Spheroid Formation Ability Research Articles

Inhibition of Stemness and PD-L1 Expression by Pien Tze Huang Enhances T Cell-Mediated Killing of Colorectal Cancer.

Pien Tze Huang (PZH) is a traditional medicinal formula consisted of four traditional Chinese medicines (TCMs) including Panax notoginseng (Burk.) F. H. Chen, Snake Gall, Calculus Bovis and Moschus, with clinical efficacy against Colorectal Cancer (CRC). However, the molecular and functional mechanisms underlying this efficacy are not fully elucidated. This study aimed to assess the impact of PZH on CRC cancer stem cells (CSCs), and evaluate the coordination effect of PZH on T cell-mediated anti-CRC with patient-derived autologous T cell co-culture. High-performance liquid chromatography (HPLC) was used to identify the main components of PZH. CCK8 and spheroid formation assays were conducted for assessing cell viability and stemness function. Western blot, immunofluorescence and immunohistochemistry were used to evaluate CSC markers and PD-L1 expression. T cell successful expansion was validated by flow cytometry. Co-culture assay was conducted to explore the activation effect of PZH on T cells. The potential mechanism of PZH in CRC was identified with transcriptomics sequencing and network pharmacology analysis. PZH reduced cell viability and spheroid formation ability in CRC, and suppressed the expression of CSC markers - LGR5, DCLK1, and CD133. Moreover, PZH enhanced T cell-mediated cytotoxicity against CRC cells by decreasing the expression of PD-L1. Furthermore, PZH with anti-PD-1 immunotherapy enhancing antitumor efficacy and increasing CD8+ T cell infiltration with decreasing expression of CSC markers and PD-L1. Notably, PZH inhibited CRC patient-derived organoids (PDOs) tumorigenesis and increased autologous T cell cytotoxicity against PDOs (n=5). Consistently, PZH decreased expression of CSC markers and PD-L1 in PDOs. RNA sequencing and network pharmacology also highlighted that PZH inhibited CRC stemness and PD-L1 to enhance T cell-mediated antitumor effects. PZH enhances T cell-mediated killing by inhibiting the expression of CRC stem cell markers and PD-L1, which warrant further investigation and clinical applications.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of the Long Noncoding RNA HOTAIR and Lysine Methyltransferase EZH2.

The persistence of cancer stem cells (CSC) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse in ovarian cancer, the fifth leading cause of cancer-related death among US women. HOXC transcript antisense RNA (HOTAIR) is a long, noncoding RNA (lncRNA) overexpressed in high-grade serous ovarian cancer and linked to chemoresistance. However, HOTAIR impacts chromatin dynamics in ovarian CSCs. Oncogenic lncRNA's contributions to drug-resistant disease are incompletely understood. Here, we generated HOTAIR knockout (KO) high-grade serous ovarian cancer cell lines using paired CRISPR guide RNA design to investigate the function of HOTAIR. We show the loss of HOTAIR function resensitized ovarian cancer cells to platinum treatment and decreased the population of ovarian CSCs. Furthermore, HOTAIR KO inhibited the development of stemness-related phenotypes, including spheroid formation ability and expression of key stemness-associated genes ALDH1A1, NOTCH3, SOX9, and PROM1. HOTAIR KO altered the cellular transcriptome and chromatin accessibility landscape of multiple oncogenic-associated genes and pathways, including the NF-kB pathway. HOTAIR functions as an oncogene by recruiting enhancer of zeste homolog 2 (EZH2) to catalyze H3K27 trimethylation to suppress downstream tumor suppressor genes, and it was of interest to inhibit both HOTAIR and EZH2. In vivo, combining a HOTAIR inhibitor with an EZH2 inhibitor and platinum chemotherapy decreased tumor formation and increased survival. These results suggest a key role for HOTAIR in ovarian CSCs and malignant potential. Targeting HOTAIR in combination with epigenetic therapies may represent a therapeutic strategy to ameliorate ovarian cancer progression and resistance to platinum-based chemotherapy.

LncRNA BCYRN1 as a Potential Therapeutic Target and Diagnostic Marker in Serum Exosomes in Bladder Cancer.

