Sphere-forming Cells Research Articles

Intrinsically Self-renewing Neuroprogenitors From the A/J Mouse Spiral Ganglion as Virtually Unlimited Source of Mature Auditory Neurons.

Nearly 460 million individuals are affected by sensorineural hearing loss (SNHL), one of the most common human sensory disorders. In mammals, hearing loss is permanent due to the lack of efficient regenerative capacity of the sensory epithelia and spiral ganglion neurons (SGN). Sphere-forming progenitor cells can be isolated from the mammalian inner ear and give rise to inner ear specific cell types in vitro. However, the self-renewing capacities of auditory progenitor cells from the sensory and neuronal compartment are limited to few passages, even after adding powerful growth factor cocktails. Here, we provide phenotypical and functional characterization of a new pool of auditory progenitors as sustainable source for sphere-derived auditory neurons. The so-called phoenix auditory neuroprogenitors, isolated from the A/J mouse spiral ganglion, exhibit robust intrinsic self-renewal properties beyond 40 passages. At any passage or freezing–thawing cycle, phoenix spheres can be efficiently differentiated into mature spiral ganglion cells by withdrawing growth factors. The differentiated cells express both neuronal and glial cell phenotypic markers and exhibit similar functional properties as mouse spiral ganglion primary explants and human sphere-derived spiral ganglion cells. In contrast to other rodent models aiming at sustained production of auditory neurons, no genetic transformation of the progenitors is needed. Phoenix spheres therefore represent an interesting starting point to further investigate self-renewal in the mammalian inner ear, which is still far from any clinical application. In the meantime, phoenix spheres already offer an unlimited source of mammalian auditory neurons for high-throughput screens while substantially reducing the numbers of animals needed.

Combined PI3K\u03b1-mTOR Targeting of Glioma Stem Cells

Glioblastoma (GBM) is the most common and lethal primary intrinsic tumour of the adult brain and evidence indicates disease progression is driven by glioma stem cells (GSCs). Extensive advances in the molecular characterization of GBM allowed classification into proneural, mesenchymal and classical subtypes, and have raised expectations these insights may predict response to targeted therapies. We utilized GBM neurospheres that display GSC characteristics and found activation of the PI3K/AKT pathway in sphere-forming cells. The PI3Kα selective inhibitor alpelisib blocked PI3K/AKT activation and inhibited spheroid growth, suggesting an essential role for the PI3Kα catalytic isoform. p110α expression was highest in the proneural subtype and this was associated with increased phosphorylation of AKT. Further, employing the GBM BioDP, we found co-expression of PIK3CA with the neuronal stem/progenitor marker NES was associated with poor prognosis in PN GBM patients, indicating a unique role for PI3Kα in PN GSCs. Alpelisib inhibited GSC neurosphere growth and these effects were more pronounced in GSCs of the PN subtype. The antineoplastic effects of alpelisib were substantially enhanced when combined with pharmacologic mTOR inhibition. These findings identify the alpha catalytic PI3K isoform as a unique therapeutic target in proneural GBM and suggest that pharmacological mTOR inhibition may sensitize GSCs to selective PI3Kα inhibition.

Abstract PO-016: Modulating cancer stem cell pathways to enhance systemic therapeutic efficacy

