Treatment Of Spheroids Research Articles

Abstract 14217: Myofibroblasts Enhance the Intrinsic Sinus Rhythm at Low Cell-Cell Coupling

Heart rhythm is initiated by the sinoatrial node (SAN). Compared to the chamber myocardium, the healthy SAN exhibits an exceedingly high content of fibroblasts (FBs). Developmental, aging and pathophysiological cues can activate FBs to myofibroblasts (MFBs) which exhibit depolarized membrane potential and stronger electrotonic coupling compared to FBs. We hypothesized that MFBs, at a low content, can paradoxically increase the pacing frequency of pacemaker cells (PMs) by contributing to the spontaneous depolarization slope.Monolayers and scaffold-free 3D spheroids of TBX18-induced PMs (iPMs) were generated as in vitro models of SAN by somatic gene transfer of TBX18 to neonatal rat cardiomyocytes. Retrograde-perfused, adult rat heart was employed to study intact SAN ex vivo. In silico modeling was performed to simulate pacing frequencies of PMs coupled to MFBs in 2D and 3D pacemaker tissues. Upon reducing electrotonic coupling with carbenoxolone (CBX, 5μM), pacing frequency of iPM monolayers either increased from 66 ± 51 to 168 ± 92 bpm (n=4/13) or decreased from 125 ± 47 to 98 ± 28 bpm (n=9/13). GFP-transduced control NRVM monolayers (n=8) showed only occasional activity, which was not affected by CBX treatment (|ΔBR| = 57 ± 69 in iPM monolayers vs. 11± 11 bpm in control, GFP monolayers, p<0.05). In culture, FBs are known to undergo spontaneous activation to MFBs via TGFβ signaling. Treatment of iPM spheroids with a TGFβ inhibitor (SB431542, 10μM) slowed their automaticity to 124 ± 89 bpm compared to 214 ± 18 bpm in untreated iPM spheroids. Sinus rhythm of the rat heart ex vivo increased upon CBX treatment from 199 ± 64 to 242 ± 80 bpm (n=3, p=0.07). Immunostaining confirmed presence of αSMA+ MFBs in both iPMs and the SAN. Computational study demonstrated that pacing frequencies of 2D and 3D PMs rose and fell with increasing gap junction conductance when coupled to MFBs (Vrest = -25 mV, g = 0 to 4.0 nS) but not to FBs (Vrest = -50 mV, g = 0 to 0.4 nS). Dynamic range of cardiac pacing was optimal when the ratio of MFB to PM was no more than one.Our data demonstrate that MFBs positively contribute to the pacing frequency and the safety factor of the sinus rhythm. We identify fibroblast activation state as a potential target to rejuvenate heart rhythm under aging and pathological conditions.

Long Noncoding RNA MIAT Modulates the Extracellular Matrix Deposition in Leiomyomas by Sponging MiR-29 Family.

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

Ginsenoside Rg4 Enhances the Inductive Effects of Human Dermal Papilla Spheres on Hair Growth Via the AKT/GSK-3β/β-Catenin Signaling Pathway.

Ginsenoside Rg4 is a rare ginsenoside that is naturally found in ginseng, and exhibits a wide range of biological activities including antioxidant and anti-inflammatory properties in several cell types. The purpose of this study was to use an in vivo model of hair follicle (HF)-mimic based on a human dermal papilla (DP) spheroid system prepared by three-dimensional (3D) culture and to investigate the effect of Rg4 on the hair-inductive properties of DP cells. Treatment of the DP spheroids with Rg4 (20 to 50 μg/ml) significantly increased the viability and size of the DP spheres in a dose-dependent manner. Rg4 also increased the mRNA and protein expression of DP signature genes that are related to hair growth including ALP, BMP2, and VCAN in the DP spheres. Analysis of the signaling molecules and luciferase reporter assays further revealed that Rg4 induces the activation of phosphoinositide 3-kinase (PI3K)/AKT and the inhibitory phosphorylation of GSK3β, which activates the WNT/β-catenin signaling pathway. These results correlated with not only the increased nuclear translocation of β-catenin following the treatment of the DP spheres with Rg4 but also the significant elevation of mRNA expression of the downstream target genes of the WNT/β-catenin pathway including WNT5A, β-catenin, and LEF1. In conclusion, these results demonstrated that ginsenoside Rg4 promotes the hair-inductive properties of DP cells by activating the AKT/GSK3β/β-catenin signaling pathway in DP spheres, suggesting that Rg4 could be a potential natural therapy for hair growth.