Bladder cancer (BC) is a common genitourinary malignancy that exhibits silent morbidity and high mortality rates because of a lack of diagnostic markers and limited effective treatments. Here, we evaluated the role of the lncRNA brain cytoplasmic RNA 1 (BCYRN1) in BC. We performed loss-of-function assays to examine the effects of BCYRN1 downregulation in T24 and BOY BC cells. We found that BCYRN1 downregulation significantly inhibited the proliferation, migration, invasion, and three-dimensional spheroid formation ability and induced apoptosis in BC cells. Additionally, gene set enrichment analysis (GSEA) using RNA sequences from tumor fractions showed that BCYRN1 downregulation decreased the expression of mRNAs associated with the cell cycle. These findings were supported by observations of G2/M arrest in flow cytometry assays. Finally, we examined the expression of serum exosomal BCYRN1 as a biomarker. Clinically, BCYRN1 expression in serum exosomes from patients with BC (n = 31) was significantly higher than that in healthy donors (n = 19; mean difference: 4.1-fold higher, p < 0.01). Moreover, in patients who had undergone complete resection of BC, serum exosomal BCYRN1 levels were significantly decreased (n = 8). Thus, serum exosomal BCYRN1 may be a promising diagnostic marker and therapeutic target in patients with BC.

Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2.

Purpurogallin carboxylic acid (PCA) is a natural phenol compound derived from Macleaya microcarpa (Maxim.) Fedde, which exerts particular antioxidant and anti-inflammatory capacities. However, the effects and mechanisms of PCA on liver cancer cells remain unknown. Therefore, network pharmacology and computer virtual docking were used to identify the target-proteins of PCA. In addition, surface plasmon resonance, protease activity and rhodamine excretion assays were carried out to evaluate the effects of PCA on the activity of ATP binding cassette subfamily G member 2 (ABCG2). The synergistic effects of PCA and 5-fluorouracil (5-FU) on liver cancer cell proliferation, cell cycle arrest, colony formation and spheroid formation abilities in vitro were determined by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, western blot analysis, colony formation and spheroid formation assays, respectively. ABCG2 was identified as a potential target of PCA, with a high docking score. The equilibrium dissociation constant of PCA for ABCG2 protein was 1.84 µM, while the median inhibitory concentration of this protein was 3.09 µM. In addition, the results demonstrated that PCA could significantly reduce the drug efflux capacity of liver cancer cells. CCK-8 assays revealed that liver cancer cell treatment with 10 µM PCA and 10 µM 5-FU exhibited the most potent synergistic effects on liver cancer cell proliferation at 48 h. Additionally, cell co-treatment with PCA and 5-FU also significantly attenuated the colony and spheroid formation abilities of liver cancer cells in vitro, while it promoted their arrest at the G1 phase of the cell cycle. Furthermore, ABCG2 silencing in liver cancer cells notably abrogated the synergistic effects of PCA and 5-FU. In conclusion, the present study demonstrated that PCA exhibited synergistic effects with 5-FU on liver cancer cells in vitro via targeting ABCG2. Therefore, PCA combined with 5-FU may be a potential strategy for liver cancer therapy.

Membrane-associated RING-CH 7 inhibits stem-like capacities of bladder cancer cells by interacting with nucleotide-binding oligomerization domain containing 1

BackgroundCancer stem-like capacities are major factors contributing to unfavorable prognosis. However, the associated molecular mechanisms underlying cancer stem-like cells (CSCs) maintain remain unclear. This study aimed to investigate the role of the ubiquitin E3 ligase membrane-associated RING-CH 7 (MARCH7) in bladder cancer cell CSCs.MethodsMale BALB/c nude mice aged 4–5 weeks were utilized to generate bladder xenograft model. The expression levels of MARCHs were checked in online databases and our collected bladder tumors by quantitative real-time PCR (q-PCR) and immunohistochemistry (IHC). Next, we evaluated the stem-like capacities of bladder cancer cells with knockdown or overexpression of MARCH7 by assessing their spheroid-forming ability and spheroid size. Additionally, we conducted proliferation, colony formation, and transwell assays to validate the effects of MARCH7 on bladder cancer CSCs. The detailed molecular mechanism of MARCH7/NOD1 was validated by immunoprecipitation, dual luciferase, and in vitro ubiquitination assays. Co-immunoprecipitation experiments revealed that nucleotide-binding oligomerization domain-containing 1 (NOD1) is a substrate of MARCH7.ResultsWe found that MARCH7 interacts with NOD1, leading to the ubiquitin–proteasome degradation of NOD1. Furthermore, our data suggest that NOD1 significantly enhances stem-like capacities such as proliferation and invasion abilities. The overexpressed MARCH7 counteracts the effects of NOD1 on bladder cancer CSCs in both in vivo and in vitro models.ConclusionOur findings indicate that MARCH7 functions as a tumor suppressor and inhibits the stem-like capacities of bladder tumor cells by promoting the ubiquitin–proteasome degradation of NOD1. Targeting the MARCH7/NOD1 pathway could be a promising therapeutic strategy for bladder cancer patients.