Abstract Chemoresistance and poor immune sensitivity are one of the major challenges to treat different cancer types including lung, bladder and head & neck. Different epigenetic regulators and cancer stem cells (CSCs) regulatory pathways play a critical role in the development of therapeutic resistance. Prolonged chemotherapy treatment enriches CSCs in the tumor microenvironment and in a previous study we have shown that inhibition of the CSCs regulatory pathway/s combined with chemotherapy decreased CSC properties and efficiently inhibit tumor growth in patient-derived xenografts (PDXs) models. The purpose of this study is to determine the chemo and immunotherapeutic efficacy in combination with several CSC pathway/s modulatory agent/agents to treat different solid tumors. To identify relevant CSC regulating pathways, we tested the therapeutic efficacy of different agents such as 5-Azacytidine (DNA methyltransferase 1 inhibitor), Vismodegib (SMO inhibitor), Romidepsin (HDAC1 inhibitor), Olaparib (PARP1 inhibitor), and GSK343 (EZH2 inhibitor) using different bladder and head & neck cancer cell lines and also determined the numerous CSCs properties. Our data indicate that in vitro treatment with these potential therapeutic agents decreases cell viability, sphere formation and wound healing that suggest that, these drugs may be effective in reducing CSCs population when combined with chemo and/or immunotherapy. Our immunoblotting experiments indicate reduced expression of some CSCs markers and factors such as such as NANOG, OCT4, CD133 and CD24. Interestingly, after treating the cells with some of these agents and array analysis, we have found differential expression of some of the key genes regulating the immunogenic response. Further, to confirm the array-based data, selected candidate immune-associated genes were analyzed by qRT-PCR. Moreover, our preliminary data suggest that combination treatment with epigenetic drugs and an immune agent may effectively reduce tumor growth in vivo compared to either of these drugs. In conclusion, although comprehensive mechanistic studies are ongoing, our initial results highlight the importance of considering combination of epigenetic and immune therapy for patients with diverse cancer types that respond poorly to immune and chemotherapy. Citation Format: Pritam Sadhukhan, Masahiro Shibata, M. Talha Ugurlu, Rachel Goldberg, Mohammad O. Hoque. Modulating cancer stem cell pathways to enhance systemic therapeutic efficacy [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-016.

Autocrine Leukemia Inhibitory Factor Promotes Esophageal Squamous Cell Carcinoma Progression via Src Family Kinase-Dependent Yes-Associated Protein Activation.

The IL6 family of cytokines, including IL6 and leukemia-inhibitory factor (LIF), are induced during inflammation and are also expressed in many types of cancer where they play an important role in tumor development. IL6 family cytokines mainly activate the JAK-STAT3 pathway via the coreceptor, gp130, and IL6 is known to activate the Src family kinase (SFK)-Yes-associated protein (YAP) pathway. The current study investigated the role of autocrine LIF in human esophageal squamous cell carcinoma (ESCC) that highly expresses LIF. LIF knockdown had various effects on cancer cells, including profound changes in gene expression, suppression of cell proliferation, migration/invasion and sphere formation, and induction of apoptosis. Similar to IL6, LIF activated the SFK-YAP pathway as well as the JAK-STAT3 pathway. LIF-induced YAP activation was more important for cancer cell proliferation than LIF-induced STAT3 activation, and concomitant YAP and STAT3 activation completely compensated for the role of LIF in human ESCC growth. We also confirmed that SFK activation and LIF expression were correlated with YAP activation in human ESCC clinical samples. Furthermore, simultaneous inhibition of the SFK-YAP and JAK-STAT3 pathways in human ESCC cells was more effective at suppressing cell proliferation than single inhibition, and autocrine LIF signaling promoted human ESCC growth in vivo. Therefore, the LIF-SFK-YAP axis may represent a new therapeutic target for human ESCC. IMPLICATIONS: Autocrine LIF signaling promotes human ESCC progression via SFK-dependent YAP activation and is a new potential target of treatment for human ESCC.

Silencing long non-coding RNA HIF1A-AS2 inhibits proliferation, invasion and migration of cervical cancer cells in vitro

To explore the inhibitory effects of silencing long non-coding RNA (LncRNA) HIF1A-AS2 on epithelialmesenchymal transition (EMT) and tumor stem cell-like phenotype in cervical cancer cells. We designed 3 shRNA constructs for silencing HIF1A-AS2 in CaSki cells, and the shRNA with the strongest interference effect was selected for subsequent experiment. CaSki cells were transfected with shRNA-NC or Sh-HIF1A-AS2, and the changes in cell viability, invasion ability, EMT, expressions of EMT-related proteins, formation of cell spheres and expressions of stem cell markers were detected. Transfection with shRNA-NC and Sh-HIF1A-AS2 did not significantly affected the viability of CaSki cells (P > 0.05). Compared with the cells transfected with shRNA-NC, the cells transfected with Sh- HIF1A-AS2 showed significantly reduced invasion ability, expressions of vimentin N-cadherin, and cell sphere formation ability. HIF1A-AS2 silencing obviously lowered the rate of ABCG2-positive cells, significantly reduced the mRNA and protein expressions of Nanog, OCT4, and SOX2, and strongly enhanced the expression of E-cadherin in CaSki cells (P < 0.05). Silencing HIF1A-AS2 can inhibit proliferation, invasion and migration of cervical cancer cells in vitro.

