Non-adherent Spheroids Research Articles

SAT272 Insulin Signaling In Hyperactivation Of The HPA Axis In Metabolic Diseases

Abstract Disclosure: D. Cozma: None. P. Siatra: None. I. Oikonomakos: None. D. Kalra: None. U.A. Friedrich: None. A. Dahl: None. S.R. Bornstein: None. S. Zeissig: None. C.L. Andoniadou: None. C. Steenblock: None. Stress can lead to an increase in body fat and obesity as main clinical conditions preceding the metabolic syndrome [1]. In metabolic stress, hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis has been observed, resulting in increased steroidogenesis and alteration of cortisol secretion [1]. This increased sensitivity of the HPA axis may be related to the development of comorbidities or severe illness in patients with metabolic diseases and vice versa. Disorders of the HPA axis, e.g., Cushing’s disease, resulting from overproduction of ACTH, share similar clinical symptoms with metabolic diseases [1]. The molecular signaling mechanisms that cause such changes in the HPA axis under metabolic stress remain elusive. Previous studies indicate a role of insulin in the regulation of adrenal steroidogenesis by upregulating the transcriptional activity of NR5A1 [2]. Here, we study the molecular mechanisms to insulin-induced hyperactivation of the HPA axis and characterize the molecular changes that lead to altered cortisol secretion. We generated non-adherent spheroids from primary adrenocortical cells in vitro and treated these with insulin. This resulted in an increase of spheroid diameter over time [Slope: 5.163 ± 0.994; P=0.0139; n=11], compared to control [Slope: -4.058 ± 0.987; P=0.0261; n=10], indicating greater cell expansion following insulin treatment. Similarly, repeating this experiment with adherent primary anterior pituitary stem cell colonies showed an increase in colony size when treated with insulin compared to control [Diameter: 1221.0 ± 290.9 µm vs. 914.5 ± 256.2 µm; P=0.0079; n=3]. This indicates that the stem cell populations of the adrenal and pituitary gland are responsive to insulin stimulation, likely leading to increased proliferation. This is complementary to our previous studies where we revealed increased secretion of aldosterone in insulin-stimulated adrenocortical cultures [3]. To identify signaling pathways related to insulin stimulation, we performed bulk RNA sequencing of in vitro primary cultures of adrenocortical and pituitary cells with and without insulin treatment. Analysis of these data sets reveals specific signaling pathways that are candidates in regulation of metabolic stress in vivo. Using mouse models of obesity and diabetes along with genetic lineage-tracing studies, we aim to decipher the role of these pathways on HPA axis stem/progenitor cells during metabolic stress. The outcomes will pave the way for a better understanding of HPA axis activity during metabolic stress adaption, and the role of the HPA axis in patients suffering from metabolic diseases.

SAT268 Regulation Of Stem/Progenitor Cells Of The HPA Axis During Stress Adaptation

Abstract Disclosure: P. Siatra: None. D. Cozma: None. I. Berger: None. I. Oikonomakos: None. M. Theodoropoulou: None. S.R. Bornstein: None. C.L. Andoniadou: None. C. Steenblock: None. Chronic stress is a pervasive concern in the modern society. A long-term hyper activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to elevated amounts of stress hormones [e.g. ACTH and glucocorticoids (GCs)]. This can incur maladaptation that eventually contributes to mental illness, cardiovascular dysfunctions, diabetes, cancer and autoimmune diseases. Emerging key players for stress adaptation of the HPA axis are stem/progenitor cell populations. In this study, we aim to unravel the mechanisms involved in the regulation of stem/progenitor cells of the HPA axis during stress adaptation. To that aim, we incorporated an in vitro system of non-adherent spheroids from primary adrenocortical mouse cells that were stimulated with ACTH. The treatment resulted in the increased growth of the spheroids compared to unstimulated controls indicating a potential role of the hormone in cell proliferation and stem cell behaviour. This result is in line with the enlarged adrenals observed in patients with Cushing’s syndrome. To assess the contribution of stem cells during stress adaptation in the adrenal cortex in vivo, we performed immobilization stress and lineage tracing studies using the tamoxifen inducible Gli1:CreERT2/R26R:eYFP mouse line. By combining these results along with single-cell RNA-seq technology, we aim to shed light into molecular factors involved in the adult adrenal cortex regeneration and remodelling. To follow the effect of GC excesses on the negative feedback loop observed during a normal stress response on the pituitary gland, we stimulated adherent primary anterior pituitary colonies with dexamethasone and corticosterone. GC treatment led to downregulation of Pomc expression, a precursor polypeptide for ACTH and other hormones, in the treated colonies compared to the control. This downregulation caused by GCs could be mediated through different gene targets of the glucocorticoid receptor as well as via paracrine signalling from pituitary stem cells. Altogether, our results will provide us with more profound knowledge of mechanisms underlying interactions between stressors and stem/progenitor cells of the HPA axis along with novel therapeutic targets for stress-associated disorders. Presentation: Saturday, June 17, 2023