Abstract A110: Targeting methionine metabolism inhibits ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the leading cause of gynecologic cancer death. Platinum (Pt)- based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to disease recurrence. In the current study, we demonstrated that platinum drugs can enrich for OCSCs. In high grade serous ovarian cancer (HGSOC) cell lines, cisplatin treatment (IC50 for 16hrs) increased the percentage of cells with high aldehyde dehydrogenase (ALDH+) activity, a known OCSCs marker, and altered methionine (Met) metabolism. Extracellular Met uptake is converted to S-adenosyl-methionine (SAM) by methionine adenosyltransferase 2A (MAT2A), the rate-limiting enzyme of Met utilization. Short-term cisplatin treatment decreased SAM level and increased expression of MAT2A. It has been reported that CSCs have a high-flux metabolic state and are dependent on Met metabolism. We observed higher MAT2A expression in ALDH+ cells compared to the whole cell population, and Met depletion blocked the cisplatin-induced increase in %ALDH+ cells and spheroid formation ability. Furthermore, both MAT2A siRNA knockdown and the MAT2A inhibitor FIDAS-5 prevented cisplatin-induced increase in %ALDH+ cells, suggesting that blocking Met metabolism phenotypically inhibited the platinum enrichment of OCSCs. MAT2A inhibition also downregulated Cyclin E and arrested HGSOC cells in G1/S phase, indicating that a lower level of Met utilization re-established G1/S checkpoint in p53-mutant OC. As SAM is the universal methyl-group donor, it was of interest to examine whether the observed cisplatin-induced alterations in SAM level resulted in changes in DNA methylation. Methylation specific-PCR analysis demonstrated that siMAT2A or FIDAS-5 treatment of HGSOC cells inhibited cisplatin-induced hypermethylation of CpG islands of BRCA1 and ALDH1L1, a folate metabolic enzyme of which promoter hypermethylation is associated to tumor progression. Collectively, these results demonstrated a crucial role of Met metabolism in cisplatin-induced OCSCs enrichment. MAT2A has been recognized as a therapeutic target for the treatment of solid tumors and Met inhibition by targeting MAT2A could represent a therapeutic approach for inhibiting CSC in ovarian cancer. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methionine metabolism inhibits ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A110.

Anti-Tumor Efficacy of Oleuropein-Loaded ZnO/Au Mesoporous Silica Nanoparticle in 5-FU-Resistant Colorectal Cancer Cells.

5-fluorouracil (5-FU) is a first-line chemotherapeutic agent used to treat colorectal cancer (CRC). However, 5-FU induces drug resistance and activation of cancer stem cells (CSCs). In the present study, we designed a novel biocompatible nanomedicine system with high efficacy and biocompatibility by synthesizing mesoporous silica nanoparticle (MSN)-structured ZnO and gold ions. Oleuropein (OLP) is a phenolic compound derived from olive leaves that exerts anti-cancer effects. Therefore, we synthesized OLP-loaded ZnO/Au MSNs (ZnO/Au/OLP MSNs) and examined their anti-cancer effects on 5-FU-resistant CRC cells. ZnO/Au MSNs were synthesized and functionalized, and their physical and chemical compositions were characterized using UV-visible spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). Their effects were assessed in terms of cellular proliferation capacity, migration and invasion ability, colony-forming ability, spheroid-forming ability, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. ZnO/Au MSNs were mostly composed of various ions containing ZnO and gold ions, had a spheroid phenotype, and exhibited no cytotoxicity. ZnO/Au/OLP MSNs reduced cell viability and CSC formation and induced apoptosis of 5-FU-resistant CRC cells via necrosis via ROS accumulation and DNA fragmentation. ZnO/Au/OLP MSNs exhibited anti-cancer activity by upregulating necrosis. These results revealed that ZnO/Au/OLP MSNs are a novel drug delivery system for 5-FU CRC therapy.