Proprotein convertases blockage up-regulates specifically metallothioneins coding genes in human colon cancer stem cells

Despite continuous exertion made, colon cancer still represents a major health problem and its incidence continues being high worldwide. There is growing evidence in support of the cancer stem cells (CSCs) being central in the initiation of this cancer, and CSCs have been the focus of various studies for the identification of new ways of treatment. Lately, the proprotein convertases (PCs) were reported to regulate the maturation and expression of various molecules involved in the malignant phenotype of colon cancer cells, however, the identity of the molecules regulated by these serine proteases in CSCs is unknown. In this study, we used the general PCs inhibitor, the Decanoyl-RVKR-chloromethylketone (Decanoyl-RVKR-CMK) that inhibits all the PCs found in the secretory pathway, and analyzed its effect on CSCs using RNA-seq analysis. Remarkably, from the only 9 up-regulated genes in the human SW620-derived sphere-forming cells, we identified 7 of the 11 human metallothioneins, all of them localized on chromosome 16, and zinc related proteins as downstream effectors of the PCs. The importance of these molecules in the regulation of cell proliferation, differentiation and chemoresistance, and their reported potential tumor suppressor role and loss in colon cancer patients associated with worse prognosis, suggests that targeting PCs in the control of the malignant phenotype of CSCs is a new potential therapeutic strategy in colon cancer.

LncRNA SNHG7 Mediates the Chemoresistance and Stemness of Breast Cancer by Sponging miR-34a.

Chemoresistance is considered to be a major cause of the recurrence and metastasis of breast cancer (BC). LncRNA SNHG7 has been reported to be upregulated in breast cancer and to promote tumor progression and metastasis. Nevertheless, the function and potential regulatory mechanism of SNHG7 in BC drug resistance are still largely unclear. This study indicated that SNHG7 was highly expressed in chemoresistant BC tissues and cells. Upregulated SNHG7 might predict a low pCR rate and poor clinical outcome in BC patients. Knockdown of SNHG7 enhanced drug sensitivity and drug-induced apoptosis in chemoresistant BC cells. In terms of the mechanism, miR-34a was found to be a target of SNHG7 and its expression in breast cancer tissues and chemoresistant cell lines was negatively correlated with SNHG7 expression. Importantly, sh-SNHG7 upregulated miR-34a expression, reduced the percentages of CD44+/CD24−cells, and inhibited sphere-formation and stem cell factor (Oct4, Nanog, SOX2) expression. Functional loss experiments showed that the repressive effect of SNHG7 knockdown on BC cell stemness was partially reversed by transfection with miR-34a inhibitors. In summary, this study indicated that SNHG7 contributed to the chemoresistance of BC and mediated chemoresistance and cancer stemness by sponging miR-34a.

The long non-coding RNA TSLC8 inhibits colorectal cancer by stabilizing puma

ABSTRACT The colorectal cancer (CRC) dictates a common malignancy with high recurrence rate. Long non-coding RNAs (lncRNAs) belong to a class of regulatory factors involved in multiple cancers. In current work, we have uncovered a novel lncRNA named TSLC8. TSLC8 was dramatically downregulated in CRC samples and cell lines. Reintroduction of TSLC8 inhibited tumor sphere formation and viability in CRC cells. In vivo experiments further confirmed the tumor suppressive function of TSLC8. Ectopic TSLC8 expression elevates puma abundance whereas this effect is mediated by TSLC8-puma binding and stabilization. FOXO1 can transcriptionally induce TSLC8 expression. Epigenetic investigation suggested that TSLC8 locus was hypermethylated in CRC leading to diminished TSLC8 expression. Our current work has identified a novel tumor suppressive function of TSLC8, whose reduced expression may facilitate malignant phenotypes during CRC progression.