Abstract P1-05-09: Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method

Abstract Purpose: Immunophenotyping is a useful tool for diagnostic and therapeutic assessment of heterogenous breast cancer cells that depend on external factors and growth conditions. The objective of this study was to characterize the phenotype of patient-derived breast cancer cells (PDBCCs) grown in different culture medium and under different conditions including 2D monolayer and non-adherent 3D spheroid culture by using flow cytometry. Methods: PDBCCs and normal breast cells were harvested from fresh surgical specimens (N=11) of breast tumor and normal tissues. PDBCCs were grown in various culture media under monolayer, spheroid and organoid conditions. Flow cytometry, qRT-PCR, ALDEFLUOR assay, β-galactosidase staining and RNA-seq were performed. Results: PDBCC growth rates were different for each patient case. As compared with full media, A83-01 removal increased EpCAM+CD24+ cells (1.8-fold, p<0.029) and decreased EpCAM-CD44high/+CD24- (0.45-fold, p=0.029). EGF removal increased EpCAM+CD49f− cells (14.1-fold, p=0.029). Removal of both EGF and A83-01 induced senescent cells with high β-galactosidase activity. EpCAM-CD44high/+CD24- cells, ALDHhigh cells, and EpCAMlow/-CD49f+ cells were enriched by 3D spheroids relative to 2D monolayer. 562 genes with 2-fold change and, p-value<0.05 were differentially expressed in 3D spheroids compared to 2D monolayers. Based on public database, many genes associated with poor prognosis in 3D spheroids were identified (Table 1). Table 1. Clinically poor prognosis-associated DEGs were selected from tumor spheroids based on recurrence-free survival analysis using Kaplan-Meier Plotter on database that includes estrogen receptor positive patients. B, coefficient of regression; HR, hazard ratio; CI, confidence interval; ER, estrogen receptor, HER2, human epidermal receptor 2; RFS, recurrent free survival; FC, fold change. Conclusion: PDBCC phenotypes can be modulated by external factors and culture methods. Cancer stem-like properties of PDBCCs can be enriched by 3D spheroids. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for heterogenous breast cancer Clinically poor prognosis-associated DEGs based on recurrence-free survival analysisGenes symbolLog2(FC) P<0.05Prognosis95% CI for Exp(B)HRlowerupperP-valueTop 30 of DEGsUpregulated genesMMP110.90Poor1.611.381.877.5e-10BMP68.85Poor1.21.021.040.024MYCN7.20Poor1.231.041.460.015COL22A17.12Poor1.571.172.110.0026CYP1A1(CP11)7.01Poor1.271.051.530.012PGBD56.78Poor1.221.051.420.011S100P6.70Poor1.491.281.733.3e-07CALML56.24Poor1.271.0810480.0034PPP2R2C6.19Poor1.491.131.970.005PMEPA16.07Poor1.181.011.40.043Downregulated geneFOSB-6.36Poor0.630.520.762e-06LINC00844-6.01Poor0.670.480.930.017DLK2-5.46Poor0.820.710.960.011IL33-5.31Poor0.650.550.773.1e-7SCN2B-5.09Poor0.80.670.950.0099CLDN19-4.80Poor0.660.490.890.0065FOS-4.67Poor0.670.570.798.5e-7EGR1-4.63Poor0.680.580.822.3e-0.5JAM2-4.62Poor0.80.680.950.0092MYH11-4.38Poor0.760.660.890.00045ALPL-4.35Poor0.820.710.960.015 Citation Format: Seungyeon RYU, Hoe Suk Kim, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Wonshik Han. Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method [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 P1-05-09.

Peritoneal Spread of Ovarian Cancer Harbors Therapeutic Vulnerabilities Regulated by FOXM1 and EGFR/ERBB2 Signaling.