Dicalcin suppresses in vitro trophoblast attachment in human cell lines

Implantation is a highly organized process that involves an interaction between a competent blastocyst and a receptive uterus. Despite significant research efforts, the molecular mechanisms governing this complex process remain elusive. Here, we investigated the effect of dicalcin, an S100-like Ca2+-binding protein, on the attachment of choriocarcinoma cells (BeWo cells) onto a monolayer of endometrial carcinoma cells (Ishikawa cells). Extracellularly administered dicalcin bound to both BeWo and Ishikawa cells. Pretreatment of BeWo spheroids with dicalcin reduced the attachment ratio of the spheroids onto the monolayer, whereas that of Ishikawa cells showed no apparent change. We identified the partial amino acid sequence of human dicalcin that exhibited maximum suppression for BeWo spheroid attachment. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide caused a dilation of the intercellular junction between BeWo and ISK cells. Peptide treatment of BeWo spheroids downregulated the expression of integrinαvβ3 in BeWo cells, and induced alterations in their phalloidin-staining pattern, as measured by the length of each F-actin fiber and the thickness of the cortical stress fiber. Thus, dicalcin affects reorganization of the intracellular actin meshwork and subsequently the intensity of attachment, functioning as a novel suppressor of implantation.

CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition.

Malignant pleural mesothelioma is an aggressive cancer, heterogeneous in its presentation and behaviour. Despite an increasing knowledge about molecular markers and their diagnostic and prognostic value, they are not used as much as they might be for treatment allocation. It has been recently reported that mesothelioma cells that lack BAP1 (BRCA1 Associated Protein) are sensitive to inhibition of the EZH2 (Enhancer of Zeste Homolog 2) histone methyltransferase. Since we observed strong H3K27me3 (histone H3 lysine 27 trimetylation) immunoreactivity in BAP1 wild-type mesothelioma biopsies, we decided to characterize in vitro the response/resistance of BAP1 wild-type mesothelioma cells to the EZH2 selective inhibitor, EPZ-6438. Here we demonstrate that BAP1 wild-type mesothelioma cells were rendered sensitive to EPZ-6438 upon SIRT1 (Sirtuin 1) silencing/inhibition or when cultured as multicellular spheroids, in which SIRT1 expression was lower compared to cells grown in monolayers. Notably, treatment of spheroids with EPZ-6438 abolished H3K27me3 and induced the expression of CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A), causing cell growth arrest. EPZ-6438 treatment also resulted in a rapid and sustained induction of the genes encoding HIF2α (Hypoxia Inducible Factor 2α), TG2 (Transglutaminase 2) and IL-6 (Interleukin 6). Loss of CDKN2 is a common event in mesothelioma. CDKN2A silencing in combination with EPZ-6438 treatment induced apoptotic death in mesothelioma spheroids. In a CDKN2A wild-type setting apoptosis was induced by combining EPZ-6438 with 1-155, a TG2 selective and irreversible inhibitor. In conclusion, our data suggests that the expression of CDKN2A predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis.

Tryptophan catabolism is dysregulated in leiomyomas

To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma. Experimental study. Academic research laboratory. Women undergoing hysterectomy for leiomyoma. Blockade of IDO1 and TDO2. Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix. Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids. These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas.

IGF1R and Src inhibition induce synergistic cytotoxicity in HNSCC through inhibition of FAK

Head and neck cancer is the sixth most common cancer worldwide with a 5-year survival of only 65%. Targeting compensatory signaling pathways may improve therapeutic responses and combat resistance. Utilizing reverse phase protein arrays (RPPA) to assess the proteome and explore mechanisms of synergistic growth inhibition in HNSCC cell lines treated with IGF1R and Src inhibitors, BMS754807 and dasatinib, respectively, we identified focal adhesion signaling as a critical node. Focal Adhesion Kinase (FAK) and Paxillin phosphorylation were decreased as early as 15 min after treatment, and treatment with a FAK inhibitor, PF-562,271, was sufficient to decrease viability in vitro. Treatment of 3D spheroids demonstrated robust cytotoxicity suggesting that the combination of BMS754807 and dasatinib is effective in multiple experimental models. Furthermore, treatment with BMS754807 and dasatinib significantly decreased cell motility, migration, and invasion in multiple HNSCC cell lines. Most strikingly, treatment with BMS754807 and dasatinib, or a FAK inhibitor alone, significantly increased cleaved-PARP in human ex-vivo HNSCC patient tissues demonstrating a potential clinical utility for targeting FAK or the combined targeting of the IGF1R with Src. This ex-vivo result further confirms FAK as a vital signaling node of this combinatorial treatment and demonstrates therapeutic potential for targeting FAK in HNSCC patients.