Differences in sphere-forming cells from keratoconic and normal corneal tissue: Implications for keratoconus pathogenesis

Keratoconus is primarily an anterior corneal disorder of unclear aetiology. Stem cells may play a role in the perpetuation of keratoconus, although this has yet to be definitively established. Sphere-forming cells from normal human donor corneas have previously been shown to be a heterogenous mix of epithelial, stromal, stem and progenitor cell components which have potential for treatment of corneal dystrophies. Our work set out to isolate and characterise sphere-forming cells from human keratoconic tissue. Keratoconic donor corneas were successfully used to culture sphere-forming cells in vitro. Time lapse imaging of these spheres on a collagen surface over 8 days revealed keratoconic spheres lack the ability to maintain a central core and have diminished ability to repopulate the surface. Immunocytochemistry showed positive labelling for the stem cell marker ‘Adenosine triphosphate-binding cassette sub-family B member 5 (ABCB5)’ indicating stem cell retention and the myofibroblast marker alpha smooth muscle actin indicating wound repair while droplet digital Polymerase Chain Reaction confirmed an increase in expression of stem and stromal cell markers in keratoconic spheres compared to spheres cultured from normal donors at day 7 post-placement. Keratoconic sphere-forming cells showed a diminished repopulation ability, a faster wound healing response and lack of central core retention. These results suggest stem cells in keratoconus may be in an elevated state of wound repair and unable to respond appropriately to further injury in corneal maintenance. Sphere forming cell populations in keratoconus appear to be different to those isolated from normal corneas and this may be an important consideration in unearthing keratoconus aetiology.

Exosomes secreted under hypoxia enhance stemness in Ewing's sarcoma through miR-210 delivery.

Intercellular communication between tumor cells within the hypoxic microenvironment promote aggressiveness and poor patient prognoses for reasons that remain unclear. Here we show that hypoxic Ewing’s sarcoma (EWS) cells release exosomes that promote sphere formation, a stem-like phenotype, in EWS cells by enhancing survival. Analysis of the hypoxic exosomal miRNA cargo identified a HIF-1α regulated miRNA, miR-210, as a potential mediator of sphere formation in cells exposed to hypoxic exosomes. Knockdown of HIF-1α in hypoxic EWS cells led to decreased exosomal miR-210 levels and reduced the capacity of hypoxic exosomes to form spheres. Inhibition of miR-210 in hypoxic spheres attenuated sphere formation and overexpression of miR-210 in normoxic spheres significantly enhanced the number of EWS spheres. Our results indicate that hypoxic exosomal miR-210 targets the proapoptotic protein CASP8AP2 in recipient cells. Moreover, the suppression of CASP8AP2 led to a reduction in apoptotic cells and increased sphere formation. Together, the findings in this study suggest that hypoxic exosomes promote stemness in EWS cells by delivering enriched miR-210 that is capable of down-regulating apoptotic pathways, resulting in the survival of cells with increased sphere formation. Future studies will further investigate the effects of EWS derived exosomal miRNAs on target genes and the role these interactions play in driving aggressiveness in hypoxic EWS tumors.

Overexpression of Cancer Upregulated Gene 2 (CUG2) Decreases Spry2 Through c-Cbl, Leading to Activation of EGFR and β-Catenin Signaling.

PurposeThe mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells.Materials and MethodsSpry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and β-catenin reporter assay were performed for downstream of Spry2 signaling.ResultsAlthough A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial–mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and β-catenin-NEK2-Yap1 signaling pathways.ConclusionCUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and β-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.

TAp63\u03b1 targeting of Lgr5 mediates colorectal cancer stem cell properties and sulforaphane inhibition

Cancer stem cells (CSCs) have an established role in cancer progression and therapeutic resistance. The p63 proteins are important transcription factors which belong to the p53 family, but their function and mechanism in CSCs remain elusive. Here, we investigated the role of TAp63α in colorectal CSCs and the effects of sulforaphane on TAp63α. We found that TAp63α was upregulated in spheres with stem cell properties compared to the parental cells. Overexpression of TAp63α promoted self-renewal capacity and enhanced CSC markers expression in colorectal sphere-forming cells. Furthermore, we showed that TAp63α directly bound to the promoter region of Lgr5 to enhance its expression and activate its downstream β-catenin pathway. Functional experiments revealed that sulforaphane suppressed the stemness of colorectal CSCs both in vitro and in vivo. Upregulation of TAp63α attenuated the inhibitory effect of sulforaphane on colorectal CSCs, indicating the role of TAp63α in sulforaphane suppression of the stemness in colorectal cancer. The present study elucidated for the first time that TAp63α promoted CSCs through targeting Lgr5/β-catenin axis and participated in sulforaphane inhibition of the stem cell properties in colorectal cancer.