Synergistic suppression of ovarian cancer by combining NRF2 and GPX4 inhibitors: in vitro and in vivo evidence

Ovarian cancer is a significant challenge in women's health due to the lack of effective screening and diagnostic methods, often leading to late detection and the highest mortality rate among all gynecologic tumors worldwide. Recent research has shown that ovarian cancer has an "iron addiction" phenotype which makes it vulnerable to ferroptosis inducers. We tested the combination of NRF2-targeted inhibitors with GPX4-targeted inhibitors in ovarian cancer through in vitro and in vivo experiment. The data showed that combination treatment effectively suppressed adherent cell growth, inhibited suspended cell spheroid formation, and restrained the ability of spheroid formation in 3D-culture. Mechanistically, the combination induced accumulation of ROS, 4-HNE, as well as activation of caspase-3 which indicates that this combination simultaneously increases cell ferroptosis and apoptosis. Notably, inhibition of GPX4 or NRF2 can suppress ovarian cancer spreading and growth in the peritoneal cavity of mice, while the combination of NRF2 inhibitor ML385 with GPX4 inhibitors showed a significant synergistic effect compared to individual drug treatment in a syngeneic mouse ovarian cancer model. Overall, these findings suggest that combining NRF2 inhibitors with GPX4 inhibitors results in a synergy suppression of ovarian cancer in vitro and in vivo, and maybe a promising therapeutic strategy for the treatment of ovarian cancer.

Deciphering head and neck cancer microenvironment: Single-cell and spatial transcriptomics reveals human papillomavirus-associated differences.

Human papillomavirus (HPV) is a major causative factor of head and neck squamous cell carcinoma (HNSCC), and the incidence of HPV- associated HNSCC is increasing. The role of tumor microenvironment in viral infection and metastasis needs to be explored further. We studied the molecular characteristics of primary tumors (PTs) and lymph node metastatic tumors (LNMTs) by stratifying them based on their HPV status. Eight samples for single-cell RNA profiling and six samples for spatial transcriptomics (ST), composed of matched primary tumors (PT) and lymph node metastases (LNMT), were collected from both HPV- negative (HPV- ) and HPV-positive (HPV+ ) patients. Using the 10x Genomics Visium platform, integrative analyses with single-cell RNA sequencing were performed. Intracellular and intercellular alterations were analyzed, and the findings were confirmed using experimental validation and publicly available data set. The HPV+ tissues were composed of a substantial amount of lymphoid cells regardless of the presence or absence of metastasis, whereas the HPV- tissue exhibited remarkable changes in the number of macrophages and plasma cells, particularly in the LNMT. From both single-cell RNA and ST data set, we discovered a central gene, pyruvate kinase muscle isoform 1/2 (PKM2), which is closely associated with the stemness of cancer stem cell-like populations in LNMT of HPV- tissue. The consistent expression was observed in HPV- HNSCC cell line and the knockdown of PKM2 weakened spheroid formation ability. Furthermore, we found an ectopic lymphoid structure morphology and clinical effects of the structure in ST slide of the HPV+ patients and verified their presence in tumor tissue using immunohistochemistry. Finally, the ephrin-A (EPHA2) pathway was detected as important signals in angiogenesis for HPV- patients from single-cell RNA and ST profiles, and knockdown of EPHA2 declined the cell migration. Our study described the distinct cellular composition and molecular alterations in primary and metastatic sites in HNSCC patients based on their HPV status. These results provide insights into HNSCC biology in the context of HPV infection and its potential clinical implications.

Orai1α and Orai1β support calcium entry and mammosphere formation in breast cancer stem cells

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells.

Scoulerine promotes cytotoxicity and attenuates stemness in ovarian cancer by targeting PI3K/AKT/mTOR axis.

In women, ovarian cancer is a common gynecological cancer associated with poor prognosis, reoccurrence and chemoresistance. Scoulerine, a benzylisoquinoline alkaloid, has been reported effective against several carcinomas. Thus, we investigated the impact of scoulerine on ovarian cancer cells (OVCAR3). Cell viability was assessed by MTT assay, migration was determined by Boyden Chamber assay, while the invasion was monitored by Boyden Chamber assay using the matrigel. The stemness properties of OVCAR3 cells were observed by tumorsphere assay. Epithelial to mesenchymal transition (EMT) and stemness-related protein markers were monitored by real-time PCR analysis and immunoblotting. Scoulerine inhibits the viability of OVCAR3 cells with the IC 50 observed at 10 µmol L-1 after 48 h treatment. Scoulerine inhibited the colony-forming ability, migration and invasiveness of OVCAR3 cells in a dose-dependent fashion. Scoulerine treatment also drastically reduced the spheroid-forming ability of OVCAR3 cells. The mesenchymal and stemness--related markers like N-cadherin, vimentin, CD-44, Oct-4, Sox-2 and Aldh1A1 were downregulated, whereas the epithelial markers like E-cadherin and CD-24 were upregulated in scoulerine-treated cells. The upstream PI3K/Akt/mTOR-axis was downregulated in scoulerine-treated cells. We concluded that scoulerine successfully perturbs the cancerous properties of OVCAR3 cells by targeting the PI3K/Akt/mTOR axis. In vivo studies revealed a substantial decrease in tumor mass and volume after scoulerine treatment. Furthermore, scoulerine treatment was found to decrease oxidative stress factors in ovarian cancer mice model. Scoulerine is a potential anticancer agent against ovarian cancer and can be considered as a lead molecule for this malignancy, provided further investigations are performed.