Curcumin Targets Both Apoptosis and Necroptosis in Acidity-Tolerant Prostate Carcinoma Cells.

Objective Curcumin, a major bioactive curcuminoid derived from the rhizome of Curcuma longa, is known to have anticancer potential and is still under investigation. In this study, we investigated the cytotoxic mechanism(s) of curcumin against acidity-tolerant prostate cancer PC-3AcT cells in lactic acid-containing medium. Methods Using 2D-monolyer and 3D spheroid culture models, MTT assay, annexin V-PE binding assay, flow cytometric analysis, measurement of ATP content, and Western blot analysis were used for this study. Results At nontoxic concentrations in normal prostate epithelial RWPE-1 and HPrEC cells, curcumin led to strong cytotoxicity in PC-3AcT cells, including increases in sub-G0/G1 peak, annexin V-PE-positive cells, and ROS levels; loss of mitochondrial membrane potential; reduction of cellular ATP content; DNA damage; and concurrent induction of apoptosis and necroptosis. A series of changes induced by curcumin were effectively reversed by reducing ROS levels or replenishing ATP. Pretreatment with apoptosis inhibitor Q-VD-Oph-1 or necroptosis inhibitor necrostatin-1 restored cell viability inhibited by curcumin. Treatment of 3D spheroids with curcumin decreased cell viability, accompanied by an increase in mediators of apoptosis and necroptosis, including cleaved caspase-3 and cleaved PARP, phospho (p)-RIP3, and p-MLKL proteins. Conclusion This study shows that curcumin simultaneously induces apoptosis and necroptosis by oxidative mitochondrial dysfunction and subsequent ATP depletion, providing a mechanistic basis for understanding the novel role of curcumin for prostate carcinoma cells.

O11: MYOFERLIN: A NOVEL BIOMARKER OF RADIOSENSITIVITY IN LOCALLY ADVANCED RECTAL CANCER

Abstract Introduction Our proteomic data has validated that high levels of the protein myoferlin confers poorer response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Myoferlin plays a role in membrane repair and VEGF signal transduction, and is associated with worse prognosis in numerous other epithelial cancers. We aim to assess the impact of myoferlin on the radiosensitivity of rectal cancer. Method Clonogenic assays were performed using immortalised colorectal cancer cells (HCT116,HT29,LIM,MDST8) to assess survival at escalating radiation doses following knockdown with myoferlin siRNA or a small molecular inhibitor(WJ460). 3D models (spheroids) were used to examine the effect of WJ460 on tumour growth. Result Quantification of myoferlin using immunoblotting demonstrated that MDST8 and LIM were higher expressors than HCT116 and HT29. Higher levels correlated with increasing radio-resistance as calculated by colony formation efficiency (CFE). Using clonogenic assays, cells treated with myoferlin siRNA or WJ460 demonstrated increased radiosensitivity compared to controls across all radiation doses, most significantly at 4Gy. Treatment of spheroids with WJ460 significantly reduced growth compared to controls at all radiation doses (p<0.05), with WJ460 limiting growth considerably more than treatment with the current gold standard 5-FU. HCT116 spheroid volume day 15; WJ460 4.96um3,5-FU 6.74um3,DMSO 24.9um3. Conclusion Inhibition of myoferlin is associated with increased radiosensitivity of colorectal cancer cells, and treatment with a small molecular inhibitor significantly reduces growth in spheroid models. Further work is required further validate its potential use as a biomarker in locally advanced rectal cancer. Take-home message We have found that myoferlin is a protein associated with poor response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Manipulation of this protein sensitises the cancer cells to radiotherapy.

O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p<0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

Inhibition of the Histone Methyltransferase EZH2 Enhances Protumor Monocyte Recruitment in Human Mesothelioma Spheroids.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes’ recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

Cold Atmospheric Plasma Increases Temozolomide Sensitivity of Three-Dimensional Glioblastoma Spheroids via Oxidative Stress-Mediated DNA Damage.