Sestrin2 Expression Has Regulatory Properties and Prognostic Value in Lung Cancer.

Lung cancer remains the most dangerous type of cancer despite recent progress in therapeutic modalities. Development of prognostic markers and therapeutic targets is necessary to enhance lung cancer patient survival. Sestrin family genes (Sestrin1, Sestrin2, and Sestrin3) are involved in protecting cells from stress. In particular, Sestrin2, which mainly protects cells from oxidative stress and acts as a leucine sensor protein in mammalian target of rapamycin (mTOR) signaling, is thought to affect various cancers in different ways. To investigate the role of Sestrin2 expression in lung cancer cells, we knocked down Sestrin2 in A549, a non-small cell lung cancer cell line; this resulted in reduced cell proliferation, migration, sphere formation, and drug resistance, suggesting that Sestrin2 is closely related to lung cancer progression. We analyzed Sestrin2 expression in human tissue using various bioinformatic databases and confirmed higher expression of Sestrin2 in lung cancer cells than in normal lung cells using Oncomine and the Human Protein Atlas. Moreover, analyses using Prognoscan and KMplotter showed that Sestrin2 expression is negatively correlated with the survival of lung cancer patients in multiple datasets. Co-expressed gene analysis revealed Sestrin2-regulated genes and possible associated pathways. Overall, these data suggest that Sestrin2 expression has prognostic value and that it is a possible therapeutic target in lung cancer.

MicroRNA‐377 acts as a suppressor in esophageal squamous cell carcinoma through CBX3‐dependent P53/P21 pathway

Stem cells play pivotal roles in esophageal squamous cell carcinoma (ESCC) recurrence and metastasis. The self-renewal ability of stem cells was associated with specific microRNAs (miRs). Herein, we identified the effects of miR-377 on ESCC stem cell activities. First, the expression of miR-377 in ESCC and adjacent normal tissues was determined. The relationship between miR-377 and chromobox protein homolog 3 (CBX3) was assessed by a dual-luciferase reporter gene assay. miR-377 was overexpressed or inhibited in ESCC stem cells to explore its role in ESCC. To further investigate the mechanism of miR-377 in ESCC, cells were introduced with short hairpin RNA against CBX3 or pifithrin-α (inhibitor of P53 pathway). Besides, the expression of P21, P53, CD133, CD13, Nanog, sex determining region Y-Box 2 (Sox2), and octamer-binding transcription factor 4 (Oct4), cell sphere formation, colony formation, and proliferation were evaluated respectively. Finally, limiting dilution assay in vivo and tumor xenograft in nude mice were conducted to confirm the roles of miR-377 in vivo. miR-377 was poorly expressed in ESCC. Overexpression of miR-377 could suppress the stem-like trait of ESCC as well as the tumor growth in vivo. miR-377 targeted CBX3 to activate the P53/P21 pathway. Besides, the expression of stem-like markers including CD133, CD13, Oct4, Sox2, and Nanog was decreased, and the abilities of cell sphere formation, colony formation, proliferation, and tumorigenicity were significantly reduced by overexpressing miR-377 or silencing CBX3. The results were reversed after inactivating the P53/P21 pathway. In summary, upregulation of miR-377 inhibits the self-renewal of ESCC stem cells by inhibiting CBX3 expression and promoting activation of the P53/P21 pathway.

Abstract 1491: Transcriptional heterogeneity identifies functional states of tumor-initiating cell differentiation in human colorectal cancer