Peritoneal spread is the primary mechanism of metastasis of ovarian cancer, and survival of ovarian cancer cells in the peritoneal cavity as nonadherent spheroids and their adherence to the mesothelium of distant organs lead to cancer progression, metastasis, and mortality. However, the mechanisms that govern this metastatic process in ovarian cancer cells remain poorly understood. In this study, we cultured ovarian cancer cell lines in adherent and nonadherent conditions in vitro and analyzed changes in mRNA and protein levels to identify mechanisms of tumor cell survival and proliferation in adherent and nonadherent cells. EGFR or ERBB2 upregulated ZEB1 in nonadherent cells, which caused resistance to cell death and increased tumor-initiating capacity. Conversely, Forkhead box M1 (FOXM1) was required for the induction of integrin β1, integrin-α V, and integrin-α 5 for adhesion of cancer cells. FOXM1 also upregulated ZEB1, which could act as a feedback inhibitor of FOXM1, and caused the transition of adherent cells to nonadherent cells. Strikingly, the combinatorial treatment with lapatinib [dual kinase inhibitor of EGFR (ERBB1) and ERBB2] and thiostrepton (FOXM1 inhibitor) reduced growth and peritoneal spread of ovarian cancer cells more effectively than either single-agent treatment in vivo. In conclusion, these results demonstrate that FOXM1 and EGFR/ERBB2 pathways are key points of vulnerability for therapy to disrupt peritoneal spread and adhesion of ovarian cancer cells. SIGNIFICANCE: This study describes the mechanism exhibited by ovarian cancer cells required for adherent cell transition to nonadherent form during peritoneal spread and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5554/F1.large.jpg.

Cryptotanshinone attenuates the stemness of non-small cell lung cancer cells via promoting TAZ translocation from nuclear to cytoplasm

BackgroundCancer stem cells (CSCs) are regarded as the root of tumor progression, thus representing an anti-cancer therapy through targeting this cell sub-population.MethodsNon-small cell lung cancer (NSCLC) CSCs were enriched by non-adherent spheroid formation analysis. Lentivirus infection was used to stably change gene expression. Cell cycle, EdU incorporation, cell apoptosis, cell viability, ALDH1 activity, spheroid formation and in vivo tumor initiation assays were performed to detect the effects of Cryptotanshinone (CT), a traditional Chinese herb medicine, on the stemness of NSCLC cells. RNA-sequencing combined qRT-PCR and western blot analysis were constructed to explore the underlying mechanism contributing to CT-mediated effects.ResultsCT could attenuate the stemness of NSCLC CSCs, as evident by the reduced spheroid formation ability, stemness marker expression and ALDH1 activity. Additionally, CT provoked NSCLC CSCs entry into the cell cycle. RNA-sequencing analysis showed that Hippo signaling pathway was highly enriched in NSCLC CSCs with CT treatment. Further experiments disclosed that CT decreased TAZ (a regulatory master of Hippo pathway) expression via promoting its nuclear-cytoplasm translocation in NSCLC CSCs. Also, overexpression of TAZ partially saved the attenuation of CT on the stemness of NSCLC CSCs. Notably, CT enhanced the sensitivity of tyrosine kinase inhibitor (TKI) and chemotherapy in NSCLC CSCs.ConclusionsThis work reveals that CT attenuates NSCLC CSC stemness, implying the possibility of CT as an adjuvant therapy for NSCLC.

Highly expressed STAT1 contributes to the suppression of stemness properties in human paclitaxel-resistant ovarian cancer cells.

Signal transducer and activator of transcription-1 (STAT1) is an important factor in various cellular processes. The cancer stem cell (CSC) is considered as a tumor-initiating cell that drives the inner hierarchy in many cancers including epithelial ovarian cancer (EOC). Here, we explored for the first time the regulation of STAT1 on stemness properties in chemoresistant EOC cells. The paclitaxel (PTX)-resistant EOC cell line (OV3R-PTX) was derived from PTX-sensitive OVCAR-3 cells treated by the PTX regimen. A single cell clone OV3R-PTX-B4 was selected by fluorescence-activated cell sorting. PTX-resistant cells grew slowly in conventional 2D and 3D cultures, but tumor xenograft with PTX-resistant cells grew fast in nude mice. Interestingly, OV3R-PTX-B4 cells shared the characteristics of CSCs and stemness properties were found to be increased in the non-adherent spheroid culture system. The PTX-resistant cells had a high expression of CSC-related markers and low expression of STAT1 that had a high methylation level of CpG in its promoter region. Overexpressed STAT1 suppressed stemness properties, cell proliferation, and colony formation and favored the overall survival of patients with EOC. In summary, these data indicate a regulatory mechanism of STAT1 underlying drug resistance and provide a potential therapeutic application for EOC patients with PTX resistance.

Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance

Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Method: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. Conclusion: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Limonin provokes hepatocellular carcinoma cells with stemness entry into cycle via activating PI3K/Akt signaling

The inhibitory roles of limonin have been revealed in various tumors. However, the roles and related mechanism of limonin in hepatocellular carcinoma (HCC) progression are still confused. Here, we collected non-adherent spheroids formed by HCC cells and found that the proportion of spheroids in G0 phase was remarkably higher than that in HCC cells. Additionally, limonin increased EdU incorporation without affecting apoptosis in spheroids. Notably, upon limonin treatment, the proportion of spheroids in G0 was decreased and S/G2/M increased. Furthermore, we found that limonin attenuated the stemness of HCC cells, characterized as the decreased ALDH1 activity, stemness marker expression and spheroid formation ability. Moreover, an integrated transcriptional analysis based on RNA-sequencing data was employed to gain mechanistic insight into limonin functioning, and we found that the most significant pathways identified centered on PI3K/Akt signaling. qRT-PCR and western blot obtained the consistent results. Overall, these data suggest that limonin attenuates the stemness of HCC cells by reducing cellular quiescence through activating PI3K/Akt signaling.

Neurturin-containing laminin matrices support innervated branching epithelium from adult epithelial salispheres

Cell transplantation of autologous adult biopsies, grown ex vivo as epithelial organoids or expanded as spheroids, are proposed treatments to regenerate damaged branching organs. However, it is not clear whether transplantation of adult organoids or spheroids alone is sufficient to initiate a fetal-like program of branching morphogenesis in which coordinated branching of multiple cell types including nerves, mesenchyme and blood vessels occurs. Yet this is an essential concept for the regeneration of branching organs such as lung, pancreas, and lacrimal and salivary glands. Here, we used factors identified from fetal organogenesis to maintain and expand adult murine and human epithelial salivary gland progenitors in non-adherent spheroid cultures, called salispheres. These factors stimulated critical developmental pathways, and increased expression of epithelial progenitor markers such as Keratin5, Keratin14, FGFR2b and KIT. Moreover, physical recombination of adult salispheres in a laminin-111 extracellular matrix with fetal salivary mesenchyme, containing endothelial and neuronal cells, only induced branching morphogenesis when neurturin, a neurotrophic factor, was added to the matrix. Neurturin was essential to improve neuronal survival, axon outgrowth, innervation of the salispheres, and resulted in the formation of branching structures with a proximal-distal axis that mimicked fetal branching morphogenesis, thus recapitulating organogenesis. Epithelial progenitors were also maintained, and developmental differentiation programs were initiated, showing that the fetal microenvironment provides a template for adult epithelial progenitors to initiate branching and differentiation. Further delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental-like program in vitro and to regenerate branching organs in vivo.

Generation of Cardiac Organoids Using Cardiomyocytes, Endothelial Cells, Epicardial Cells, and Cardiac Fibroblasts Derived From Human Induced Pluripotent Stem Cells