Simple SummaryCold atmospheric plasma (CAP) is gaining increasing interest for cancer treatment, for a wide range of cancer types. The studies performed with CAP as a standalone treatment modality serve as evidence that it can also be a suitable candidate for combination therapy. Temozolomide (TMZ) is used as the gold standard drug for glioblastoma treatment, one of the most aggressive malignant brain tumors in adults that remains incurable despite treatment advances. In this study, we explore whether CAP, a cocktail of reactive oxygen and nitrogen species, can amplify the cytotoxic effect on both TMZ-sensitive and TMZ-resistant glioblastoma multiforme (GBM) in three-dimensional tumor-like tissues through inhibiting the glutathione (GSH)/ glutathione peroxidase 4 (GPX4) antioxidant machinery, which can further lead to DNA damage.Glioblastoma multiforme (GBM) is the most frequent and aggressive primary malignant brain tumor in adults. Current standard radiotherapy and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ) yield poor clinical outcome. This is due to the stem-like properties of tumor cells and genetic abnormalities in GBM, which contribute to resistance to TMZ and progression. In this study, we used cold atmospheric plasma (CAP) to enhance the sensitivity to TMZ through inhibition of antioxidant signaling (linked to TMZ resistance). We demonstrate that CAP indeed enhances the cytotoxicity of TMZ by targeting the antioxidant specific glutathione (GSH)/glutathione peroxidase 4 (GPX4) signaling. We optimized the threshold concentration of TMZ on five different GBM cell lines (U251, LN18, LN229, U87-MG and T98G). We combined TMZ with CAP and tested it on both TMZ-sensitive (U251, LN18 and LN229) and TMZ-resistant (U87-MG and T98G) cell lines using two-dimensional cell cultures. Subsequently, we used a three-dimensional spheroid model for the U251 (TMZ-sensitive) and U87-MG and T98G (TMZ-resistant) cells. The sensitivity of TMZ was enhanced, i.e., higher cytotoxicity and spheroid shrinkage was obtained when TMZ and CAP were administered together. We attribute the anticancer properties to the release of intracellular reactive oxygen species, through inhibiting the GSH/GPX4 antioxidant machinery, which can lead to DNA damage. Overall, our findings suggest that the combination of CAP with TMZ is a promising combination therapy to enhance the efficacy of TMZ towards the treatment of GBM spheroids.

Prediction of Residual Mg Contents in Ladle and Product after Graphite Spheroidizing Treatment by Using Artificial Neural Network

Prediction of Residual Mg Contents in Ladle and Product after Graphite Spheroidizing Treatment by Using Artificial Neural Network

Accelerated Spheroidization of Cementite in Sintered Ultrahigh Carbon Steel by Warm Deformation

Evolution of microstructure and hardness in quenched ultrahigh carbon steel Fe-0.85Mo-0.6Si-1.4C by warm compression on a Bähr plastometer-dilatometer at 775 °C and at 0.001 to 1 s−1 strain rate range is reported. The material was prepared via powder metallurgy: cold pressing and liquid phase sintering. Independent of strain rate, the initial martenstic microstructure was transformed to ferrite and spheroidized cementite. Strain rate had an effect on size and shape of spheroidized Fe3C precipitates: the higher the strain rate, the smaller the precipitates. Morphology of the spheroidized carbides influenced hardness, with the highest hardness, 362 HV10, for strain rate 1 s−1 and the lowest, 295 HV10, for the lowest strain rate 0.001 s−1. Resultant microstructure and ambient temperature mechanical properties were comparable to those of the material that had undergone a fully spheroidizing treatment with increased time and energy consumption, indicating that it can be dispensed with in industrial processing. All our results are consistent with the Hall–Petch relation developed for spheroidized steels.

Doppler imaging detects bacterial infection of living tissue

Living 3D in vitro tissue cultures, grown from immortalized cell lines, act as living sentinels as pathogenic bacteria invade the tissue. The infection is reported through changes in the intracellular dynamics of the sentinel cells caused by the disruption of normal cellular function by the infecting bacteria. Here, the Doppler imaging of infected sentinels shows the dynamic characteristics of infections. Invasive Salmonella enterica serovar Enteritidis and Listeria monocytogenes penetrate through multicellular tumor spheroids, while non-invasive strains of Escherichia coli and Listeria innocua remain isolated outside the cells, generating different Doppler signatures. Phase distributions caused by intracellular transport display Lévy statistics, introducing a Lévy-alpha spectroscopy of bacterial invasion. Antibiotic treatment of infected spheroids, monitored through time-dependent Doppler shifts, can distinguish drug-resistant relative to non-resistant strains. This use of intracellular Doppler spectroscopy of living tissue sentinels opens a new class of microbial assay with potential importance for studying the emergence of antibiotic resistance.