Hypericum roeperianum bark extract suppresses breast cancer proliferation via induction of apoptosis, downregulation of PI3K/Akt/mTOR signaling cascade and reversal of EMT

Ethnopharmacological relevanceHypericum roeperianum is a medicinal spice traditionally used in West Africa to treat female sterility, fungal infections, and cancer. It has previously been reported that H. roeperianum exhibits cytotoxic potential by reducing the viability of cancer cells involving multidrug-resistant phenotypes, but its underlying molecular mechanism remains unknown. Aim of the studyThe mechanistic involvement of H. roeperianum methanolic crude extract (HRC) in attenuating breast cancer progression by exploring the effects on mitochondrial apoptosis and epithelial-mesenchymal transition (EMT) was investigated. Materials and methodsIn the present study, we examined the anticancer properties of HRC through MTT assay, colony formation, wound healing assay, spheroid formation, DNA fragmentation and flow cytometry for cell cycle arrest, apoptosis (Annexin V/PI staining) and mitochondrial membrane potential (MMP) (JC-1) detection. In addition, western blot analysis of various proteins and quantitative real time PCR of various genes involved in apoptosis, EMT and the PI3K/Akt/mToR signal transduction pathway were performed. ResultsThis study revealed that HRC treatment significantly decreased breast cancer cell viability, colony forming efficiency and reduced the ability of cell migration and spheroid formation. HRC also induced apoptosis in MDA-MB-231 and MCF-7 via promoting G0/G1 cell cycle arrest, disruption of mitochondrial membrane potential and induction of DNA damage. The crude extract induced apoptosis by activating the intrinsic pathway with a stronger effect that relies on the combined potency of associated molecular markers including Bax, Bad, Bcl-2, cytochrome C, caspase-9, and cleaved-PARP. It was also found that HRC regulates the PI3K/Akt/mToR pathway. In addition, HRC inhibited EMT by expressional alteration of Vimentin and E-cadherin, as well as the regulatory transcription factors such as Snail and Slug. The in vitro findings reflected similar mechanistic approach in 4T1 cell induced syngeneic mice model, indicating the reduction of tumor volume along with the significant expressional alteration of EMT and apoptotic markers. ConclusionTaken together the findings concluded that H. roeperianum is a potential source of cytotoxic phytochemicals that exhibit abortifacient effect on breast cancer, both in vitro and in vivo, thus could further be utilized in breast cancer therapy.

A dual-action niclosamide-based prodrug that targets cancer stem cells and inhibits TNBC metastasis

Chemotherapy typically destroys the tumor mass but rarely eradicates the cancer stem cells (CSCs) that can drive metastatic recurrence. A key current challenge is finding ways to eradicate CSCs and suppress their characteristics. Here, we report a prodrug, Nic-A, created by combining a carbonic anhydrase IX (CAIX) inhibitor, acetazolamide, with a signal transducer and transcriptional activator 3 (STAT3) inhibitor, niclosamide. Nic-A was designed to target triple-negative breast cancer (TNBC) CSCs and was found to inhibit both proliferating TNBC cells and CSCs via STAT3 dysregulation and suppression of CSC-like properties. Its use leads to a decrease in aldehyde dehydrogenase 1 activity, CD44high/CD24low stem-like subpopulations, and tumor spheroid-forming ability. TNBC xenograft tumors treated with Nic-A exhibited decreased angiogenesis and tumor growth, as well as decreased Ki-67 expression and increased apoptosis. In addition, distant metastases were suppressed in TNBC allografts derived from a CSC-enriched population. This study thus highlights a potential strategy for addressing CSC-based cancer recurrence.