Two‐dimensional (2D) cell culture models fail to recapitulate the complex tissue architectures, extracellular matrices, and cellular crosstalk that occur in vivo. Three‐dimensional models (3D), including scaffold‐free organoids, derived from a single human induced pluripotent stem cell (hiPSC) cell source can more accurately model cell‐cell and cell‐matrix interactions to study specific genetic and organ‐level diseases that are difficulty to study in vitro. Additionally, 3D culture methods utilizing simulated microgravity environments have been shown to improve (iPSC)‐derived cardiomyocyte (CM) maturity, differentiation efficiency, and function, while promoting self‐organization in models that include multiple cardiac cell types. However, current published models have incorporated only one or two cell types and have used cells from a mixture of primary and hiPSC‐derived sources, primarily due to inefficient hiPSC differentiation protocols, especially for epicardial cells (epiC) and cardiac fibroblasts (CF). This study investigates and compares the cellular organization and function of cardiac organoids consisting of all four predominant cardiac cell types (CM, epiC, endothelial, and CF) differentiated from a single hiPSC source using two differentiation methods for cardiac fibroblasts and two 3D culture techniques. hiPSCs were differentiated into cardiomyocytes, endothelial cells (EC), and epiC using established methods. hiPSC‐CF were differentiated two ways: differentiation of hiPSC‐epiC to hiPSC‐CF and isolation of vimentin‐positive, cardiac troponin t‐negative cells from standard CM differentiation. All cells were singularized with Accutase, mixed at a 5:2:2:1 ratio (CM:CF:epiC:EC) as proposed by the developing cardiac model, and cultured in a non‐adherent spheroid plate (Aggrewell, StemCell Tech) for two days. The spheroids were then cultured for seven days in standard gravity (suspension) or simulated microgravity (rotating bioreactor) environment, with media change every two days. After seven days the organoids were analyzed for organization through immunofluorescence, maximum pacing frequency, calcium transients, and motion contraction velocities. Cardiac organoids containing cells from both hiPSC‐CF differentiation methods were compact and viable, but organoids did not form when no CF was included. This suggests non‐CM cells (cardiac troponin t negative) from CM differentiations are functional cardiac fibroblasts for organoid formation and ECM production. Organoid formation was further supported by the positive staining of cTnT, CD31, WT1, Cx‐43, and alpha‐SMA. Therefore, this study demonstrates a method of generating cardiac organoids containing all four cardiac cell types derived from the same hiPSC with tissue‐level organization and function for the in vitro study of genetic diseases targeting cardiac tissue.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA binding protein PUM2 promotes the stemness of breast cancer cells via competitively binding to neuropilin-1 (NRP-1) mRNA with miR-376a

Others and ours studies have established the promoting roles of NRP-1 (neuropilin-1) in breast cancer, however, the underlying mechanisms by which NRP-1 is regulated are still confused. Here, bioinformatics analysis indicated that RNA binding protein PUM2 could bind to NRP-1 mRNA. Clinical samples showed that PUM2 expression was significantly increased in breast cancer tissues, negatively correlated with the overall survival and relapse-free survival of breast cancer patients, and positively correlated with NRP-1 expression. Meanwhile, PUM2 expression was remarkably increased in non-adherent spheroids. in vitro experiments demonstrated that PUM2 knockdown attenuated the stemness of breast cancer cells, evident by the decrease of spheroid formation capacity, ALDH1 activity and stemness marker expression. Mechanistically, RNA immunoprecipitation (RIP) and luciferase reporter analysis indicated that PUM2 competitively bound to NRP 3′UTR with miR-376a, which had been previously confirmed by us to suppress the stemness of breast cancer cells, and increased NRP-1 mRNA stability and expression. Furthermore, ectopic expression of NRP-1 or miR-376a knockdown rescued the inhibitory effects of NRP-1 knockdown on the stemness of breast cancer cells. Thus, our results suggest that PUM2 could facilitate the stemness of breast cancer cells by competitively binding to NRP-1 3′UTR with miR-376a.

Abstract 167: Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation

Abstract Over 70% of patients with advanced stage ovarian cancer relapse within 2 years, with long term overall survival being only 25%. Tumor-initiating cells (TICs) are thought to contribute to this high rate of recurrence as they can resist killing by platinum-based chemotherapy. In this study we sought to identify pathways unique to TICs and to disrupt these pathways in order to more effectively target this population of cells. We conducted an siRNA screen which identified NF-kappaB signaling as an important pathway in the maintenance of ovarian cancer cells grown in non-adherent spheroid cultures that mimic TICs. Downregulation of several known NF-kappaB target genes, including NXF1, RAC2, CDC42, BCL2L1 and SERPINB3, significantly decreased growth of TIC cultures but not the corresponding adherent cultures, thereby indicating that expression of these genes is necessary for growth in TIC cultures. These findings are consistent with previous studies which showed that higher NF-kappaB activity is linked with drug resistance in patients. In parallel, we conducted a drug screen to identify compounds that target TICs. One clear candidate was bardoxolone methyl, which has been shown to inhibit IKK, an upstream NF-kappaB signaling protein. We confirmed the results of the drug screen in a panel of ovarian cancer cell lines, showing that bardoxolone methyl decreased known TIC markers CD133 and ALDH by flow cytometry, and decreased NF-kappaB proteins by western blot. To further elucidate the mechanism of bardoxolone methyl on TICs we conducted an RNAi screen +/- bardoxolone methyl. Interestingly, the knockdown of genes including BRAF, MAP3K8, LMNA and SMAD6 acted in a synthetically lethal manner with bardoxolone methyl in the TICs. To begin translating to a clinically relevant model, we proceeded to test whether bardoxolone methyl could eliminate TICs that remained after treatment with platinum-based chemotherapy. We confirmed in vitro that bardoxolone methyl in combination with chemotherapy reduced TIC populations. Our ongoing studies will address whether this drug is effective in preventing relapse in vivo after chemotherapy treatment of ovarian cancer in mouse models. Citation Format: Michelle K. Ozaki, Carrie D. House, Krystyna Mazan-Mamczarz, Madhu Lal-Nag, Craig Thomas, Christina M. Annunziata. Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 167.