BNO 1095, a Standardized Dry Extract from the Fruits of Vitex agnus-castus, Impairs Angiogenesis-related Endothelial Cell Functions In Vitro.

BNO 1095, a standardized dry extract from the fruits of Vitex agnus-castus, represents an approved herbal medicinal product for the treatment of premenstrual syndrome. Angiogenesis, the formation of new blood vessels from pre-existing capillaries, plays a major role in physiological situations, such as wound healing or tissue growth in female reproductive organs, but it is also of great importance in pathophysiological conditions such as chronic inflammatory diseases or cancer. Angiogenesis is a highly regulated multi-step process consisting of distinct key events that can be influenced pharmacologically. Few studies suggested anti-angiogenic actions of V.agnus-castus fruit extracts in in vivo and ex vivo models. Here, we provide for the first time profound in vitro data on BNO 1095-derived anti-angiogenic effects focusing on distinct angiogenesis-related endothelial cell functions that are inevitable for the process of new blood vessel formation. We found that V.agnus-castus extract significantly attenuated undirected and chemotactic migration of primary human endothelial cells. Moreover, the extract efficiently inhibited endothelial cell proliferation and reduced the formation of tube-like structures on Matrigel. Of note, the treatment of endothelial cell spheroids almost blocked endothelial sprouting in a 3D collagen gel. Our data present new and detailed insights into the anti-angiogenic actions of BNO 1095 and, therefore, suggest a novel scope of potential therapeutic applications of the extract for which these anti-angiogenic properties are required.

CXCR4 Inhibition Counteracts Immunosuppressive Properties of Metastatic NSCLC Stem Cells.

Cancer stem cells (CSCs) are functionally defined as the cell subset with greater potential to initiate and propagate tumors. Within the heterogeneous population of lung CSCs, we previously identified highly disseminating CD133+CXCR4+ cells able to initiate distant metastasis (metastasis initiating cells-MICs) and to resist conventional chemotherapy. The establishment of an immunosuppressive microenvironment by tumor cells is crucial to sustain and foster metastasis formation, and CSCs deeply interfere with immune responses against tumors. How lung MICs can elude and educate immune cells surveillance to efficiently complete the metastasis cascade is, however, currently unknown. We show here in primary tumors from non-small cell lung cancer (NSCLC) patients that MICs express higher levels of immunoregulatory molecules compared to tumor bulk, namely PD-L1 and CD73, an ectoenzyme that catalyzes the production of immunosuppressive adenosine, suggesting an enhanced ability of MICs to escape immune responses. To investigate in vitro the immunosuppressive ability of MICs, we derived lung spheroids from cultures of adherent lung cancer cell lines, showing enrichment in CD133+CXCR4+MICs, and increased expression of CD73 and CD38, an enzyme that also concurs in adenosine production. MICs-enriched spheroids release high levels of adenosine and express the immunosuppressive cytokine IL-10, undetectable in an adherent cell counterpart. To prevent dissemination of MICs, we tested peptide R, a novel CXCR4 inhibitor that effectively controls in vitro lung tumor cell migration/invasion. Notably, we observed a decreased expression of CD73, CD38, and IL-10 following CXCR4 inhibition. We also functionally proved that conditioned medium from MICs-enriched spheroids compared to adherent cells has an enhanced ability to suppress CD8+ T cell activity, increase Treg population, and induce the polarization of tumor-associated macrophages (TAMs), which participate in suppression of T cells. Treatment of spheroids with anti-CXCR4 rescued T cell cytotoxic activity and prevented TAM polarization, likely by causing the decrease of adenosine and IL-10 production. Overall, we provide evidence that the subset of lung MICs shows high potential to escape immune control and that inhibition of CXCR4 can impair both MICs dissemination and their immunosuppressive activity, therefore potentially providing a novel therapeutic target in combination therapies to improve efficacy of NSCLC treatment.

Marked synergy by vertical inhibition of EGFR signaling in NSCLC spheroids shows SOS1 is a therapeutic target in EGFR-mutated cancer.

Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR- and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.