Abstract 2102: Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model

Abstract Esophageal squamous cell carcinoma (ESCC) metastatic-initiating cells are heterogenous subpopulations that are hypothesized to promote distal metastasis. Pulmonary metastatic human-origin ESCC mice model was established in our laboratory, chronological flow cytometry collection of tumor cells isolated from metastatic mice lungs at different timepoints inoculated via intravenous injection was followed by 10X genomics multiplex single-cell transcriptomic sequencing (scRNA-seq). Bioinformatics analyses denoted a 7-signature panel previously introduced with preliminary data, follow-up validations of signature-enriched subpopulation in silico, in vitro, and in vivo were performed in this study. In silico bulk transcriptomic sequencing (RNA-seq) of single signature-enriched KYSE30 subpopulation followed by gene ontology (GO) enrichment analyses indicated significantly (FDR&amp;lt;0.05) enhanced biological processes involved in cell motility, cell migration and cell adhesion, compared to signature-low subpopulation. In vitro western blotting showed elevated mesenchymal markers expression, while diminished epithelial markers expression. In vitro real-time qPCR revealed single signature-enriched subpopulation has co-enrichment of other signature marker RNAs compared to control group. Further in vitro functional assays demonstrated signature-enriched subpopulation possesses strengthened spheroid formation ability (p&amp;lt;0.05) and soft agar colony formation ability (p&amp;lt;0.05) as compared to signature-low subpopulation. In addition to previous preliminary data on early survival ability in vivo, signature-enriched subpopulation was significantly more favorable in sustained cell survival and proliferation, and formation of larger size of metastatic lesions in mice lungs at 2 months post-intravenous injection as illustrated by flow cytometry (p&amp;lt;0.05) and immunohistochemistry (IHC) staining analyses (p&amp;lt;0.05) respectively as compared to signature-low subpopulation. To validate the protein expression of signature markers, 6-colour multiplex IHC staining was performed on KYSE30 cell line and metastatic mice lungs collected at different timepoints. Multiplex IHC staining exhibited that these signature markers were expressed in subpopulation of cells as well as expressed on inoculated tumor cells in metastatic mice lungs with timeline pattern. In summary, signature-enriched subpopulation manifested the metastatic-initiating properties in ESCC via various in silico, in vitro, and in vivo experimental analyses. Further multiplex staining in clinical tissue microarray would be performed and analyzed to examine feasibility of adopting these signature markers for facilitating clinical diagnosis and molecular targeting of metastatic ESCC. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Kwong Yiu Lam, Hongyu Zhou, Xinyuan Guan. Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2102.

Abstract 1739: Reversal of lenvatinib resistance by curcumin via EGFR signaling pathway in hepatocellular carcinoma

Abstract Background: Patients with unresectable advanced hepatocellular carcinoma (HCC) are often treated with systemic therapies, and lenvatinib is currently used as one of the first-line therapies. However, the overall response rate from lenvatinib treatment is only 24.1%, and even the patients who initially respond to this treatment often subsequently acquire therapeutic resistance to this drug. Although the mechanism of lenvatinib resistance remains unclear, recent studies have suggested the involvement of epidermal growth factor receptor (EGFR) activation, indicating that a combination of lenvatinib and EGFR inhibitors might have potential clinical applications in HCC. Curcumin is one of the most widely studied natural phytochemicals as an anti-cancer compound. Many previous studies have revealed that it also functions as a potent tyrosine kinase inhibitor that targets EGFR. Accordingly, herein we hypothesized that the anti-EFGR potential of curcumin might help overcome lenvatinib resistance in HCC. Methods: We first developed two lenvatinib-resistant cell lines and performed a series of cell line experiments, including cell proliferation, invasion, sphere formation, and apoptosis assays, to determine the impact of curcumin in overcoming this resistance. We evaluated EGFR and its downstream signaling pathway to clarify its mechanistic role in lenvatinib resistance and its subsequent reversal mediated by curcumin. Results: Among five HCC cell lines, the most sensitive and relatively insensitive cells were Huh-7 (IC50: 2.0 µM) and PLC-PRF-5 (IC50: 50.0 µM), respectively. Accordingly, we successfully established lenvatinib-resistant cells, which had significantly higher IC50 concentrations; resistant Huh-7 (IC50: 50.0 µM) and resistant PLC-PRF-5 (IC50: &amp;gt;100.0 µM). We noted that co-administration of curcumin with lenvatinib inhibited cell proliferation and invasion compared to lenvatinib or curcumin monotherapy. Combination therapy also promoted apoptosis in resistant Huh-7 (lenvatinib, curcumin vs. combination (9.7%, 21.8%, vs. 30.2%) and resistant PLC-PRF-5 (12.2%, 23.1%, vs. 38.2%). This combination treatment also significantly diminished the spheroid-forming ability of cancer cells via suppression of cancer stemness markers such as CD44 (p&amp;lt;0.01) and CD133 (p&amp;lt;0.01). EGFR expression was significantly upregulated in resistant cell lines, suggesting that activation of the EGFR signaling cascade might trigger lenvatinib resistance. Following treatment with a curcumin and lenvatinib combination, EGFR expression in resistant cell lines was significantly downregulated, suppressing the PI3K-AKT-mTOR axis in HCC cells. Conclusions: We provide the first evidence that curcumin could overcome lenvatinib resistance in HCC cells via suppression of EGFR and its downstream targets, which can offer a novel therapeutic strategy in HCC. Citation Format: Katsuki Miyazaki, Yuji Morine, Caiming Xu, Mitsuo Shimada, Ajay Goel. Reversal of lenvatinib resistance by curcumin via EGFR signaling pathway in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1739.