Novel glycolipid agents for killing cisplatin-resistant human epithelial ovarian cancer cells

BackgroundChemotherapy resistance is one of the major factors contributing to mortality from human epithelial ovarian cancer (EOC). Identifying drugs that can effectively kill chemotherapy-resistant EOC cells would be a major advance in reducing mortality. Glycosylated antitumour ether lipids (GAELs) are synthetic glycolipids that are cytotoxic to a wide range of cancer cells. They appear to induce cancer cell death in an apoptosis-independent manner.MethodsHerein, the effectiveness of two GAELs, GLN and MO-101, in killing chemotherapy-sensitive and –resistant EOC cells lines and primary cell samples was tested using monolayer, non-adherent aggregate, and non-adherent spheroid cultures.ResultsOur results show that EOC cells exhibit a differential sensitivity to the GAELs. Strikingly, both GAELs are capable of inducing EOC cell death in chemotherapy-sensitive and –resistant cells grown as monolayer or non-adherent cultures. Mechanistic studies provide evidence that apoptotic-cell death (caspase activation) contributes to, but is not completely responsible for, GAEL-induced cell killing in the A2780-cp EOC cell line, but not primary EOC cell samples.ConclusionsStudies using primary EOC cell samples supports previously published work showing a GAEL-induced caspase-independent mechanism of death. GAELs hold promise for development as novel compounds to combat EOC mortality due to chemotherapy resistance.

Trabectedin Is Active against Malignant Pleural Mesothelioma Cell and Xenograft Models and Synergizes with Chemotherapy and Bcl-2 Inhibition In Vitro.

Malignant pleural mesothelioma (MPM) is characterized by widespread resistance to systemic therapy. Trabectedin is an antineoplastic agent targeting both the malignant cells and the tumor microenvironment that has been approved for the treatment of advanced soft tissue sarcoma and ovarian cancer. In this preclinical study, we evaluated the antineoplastic potential of trabectedin as a single agent and in drug combination approaches in human MPM. Therefore, we utilized an extended panel of MPM cell lines (n = 6) and primary cell cultures from surgical MPM specimens (n = 13), as well as nonmalignant pleural tissue samples (n = 2). Trabectedin exerted a dose-dependent cytotoxic effect in all MPM cell cultures in vitro when growing as adherent monolayers or nonadherent spheroids with IC50 values ≤ 2.6 nmol/L. Nonmalignant mesothelial cells were significantly less responsive. The strong antimesothelioma activity was based on cell-cycle perturbation and apoptosis induction. The activity of trabectedin against MPM cells was synergistically enhanced by coadministration of cisplatin, a drug routinely used for systemic MPM treatment. Comparison of gene expression signatures indicated an inverse correlation between trabectedin response and bcl-2 expression. Accordingly, bcl-2 inhibitors (Obatoclax, ABT-199) markedly synergized with trabectedin paralleled by deregulated expression of the bcl-2 family members bcl-2, bim, bax, Mcl-1, and bcl-xL as a consequence of trabectedin exposure. In addition, trabectedin exerted significant antitumor activity against an intraperitoneal MPM xenograft model. Together, these data suggest that trabectedin exerts strong activity in MPM and synergizes with chemotherapy and experimental bcl-2 inhibitors in vitro Thus, it represents a promising new therapeutic option for MPM. Mol Cancer Ther; 15(10); 2357-69. ©2016 AACR.