Abstract A24: Cell adhesion molecule (CAM)-related downregulated by oncogenes (CDON) promotes ovarian cancer adhesion and survival

Abstract Ovarian carcinoma (OC) broadly describes epithelial tumors involving the ovary and represents the most lethal gynecologic malignancy. The most frequently diagnosed OCs are high-grade serous carcinomas (HGSC), and the majority of these cancers are diagnosed at late stage, stage III or IV (51% and 29% respectively), with widespread disease dissemination. In addition, a significant number of patients develop malignant ascites. A key feature of tumor cells in present in ascites is the capacity to form multicellular aggregates that promote tumor cell survival and continued disease growth and spread. Cell adhesion molecule (CAM)-related downregulated by oncogenes (CDON) is a cell surface glycoprotein with established roles in normal development and associated with some tumor types. CDON signaling occurs via ligand-dependent mechanisms as a hedgehog co-receptor and ligand-independent mechanisms via interactions with cadherins. Little is known about the role of CDON in ovarian cancer, but given the importance of cadherin-mediated cell adhesion in disease dissemination, we asked whether CDON plays a role in HGSC growth and progression. We find that CDON is expressed in ovarian carcinoma (OC) cells, and protein levels are elevated by expression of oncogenes or by growth under nonadherent conditions. Depletion of CDON expression revealed that it plays an important role in HGSC cells regardless of growth conditions, resulting in decreased growth (proliferation) in cells grown under nonadherent conditions as multicellular aggregates or under adherent conditions as 2D monolayers. Alterations in signaling related to both cell adhesion and survival occur upon CDON downregulation, including decreased cadherin protein expression, integrin expression, and focal adhesion kinase activation. Depletion of CDON in OC cells results in significant tumor growth inhibition in xenograft and allograft models, suggesting that CDON plays an important role in promoting ovarian tumor growth. Treatment of OC spheroids with novel anti-CDON antibodies results in collapse of 3D cellular structures and induction of apoptosis, supporting a key role for CDON in OC cellular adhesion in tumor cell survival. These data indicate that CDON may play an essential role in OC spread since altered cell-cell adhesion and survival as multicellular aggregates is crucial for disease dissemination in patients. Taken together, our results nominate CDON as an important protein promoting HGSC growth and progression and as a novel therapeutic target for the treatment of this disease. Citation Format: Valerie L. Sodi, Shane W. O'Brien, Chao Wu, Roland L. Dunbrack, Tiffiney R. Hartman, Alana M. O'Reilly, Denise C. Connolly. Cell adhesion molecule (CAM)-related downregulated by oncogenes (CDON) promotes ovarian cancer adhesion and survival [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A24.

Abstract B14: Atovaquone targets STAT3 in ovarian cancer spheroids

Abstract Ovarian cancer remains a deadly disease for countless women. Recent evidence demonstrates that ovarian cancer cell clusters, spheroids, are important in promoting ovarian recurrence and metastasis. Often these spheroids are resistant to therapy. Therefore, we were interested in identifying drugs that could target ovarian cancer spheroids. To do this, we wanted to identify pathways that were activated in cells growing in three-dimensional (3D) clusters. Analysis of gene expression from 2D and 3D growing cells identified STAT3 as a pathway enriched in 3D growing cells. STAT3 is a transcription factor that is activated by tyrosine phosphorylation. In ovarian cancer cells, activation usually occurs from interleukin 6 (IL-6) binding to the IL-6 receptor and recruiting the signal transducer molecule (GP130, IL6ST). This activates Jak kinases that then tyrosine phosphorylates STAT3. Once activated, STAT3 dimerizes, translocates into the nucleus, and regulates the expression of target genes. Using shRNA targeting STAT3 and the upstream signaling molecule, GP130, we found that reduction of both STAT3 and its upstream signaling molecule GP130 reduced the growth of spheroids. This suggested that targeting STAT3 or an upstream signaling molecule might be a viable treatment option. Atovaquone, used normally for the treatment of malaria or the prevention of pneumocystis pneumonia, inhibits STAT3 activation by inhibiting the expression of GP130 on the cell surface. Therefore, we hypothesized that atovaquone would have efficacy in ovarian cancer spheroids. Treatment of ovarian spheroids with atovaquone reduced their growth and viability. Importantly, atovaquone inhibited STAT3 tyrosine phosphorylation and target gene expression, demonstrating that atovaquone is inhibiting STAT3 in ovarian spheroids. Preliminary data suggest that overexpression of STAT3 partially rescues the effects of atovaquone on spheroid growth. As spheroids are also involved in ovarian cancer metastasis, we are currently assessing the effects of atovaquone on spheroid-mediated ovarian cancer metastasis. Taken together, our data suggest that atovaquone may be a beneficial treatment for ovarian cancer patients. Citation Format: Kayli E. Neil, Sarah J. Lacroix, David F. Walker, Suhu Liu, David A. Frank, Sarah R. Walker. Atovaquone targets STAT3 in ovarian cancer spheroids [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B14.