TRRAP Enhances Cancer Stem Cell Characteristics by Regulating NANOG Protein Stability in Colon Cancer Cells.

NANOG, a stemness-associated transcription factor, is highly expressed in many cancers and plays a critical role in regulating tumorigenicity. Transformation/transcription domain-associated protein (TRRAP) has been reported to stimulate the tumorigenic potential of cancer cells and induce the gene transcription of NANOG. This study aimed to investigate the role of the TRRAP-NANOG signaling pathway in the tumorigenicity of cancer stem cells. We found that TRRAP overexpression specifically increases NANOG protein stability by interfering with NANOG ubiquitination mediated by FBXW8, an E3 ubiquitin ligase. Mapping of NANOG-binding sites using deletion mutants of TRRAP revealed that a domain of TRRAP (amino acids 1898-2400) is responsible for binding to NANOG and that the overexpression of this TRRAP domain abrogated the FBXW8-mediated ubiquitination of NANOG. TRRAP knockdown decreased the expression of CD44, a cancer stem cell marker, and increased the expression of P53, a tumor suppressor gene, in HCT-15 colon cancer cells. TRRAP depletion attenuated spheroid-forming ability and cisplatin resistance in HCT-15 cells, which could be rescued by NANOG overexpression. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model, which could be reversed by NANOG overexpression. Together, these results suggest that TRRAP plays a pivotal role in the regulation of the tumorigenic potential of colon cancer cells by modulating NANOG protein stability.

CHEMORESISTANCE RELATED TO HYPOXIA ADAPTATION INMESOTHELIOMA CELLS FROM TUMOR SPHEROIDS.

Hypoxia has been noted as a key factor for induction and maintenance of cancer stemness thereby leading to therapy resistance. Three-dimensional (3D) spheroid models demonstrate a heterogeneity of hypoxic regions replicating the in vivo situation within tumors. Utilizing an established 3D spheroid model, we investigated whether extrinsic hypoxia reinforced chemoresistance in malignant pleural mesothelioma (MPM) spheroids. Tumor spheres were generated from Meso-1 (a typical human MPM cell line) cells having high spheroid-forming ability. To induce hypoxia condition, we utilized a hypoxia chamber with regulation of O2and CO2levels. Cell viability was estimated by a WST-8assay. Real-time polymerase chain reaction and Western blot were performed to evaluate the expression at mRNA and protein levels. Compared with cells cultured in the two-dimensional monolayer model, tumor sphere cells showed elevated mRNA levels of cancer stemness markers (CD26, CD44and ABCG2) and protein levels of the stemness and hypoxia adaptation markers (ABCG2, ALDH1A1and HIFs). Correlating with this, 3D spheroid cells were more resistant to permetrexed and topotecan than the two-dimensional cells, indicative of their potential for hypoxic adaptation. Furthermore, significantly stronger resistance to both chemotherapeutic agents was observed in spheroid cells upon hypoxic challenge compared to spheroid cells under normoxia. From the present data, it is concluded that hypoxia adaptation of MPM cells from tumor spheres could enhance their chemoresistance.

Ror1 is expressed inducibly by Notch and hypoxia signaling and regulates stem cell-like property of glioblastoma cells.