Abstract Tumor progression in colorectal cancer (CRC) is driven by a subpopulation of cells with tumor-initiating cell (TIC) activity. In the past, bulk experiments provided strong evidence for functional heterogeneity of the CRC TIC compartment with distinct types of TICs driving CRC progression. However, in these retrospective experiments direct assignment of functional states to individual functionally relevant cell types in CRC has not been possible. We here asked whether functional states of individual cells can be assigned to specific CRC cell subpopulations. Therefore, 4,663 single-cell profiles were generated by single-cell RNA-sequencing (scRNA-seq) of patient-derived CRC spheroid cultures (n=12). Non-negative matrix factorization identified 13 gene expression modules linked to cell states or cell types that resemble differentiation states of normal intestinal epithelial cells. To evaluate their presence in CRC patient tumors, the identified transcriptional programs were applied to publicly available expression data (TCGA cohort; n=459). Clustering of samples based on the contribution of individual expression signatures revealed six patient clusters with significantly different progression-free survival (p=0.043) and a trend towards different overall survival (p=0.12), indicating a relevance for patient outcome. Comparing cell state meta-signature expression in cell type-associated subpopulations revealed differences in functional states, i.e. proliferative and metabolic activity. Interestingly, stem-like cells showed significantly increased oxidative phosphorylation (OXPHOS) and decreased glycolysis scores, suggesting a link between metabolic states and cell differentiation. scRNA-seq of patient-derived organoid and xenograft models as well as primary tumors revealed similar trends. To assess whether metabolic states are linked to TIC activity, CRC spheroid cells were sorted according to mitochondrial membrane potential (MMP), and sphere forming cell (SFC) as well as TIC frequency were assessed by limiting dilutions. Strikingly, MMPhigh cells exhibited a pronounced increase of SFC frequencies in vitro compared to MMPlow cells in 80% of patients (n=5). In addition, TIC frequencies were strongly increased in the MMPhigh compared to the MMPlow subpopulation in vivo (n=2; 1/46,535 vs. 1/211,305 and 1/249 vs. 1/2,089), demonstrating that TIC activity relies on OXPHOS. In line with this, pretreatment with the OXPHOS inhibitor CCCP decreased SFC frequencies (n=3), indicating targetable metabolic vulnerability of CRC spheroid cells. In summary, we here show that transcriptional heterogeneity identifies functional states during CRC TIC differentiation. Targeting context-dependent regulation of tumor cell differentiation might unravel novel vulnerabilities for therapeutic intervention in human CRC. Citation Format: Martina K. Zowada, Stephan M. Tirier, Sebastian M. Dieter, Teresa Krieger, Ava Oberlack, Robert L. Chua, Mario Huerta, Foo Wei Ten, Karin Laaber, Jeongbin Park, Katharina Jechow, Torsten Müller, Mathias Kalxdorf, Mark Kriegsmann, Katharina Kriegsmann, Friederike Herbst, Jeroen Krijgsveld, Martin Schneider, Roland Eils, Hanno Glimm, Christian Conrad, Claudia R. Ball. Transcriptional heterogeneity identifies functional states of tumor-initiating cell differentiation in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1491.

Abstract 141: G9a regulates tumorigenicity and stemness through genome-wide DNA methylation reprogramming in non-small cell lung cancer

Abstract Eukaryotic Histone Methyltransferase 2 (EHMT2 or G9A) has been regarded as a potential target for Non-Small Cell Lung Cancer (NSCLC) therapy. To investigate the regulatory roles of G9A in tumorigenesis, and stemness, we isolated and enriched tumor-initiating cells (TIC) from surgically resected NSCLC tissues by FACS and sphere formation assays. We then knocked down G9A using shRNA and carried out genome-wide 850K methylation array and RNA sequence analyses. We identified 67 genes hypomethylated and 143 genes upregulated following G9A knockdown of which 43 genes were both hypomethylated and upregulated. We selected 6 genes (CDYL2, DPP4, SP5, FOXP1, STAMBPL1 and ROBO1) for validation. These genes are involved in epigenetic regulation in human cancers, signaling pathways, other biological processes and have roles in patients' prognosis. In addition, G9A expression was higher in TICs and targeting G9a by shRNA knockdown or by selective inhibitor UNC0638 significantly inhibited the expression of cancer stem cell markers and sphere forming capacity, in vitro proliferation and in vivo growth. Further, transient overexpression of FOXP1 in TICs resulted in downregulation of stem cell markers and sphere forming capacity and cell proliferation in vitro indicating that the genes we identified are directly regulated by G9A through aberrant DNA methylation and subsequent expression. These data suggest that G9A contributes to epigenetic regulation by maintaining DNA methylation of multiple genes genome-wide and their expression. Citation Format: Rajendra P. Pangeni, Lu Yang, Keqiang Zhang, Jinhui Wang, Wendong Li, Chao Guo, Xinwei Yun, Ting Sun, Jami Wang, Dan J. Raz. G9a regulates tumorigenicity and stemness through genome-wide DNA methylation reprogramming in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 141.

