is overexpressed in many cancer cells.&#x0D; Objective: The gold nanoclusters conjugated with a single domain antibody targeting CEACAM6 of 2A3 (2A3-AuNCs) were synthesized for the inhibition of cancer cells.&#x0D; Methods: 2A3-AuNCs were prepared via a facile hydrothermal approach. The cell viability was measured by resazurin dye reduction assay. The cell death was analyzed by fluorescence imaging.&#x0D; Results: Structural and optical characterizations demonstrated the successful synthesis of 2A3-AuNCs with a roughly spherical shape and a size of 2.35 nm. The 2A3-AuNCs revealed a maximum fluorescence intensity at 350 nm with a fluorescence quantum yield of 4.0%. The cell viability assay indicated that 2A3-AuNCs could inhibit the growths of cancer cells with overexpressed CEACAM6, including breast cancer MDA-MB-231 and MDA-MB-468 cells. The fluorescence imaging results also demonstrated that 2A3-AuNCs could inhibit the growth of cancer cells with MDA-MB-231 and MDA-MB-468 cells.&#x0D; Conclusion: Combination with the results of cell viability assay and fluorescence imaging, the surface ligand of 2A3 antibody on 2A3-AuNCs exhibited promising inhibition of CEACAM6 overexpressed cancer cells. Our work provides a potential application of AuNCs in cancer therapy.

Abstract OT1-03-01: A phase 1, first in human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in subjects with HER2 overexpressing solid tumors

Abstract Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically evince immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR on the macrophages confers the ability to selectively recognize and phagocytose antigen overexpressing cancer cells. Additionally, CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis and is seen in many cancers, including but not limited to breast and gastroesophageal cancers (Table 1). CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreasing tumor burden and prolonging survival in relevant models. CT-0508 cells were safe and effective in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 in approximately 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2, who have failed available therapies including anti-HER2 therapies where indicated.Filgrastim is being used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product is manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. The study is enrolling Group 1 subjects, who receive CT-0508 infusion split over D1, 3 and 5. Subjects will be continually assessed for acute and cumulative toxicity. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will follow, and will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and in the tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Clinical trial registry number: NCT04660929 Table 1.HER2 Positivity Frequencies Across Tumor TypesTumor typeHER2 positivity (%)ReferenceBladder cancer8-70Gandour-Edwards et al, 2002;Caner et al, 2008;Laé et al, 2010; Fleischmann et al, 2011;Charfi et al, 2013;Yan et al, 2015Breast cancer11.0-25.0Varga et al, 2013;Stenehjem et al, 2014Cervical cancer2.8-3.9Chavez-Blanco et al, 2004;Yan et al, 2015Colorectal cancer1.6-5.0Schuell et al, 2006;Ingold Heppner et al, 2014;Seo et al, 2014Esophageal cancer12.0-14.0König et al, 2013;Yoon et al, 2013;Wang et al, 2014Extrahepatic Cholangiocarcinoma6.3-9.0Yoshikawa et al, 2008;Yan et al, 2015Gallbladder cancer9.8-12.8Roa et al, 2014;Yan et al, 2015Gastric adenocarcinoma7.0-34.0Rüschoff et al, 2012;Hofmann et al, 2008Ovarian cancer26Slamon et al, 1989Salivary mucoepidermoid carcinomas17.6Glisson et al, 2004Salivary duct carcinoma30-40Skálová et al, 2003; Cornolti et al, 2007; Nardi et al, 2013Testicular cancer2.4Yan et al, 2015Uterine cancer3.0Yan et al, 2015 Citation Format: Yara George Abdou, Debora Barton, Amy Ronczka, Daniel Cushing, Michael Klichinsky, Kim Reiss Binder. A phase 1, first in human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in subjects with HER2 overexpressing solid tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-03-01.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A phase 1, first-in-human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in subjects with HER2 overexpressing solid tumors.

TPS668 Background: Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and are abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically display immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR on the macrophages allow them to selectively recognize and phagocytose antigen overexpressing cancer cells. CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis in many cancers (Table). CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden and prolonged survival in relevant models. CT-0508 cells were safe and effective in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. Methods: This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking and preliminary evidence of efficacy of investigational product CT-0508 in 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2, who have failed available therapies, including anti-HER2 therapies when indicated. Filgrastim is being used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product is manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. Group 1 subjects receive CT-0508 infusion split over D1, 3 and 5. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and in the tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Clinical trial information: NCT04660929. [Table: see text]