Abstract 3344: Targeting the treatment-induced developmental reprogramming process that facilitates progression of prostate cancer

Abstract Metastatic prostate cancer (PCa) develops resistance to hormone therapies through androgen receptor (AR) dependent and independent mechanisms. Previously we described a remarkable and reversible adaptive cellular plasticity that permits adherent AR+ PCa cells (LNCaP, VCaP, LAPC4 and CWR22rv1) to efficiently reprogram to non-adherent AR-/lo cells by exposure to an androgen-free, custom-formulated stem cell medium (STM). STM-reprogrammed cells grew as rosettes/spheroids on conventional tissue culture treated dishes, were resistant to enzalutamide compared to parental cells and had high tumor forming efficiency when xenografted into castrated and intact male mice. Reprogrammed cells overexpressed genes associated with the stem cell state, expressed cell surface antigens characteristic of neural crest stem cells (NRCAM, HNK-1, CD271 and CD29) and showed increased in vitro invasive behaviour through a matrix-coated membrane and increased invasion in vivo into zebrafish following yolk-sac xenografting. When re-exposed to serum-containing mediums, reprogrammed cells could be re-differentiated back to AR+ prostate-like cancer cells or to other differentiated cell lineages derived from neural/neural crest stem cells (neural-, oligodendrocyte-, glia- or osteoblast-like). Gene expression profiling data from reprogrammed cells was analyzed to identify activation of key signalling pathways associated with this transition and outcomes were confirmed by qPCR (for mRNAs) and Western blotting (for protein levels). Inhibitors of Bmi1 (PTC-209), PI-3-kinase (LY294002), Akt (MK2206) or NF-κB (Copper-DDC) were employed to determine whether they interfered with acquired non-adherent (spheroidal) colony formation in the LNCaP model system exposed to STM. Results indicated that key molecules in the non-classical NF-κB pathway (RelB and p100) were upregulated in the transition to AR-/lo stem-like cells and that Bmi1 and phospho-Akt proteins were also consistently overexpressed in all cell models in response to reprogramming. Inhibitors of Bmi1, PI-3-kinase, and Akt significantly suppressed the ability of LNCaP cells to form non-adherent spheroids in the STM, while an inhibitor of NF-κB was able to reduce the number and size of spheroids. The results support the idea that this adaptive and reversible tumor cell plasticity that generates resistance to androgen deprivation is targetable by agents directed at key genes associated with the developmental reprogramming process described previously. Citation Format: Mannan Nouri, Amy Anne Lubik, Josselin Caradec, Na Li, Sarah Truong, Brett G. Hollier, Ralph Buttyan. Targeting the treatment-induced developmental reprogramming process that facilitates progression of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3344.

Abstract 4295: A 3D culture model for screening of cancer therapeutics

Abstract 3D cell culture models are currently of great interest to the bioscience community. This is most notable in the cancer biology area, where a growing body of evidence suggests that more relevant and translational observations can be made by testing compounds in a physiological relevant 3D model compared to a 2D model. In this study, we take a kinetic, live-cell imaging approach to measure the growth or shrinkage of non-adherent tumor spheroids using Ultra Low Attachment (ULA) 96-well and 384-well microtiter plates (Corning®). These round-bottomed, hydrogel coated plates facilitate the formation of spheroids by promoting self-adherence as opposed to adherence to the plate surface. In a single cell type, monoculture model, A549 lung epithelial carcinoma cells developed into single spheroid structures in each well of 96 and 384-well plates within 48-96 hours in culture. In both plate types, the resulting spheroid size, ranging from 330 µm - 600 µm, was proportional to the number of cells (1K - 5K cells per well). Post formation, spheroids remained untreated or were subjected to three test agents, staurosporine, oxaliplatin or cycloheximide. The increase or decrease in spheroid size was monitored over 3 - 10 days using the IncuCyte™ ZOOM Live Content Imaging system. Using the red fluorescent channel, spheroid growth was analyzed using fluorescent area and intensity metrics. Whereas staurosporine and oxaliplatin significantly decreased spheroid size at high concentrations, cycloheximide treatment resulted in a cytostatic response, effectively inhibiting spheroid growth in a concentration dependent manner, while not reducing total spheroid area at any tested concentration. Interestingly, in side-by-side comparisons of kinetic 2D and 3D assays, we observed a significant, 10-fold (one log) shift in the pharmacology of cycloheximide. In a 3D co-culture breast cancer model, this study also illustrates the effect of Lapatinib on SK-BR-3 cells grown in the presence of mammary derived stromal cells (fibroblasts). Together, this study describes a kinetic, live-cell approach to measuring spheroid size over time, illustrates a differential response of cycloheximide in a 3D system compared to identically treated cells grown in a 2D monolayer, and details a complex co-culture method for elucidating the effect of Lapatinib in a physiologically relevant 3D model of breast cancer. Citation Format: Kalpana Patel, Belinda O'Clair, Tim O'Callaghan, Daniel M. Appledorn, Derek Trezise. A 3D culture model for screening of cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2014-4295