Abstract 3678: The role of SCAMP3 in EGFR trafficking and breast cancer cell response

Abstract Breast cancer (BC) affects approximately 1 in 8 women and is the second leading cause of cancer among women in the United States. BC tumors frequently overexpress Epidermal Growth Factor Receptor (EGFR) which contributes to BC progression and poor clinical outcomes. Studies associate the endocytosis related protein, Secretory Carrier Membrane Protein 3 (SCAMP3), with the regulation of EGFR recycling and degradation. Recently, we published that SCAMP3 is overexpressed in BC cells and tumors. Others have identified SCAMP3 as an indicator of poor prognosis in hepatocellular carcinoma. Therefore, we hypothesize that SCAMP3 promotes the trafficking of EGFR, induces EGFR signaling and BC progression. To evaluate whether SCAMP3 has a direct interaction with EGFR, we treated SUM-149 (EGFR+) and MDA-MB-468 (EGFR+++) BC cells with 100ng/mL EGF at different time points, then we evaluated the localization of both proteins by immunofluorescence. Results showed that SCAMP3 co-localizes with EGFR at the cytoplasm at early stimulation times and the perinuclear area after 30 mins in both cell lines. To evaluate if SCAMP3 has a role in BC progression, we knocked down SCAMP3 in SUM-149 cells using the CRISPR/Cas9 technique, and we assessed the effects in cell viability, colony, and sphere formation capacity and EGFR signaling. Knockdown (KO) and wild type (WT) cells were treated with the ligand at the same concentration described above for 30 min. Our results showed that SCAMP3 knockdown decreased cell viability, colony number and the area of formed spheres. The activation of ERK1/2 was also reduced in SCAMP3 KO cells. Taken together, SCAMP3 is involved in the internalization of EGFR, cell viability, proliferation, and tumor sphere formation. We conclude that SCAMP3 has an essential role in EGFR trafficking and BC progression through EGFR downstream signaling modulation. Citation Format: Ariana C. Acevedo Diaz, Jael Reyes- Chea, Michelle M. Martinez-Montemayor, Ivette J. Suárez-Arroyo. The role of SCAMP3 in EGFR trafficking and breast cancer cell response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3678.

Abstract 1446: Mitochondrial transfer in the ovarian tumor microenvironment contributes to tumor stem cell phenotypes

Abstract Introduction: Ovarian cancer is the deadliest gynecologic malignancy in the USA, and many patients incur diffuse metastases before diagnosis. In order to improve prognoses and reveal novel therapeutics, we must understand the mechanisms of ovarian tumor metastasis. Previous studies demonstrate that interactions between ovarian tumor cells (TCs) and Carcinoma-Associated Mesenchymal Stem Cells (CA-MSCs) are important drivers of metastasis. In addition, preliminary data exposes mitochondrial transfer between CA-MSCs and ovarian TCs as a potential contributor to cancer stem cell (CSC) development. Since CSCs contribute to cancer persistence, these new findings motivate current research exploring whether mitochondrial gain enhances TC metastatic fitness and CSC formation. Methods: CA-MSC mitochondria were transiently stained with MitoTracker Green, and GFP+ CA-MSCs were co-cultured with RFP+ TCs for various durations. Immunofluorescence visualized mitochondrial transfer following co-culture. Mitochondrial transfer from CA-MSCs to TCs was quantified with flow cytometry, separating GFP+ TCs from GFP-TCs. To examine whether mitochondrial transfer requires direct cell-cell contact, CA-MSC were separated from TCs with 0.4um Transwell membranes during co-culture. To elucidate functional phenotypic differences associated with mitochondrial gain, TCs and GFP+ CA-MSCs were directly co-cultured and TCs that acquired GFP+ mitochondria were sorted out via FACS. The TC populations that did or did not gain mitochondria were compared for cell proliferation rates, chemotherapy resistance, and stem-like phenotypes via cell viability and sphere formation assays. Results: We obtained fluorescent images illustrating that CA-MSC mitochondria transferred to adjacent TCs within 4 hours of direct co-culture. When CA-MSCs and TCs were indirectly co-cultured using Transwell membranes, mitochondria were not transferred as robustly as they were via direct co-culture. Interestingly, TCs that gained mitochondria appeared to grow more slowly and were less resistant to cisplatin treatment when compared to TCs that did not gain mitochondria. However, the TCs that gained mitochondria appeared to form more spheres and thus demonstrate a more CSC-like phenotype than TCs that did not gain mitochondria. Conclusions: These data suggest that ovarian TCs receive mitochondria from adjacent CA-MSCs via direct interaction. The TCs that obtain mitochondria may functionally diverge from TCs that don't gain mitochondria, exhibiting phenotypes consistent with CSCs. Future experiments will further delineate the functional difference and translational significance attributable to TC mitochondrial gain, potentially demonstrating that mitochondrial transfer in ovarian tumors can be therapeutically exploited to reduce metastases. Citation Format: Catherine Pressimone, Huda Issa Atiya, Leonard Frisbie, Lan Coffman. Mitochondrial transfer in the ovarian tumor microenvironment contributes to tumor stem cell phenotypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1446.