Ror1 plays a crucial role in cancer progression by regulating cell proliferation and migration. Ror1 is expressed abundantly in various types of cancer cells and cancer stem-like cells. However, the molecular mechanisms regulating expression of Ror1 in these cells remain largely unknown. Ror1 and its putative ligand Wnt5a are expressed highly in malignant gliomas, especially in glioblastomas, and the extents of Ror1 expression are correlated positively with poorer prognosis in patients with gliomas. We show that Ror1 expression can be upregulated in glioblastoma cells under spheroid culture, but not adherent culture conditions. Notch and hypoxia signaling pathways have been shown to be activated in spheroid-forming glioblastoma stem-like cells (GSCs), and Ror1 expression in glioblastoma cells is indeed suppressed by inhibiting either Notch or hypoxia signaling. Meanwhile, either forced expression of the Notch intracellular domain (NICD) in or hypoxic culture of glioblastoma cells result in enhanced expression of Ror1 in the cells. Consistently, we show that both NICD and hypoxia-inducible factor 1 alpha bind to upstream regions within the Ror1 gene more efficiently in GSCs under spheroid culture conditions. Furthermore, we provide evidence indicating that binding of Wnt5a to Ror1, upregulated by Notch and hypoxia signaling pathways in GSCs, might promote their spheroid-forming ability. Collectively, these findings indicate for the first time that Notch and hypoxia signaling pathways can elicit a Wnt5a-Ror1 axis through transcriptional activation of Ror1 in glioblastoma cells, thereby promoting their stem cell-like property.

Identification of a Novel Potent CYP4Z1 Inhibitor Attenuating the Stemness of Breast Cancer Cells through Lead Optimization.

Pharmacological targeting cancer stem cells are emerging as a novel therapeutic modality for cancer treatment and prevention. Human cytochrome P450 enzyme CYP4Z1 represents a promising target for its potential role in attenuating the stemness of breast cancer cells. In order to develop potent and selective CYP4Z1 inhibitors, a series of novel N-hydroxyphenylformamidines were rationally designed and synthesized from a pan-CYP inhibitor HET0016. CYP4Z1 inhibitory activities of the newly synthesized derivatives were evaluated, and the structure-activity relationships (SARs) were summarized. Among them, compound 7c exhibited the best inhibitory activity with an IC50 value of 41.8 nM. Furthermore, it was found that 7c decreased the expression of stemness markers, spheroid formation, and metastatic ability as well as tumor-initiation capability in a concentration-dependent manner in vitro and in vivo. Altogether, compound 7c might be a potential lead compound to develop CYP4Z1 inhibitor with more favorable druggability for clinical application to treat breast cancer.

ABCG2, CD44 and SOX9 are increased with the acquisition of drug resistance and involved in cancer stem cell activities in head and neck squamous cell carcinoma cells.

Cancer stem cells are a sub-population of cancer cells with self-renewal activity that play key roles in tumor resistance to chemotherapy and radiation. Several cancer stem cell markers have been identified to correlate with clinical prognosis. However, which marker is associated with which cancer stem cell characteristic is unclear. The present study aimed to clarify the relationship between cancer stem cell markers associated with drug resistance acquisition and the characteristics of cancer stem cells. We generated cisplatin-resistant head and neck squamous cell carcinoma cells by culturing cells in increasing concentrations of cisplatin. The cisplatin-resistant head and neck squamous cell carcinoma cells also acquired multidrug resistance and were named resistant HSC-3 (R HSC-3) cells. R HSC-3 showed no differences in cell proliferation or cell cycle distributions compared with parental cells. R HSC-3 cells showed increased drug excretion ability and elevated expression of ATP-binding cassette subfamily G member 2 (ABCG2), a drug excretion pump. R HSC-3 cells also highly expressed CD44, a cancer stem cell marker, and exhibited enhanced cell invasion and spheroid formation abilities. Furthermore, the stem cell-related factor SRY-box transcription factor 9 (SOX9) was identified as increased in R HSC-3 cells by microarray analysis. Knockdown experiments showed that SOX9 and ABCG2 were involved in the drug excretion ability of R HSC3 cells and ABCG2 was involved in the spheroid formation ability of R HSC-3 cells. These results indicate that CD44, SOX9 and ABCG2 expression levels were enhanced in head and neck squamous cell carcinoma cells that acquired multidrug resistance and that these molecules are important for maintaining cancer stem cell characteristics. Overall, regulating CD44, SOX9 and ABCG2 may be a strategy to inhibit cancer stem cells.