Betaglycan blocks metastatic behaviors in human granulosa cell tumors by suppressing NFκB-mediated induction of MMP2

Metastatic ovarian granulosa cell tumors (GCT) exhibit loss of betaglycan. Here we test the hypothesis that betaglycan blocks GCT metastasis by suppressing NFκB/TGFβ2-induced matrix metalloprotinease-2 (MMP2). Human GCT and a human GCT cell model demonstrated prominent MMP2 expression, which was dependent on NFκB activity and stimulated by TGFβ2 in an NFκB-dependent manner. Betaglycan suppressed both basal and TGFβ2-induced MMP2 expression and countered metastatic behaviors of GCT cells in non-adherent spheroid culture and in vivo xenograft models of metastasis. These data suggest that NFκB/TGFβ2 promotes, and betaglycan impedes, the early stages of GCT metastasis, when tumor cells first invade the peritoneum.

Linking differential radiation responses to glioma heterogeneity.

The phenotypic and genetic diversity that define tumor subpopulations within high-grade glioma can lead to therapeutic resistance and tumor recurrence. Given that cranial irradiation is a frontline treatment for malignant glioma, understanding how irradiation selectively effects different cellular subpopulations within these heterogeneous cancers should help identify interventions targeted to better combat this deadly disease. To analyze the radiation response of distinct glioma subpopulations, 2 glioma cells lines (U251, A172) were cultured under conditions that promoted either adherence or non-adherent spheroids. Past work has demonstrated that subpopulations derived from defined culture conditions exhibit differences in karyotype, proliferation, gene expression and tumorigenicity. Spheroid cultures from each of the glioma cell lines were found to be more radiosensitive, which was consistent with higher levels of oxidative stress and lower levels of both oxidative phosphorylation and glycolytic metabolism 1 week following irradiation. In contrast, radioresistant non-spheroid parental cultures showed increased glycolytic activity in response to irradiation, while oxidative phosphorylation was affected to a lesser extent. Overall these data suggest that prolonged radiation-induced oxidative stress can compromise the metabolic state of certain glioma subpopulations thereby altering their sensitivity to an important therapeutic intervention used routinely for the control of glioma.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Abstract 248: Stearoyl-CoA desaturase 1 is a key factor for lung cancer stem cell survival and expansion.

Abstract In recent years studies of cancer development and recurrence have been significantly influenced by the Cancer Stem Cell (CSC) hypothesis. According to this, cancer is sustained by highly-positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after treatment with debulking agents. Growth of cancer cells as non-adherent spheroids in non differentiating conditions is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from Malignant Pleural Effusions (MPE) of patients affected by adenocarcinoma of the lung are able to form spheroids in non-adherent conditions supplemented with selected growth factors, that these spheroids upregulate markers associated with stemness, can be serially propagated in vitro through several passages and, finally, give rise efficiently to tumors, which reproduce the same histopathological features of the original human tumor when implanted in immunodeficient mice. By expression profiling we also identified a set of genes whose expression is significantly upregulated in lung tumor spheroids vs adherent cultures. One of the most strongly upregulated gene was Stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study we show both by RNA interference or through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in primary tumor cultures. SCD1 inhibition causes a dramatic impairment of survival of cells which express ALDH and other markers of stemness in vitro. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem like properties. Furthermore, SCD1-inhibited lung cancer spheroids were strongly impaired in their in vivo tumorigenicity. These results suggest that SCD1 is a critical target in lung cancer tumor initiating cells. Citation Format: Gennaro Ciliberto, Alessia Noto, Claudia De Vitis, Roberta Barzellotti, Giuseppe Roscilli, Luigi Aurisicchio, Salvatore Raffa, Alberto Ricci, Salvatore Mariotta, Maria Rosaria Torrisi, Maria Rosaria Giovagnoli, Rita Mancini. Stearoyl-CoA desaturase 1 is a key factor for lung cancer stem cell survival and expansion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 248. doi:10.1158/1538-7445.AM2013-248