Three-dimensional culture of a pancreatic cancer cell line, SUIT-58, with air exposure can reflect the intrinsic features of the original tumor through electron microscopy

Mini-abstract: Application of a three-dimensional culture system with air exposure facilitates the formation of large cell spheres possessing cribriform glands and producing mucin in the collagen gel. Transmission electron microscopy revealed the formation of microvilli and junctional complexes at the apical side of the cell. This study aimed to reproduce the characteristics of original adenocarcinoma tumors in vitro. The pancreatic cell line, SUIT-58, derived from a moderately differentiated adenocarcinoma of metastatic pancreatic cancer was used. The cells have a sheet structure in conventional cell culture without forming glands or exhibiting mucin production in the lumen. First, the necessity of scaffolds to create an adenocarcinoma-like microenvironment for SUIT-58 pancreatic cancer cells was assessed. Compared with conventional culture plates, the use of type I collagen as a scaffold played an important role in the formation of densely congested microvilli, as observed through scanning electron microscopy. As gland formation is one of the features of adenocarcinoma, we also assessed gland formation. Use of a recently developed three-dimensional culture system with air exposure resulted in the formation of large cell spheres possessing cribriform glands, which released mucin into the lumen. Transmission electron microscopy also revealed the formation of microvilli in the lumen of the glands and junctional complex at the intercellular part, which were similar to those observed in xenografts. These findings indicate that an in vitro three-dimensional culture system with air exposure reflects the intrinsic features of the original tumor, suggesting that this culture system could be useful for preliminary research of certain cancers.

The Down-Regulation of TrkB Alleviates the Malignant Biological Behavior and Cancer Stem-Like Property of Laryngeal Cancer.

BackgroundThis study aimed to evaluate the effect of TrkB down-regulation on the malignant biological behavior and stem-like characteristics of laryngeal cancer.MethodsThe relationship was analyzed between TrkB and clinicopathological parameters in patients with laryngeal cancer. The mRNA expressive levels of TrkB and miR-10a-5p were detected by qRT-PCR in laryngeal cancer tissues and cell lines. In vitro, Hep-2 and AMC-HN-8 cell proliferation, apoptosis and stem-like properties were detected by colony formation assay, flow cytometry, sphere formation, and Western blot, respectively. In vivo, the BALB/c nude mice model was used to evaluate the effect of TrkB on tumor growth.ResultsThe results showed that TrkB was related to smoking history, clinical stage, and lymph node metastasis, but had nothing to do with the gender, age, and tumor location of patients with laryngeal cancer. TrkB was highly expressed and miR-10a-5p was lowly expressed in laryngeal cancer tissues and cell lines. Down-regulation of TrkB inhibited Hep-2 and AMC-HN-8 cell proliferation and sphere formation as well as enhanced apoptosis, The result showed that miR-10a-5p bound to the 3ʹ-UTR of BDNF by a dual-luciferase reporter assay. Down-regulation of miR-10a-5p induced up-regulation of TrkB promoting development of laryngeal cancer. In vivo, down-regulation of TrkB suppressed tumor growth and inhibited the expression of stem-like marker proteins and promoted apoptosis.ConclusionIn conclusion, down-regulation of TrkB plays an important role in laryngeal cancer and is a promising target for future intervention strategies.