Expression Of Stem Cell Markers Research Articles

Continuous TNF-α exposure in mammary epithelial cells promotes cancer phenotype acquisition via EGFR/TNFR2 activation.

Tumor necrosis factor alpha (TNF-α), an abundant inflammatory cytokine in the tumor microenvironment (TME), is linked to breast cancer growth and metastasis. In this study, we established MCF10A cell lines incubated with TNF-α to investigate the effects of continuous TNF-α exposure on the phenotypic change of normal mammary epithelial cells. The established MCF10A-LE cell line, through long-term exposure to TNF-α, displayed cancer-like features, including increased proliferation, migration, and sustained survival signaling even in the absence of TNF-α stimulation. Unlike the short-term exposed cell line MCF10A-SE, MCF10A-LE exhibited elevated levels of epidermal growth factor receptor (EGFR) and subsequent TNF receptor 2 (TNFR2), and silencing of EGFR or TNFR2 suppressed the cancer-like phenotype of MCF10A-LE. Notably, we demonstrated that the elevated levels of NAD(P)H oxidase 4 (NOX4) and the resulting increase in reactive oxygen species (ROS) were associated with EGFR/TNFR2 elevation in MCF10A-LE. Furthermore, mammosphere-forming capacity and the expression of cancer stem cell (CSC) markers increased in MCF10A-LE. Silencing of EGFR reversed these effects, indicating the acquisition of CSC-like properties via EGFR signaling. In conclusion, our results reveal that continuous TNF-α exposure activates the EGFR/TNFR2 signaling pathway via the NOX4/ROS axis, promoting neoplastic changes in mammary epithelial cells within the inflammatory TME.

The Power of Synergism: A Novel Drug Combination for Improved Therapy of Glioblastoma

AbstractOne of the biggest obstacles in treating glioblastoma multiforme (GBM) is the plasticity and adaptability of GBM cells, which renders monotherapy ineffective. Novel drug combinations that target multiple pathways are required to overcome this challenge. In the current study, the PI3K/AKT/mTOR pathway is identified as a crucial component in GBM cell survival using the KEGG database and literature search. Hence, it is hypothesized that a potential synergistic therapy can be achieved by targeting different stages of this pathway by combining disulfiram (DSF) and 7‐ethyl‐10‐hydroxycamptothecin (SN‐38). The efficacy of this drug combination is evaluated using 2D and 3D in vitro assays, which exhibited a significant synergism with 5 × 104 times lower IC50 than that of temozolomide (TMZ), the gold‐standard drug. The combination treatment increases intracellular ROS (reactive oxygen species) production, and ROS inhibition by using N‐Acetyl Cysteine (NAC) reduced the cytotoxicity substantially, indicating that ROS played a crucial role in the synergistic cytotoxicity. The combination treatment inhibited GBM cell proliferation, migration, and stem cell marker expression. Mechanistically, the DSF+SN‐38 combination is found to increase p53 expression, inhibit PI3K signaling, and activate caspase 9. Altogether, this study demonstrates that the DSF+SN‐38 combination can present a promising therapeutic strategy for treating GBM.

Electrospun poly(l-lactide-co-dl-lactide) nanofibrous scaffold as substrate for ex vivo limbal epithelial cell cultivation

Ultrathin electrospun poly(l-lactide-co-dl-lactide) nanofibrous membranes coated with fibronectin were explored as scaffolds for the ex vivo cultivation of limbal epithelial cells (LECs) for the treatment of limbal stem cell deficiency. The developed scaffolds were compared with the “gold-standard” fibrin gel. The resulting membranes composed of nanofibers possessed a very low thickness of 4 μm and allowed very good optical transparency in the wet state. The fibronectin-coated nanofibrous scaffolds demonstrated LEC expansion and successful cultivation similar to that on fibrin gel. Unlike the regular cobblestone epithelial cell morphology on the fibrin gel, the nanofibrous scaffold presented a mostly irregular epithelial morphology with a shift to a mesenchymal phenotype, as confirmed by the upregulation of profibroblastic genes: ACTA2 (p = 0.023), FBLN1 (p < 0.001), and THY1 (p < 0.001). Both culture conditions revealed comparable expression of stem cell markers, including KLF4, ΔNp63α and ABCG2, emphasizing the promise of polylactide-based nanofibrous membranes for further investigations.

Oleic acid inhibits the migration and invasion of breast cancer cells with stemness characteristics through oxidative stress-mediated attenuation of the FAK/AKT/NF-κB pathway

Oleic acid, found in a variety of food sources, is beneficial to health. However, studies on its anticancer effects on cancer stem cells resistant to conventional treatments and on the mechanisms underlying such effects are scarce. Here, we evaluated the anticancer effects of oleic acid against breast cancer cells with stemness characteristics. Oleic acid inhibited the proliferation of breast cancer stem-like cells more significantly than that of their respective parental cells, and reduced the expression of stem cell markers and migration and invasion propensity. It also repressed the phosphorylation of FAK, AKT, and NF-κB, proteins involved in cell migration and invasion. Oleic acid-induced increase in reactive oxygen species levels mediated the inhibition of the FAK/AKT/NF-κB signaling axis. Our study demonstrates that oleic acid-induced oxidative stress plays an important role in inhibiting the metastatic behavior of breast cancer cells with stemness characteristics and is relevant for management of treatment-resistant breast cancer.

Urine-derived stem cells reverse bleomycin‑induced experimental pulmonary fibrosis by inhibition of the TGF-β1-Smad2/3 pathway

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung interstitial lesions with the disease pathophysiology incompletely understood, which is a serious and fatal disorder with limited treatment options. Mesenchymal stem cells (MSCs) have exhibited promising therapeutic capability for IPF. While most types of MSCs are obtained invasively, urine-derived stem cells (USCs) can be gained in a safe, noninvasive and inexpensive procedure, which are readily available and reported to exhibit no risk of teratoma formation or oncogenic potential in vivo, sounding alternative to other MSCs. This study aims to investigate the therapeutic effect and mechanism of USCs on IPF, using a bleomycin (BLM)-induced IPF model in mice. Methods: Cell surface marker examination by flow cytometry analysis and cell differentiation culture were used to characterize USCs obtained from healthy individuals. BLM was instilled endotracheally in adult C57BL/6 mice, followed by USCs or human bone marrow-derived mesenchymal stem cells (BMSCs) treatment by tail vein injection on day 14. Mice were euthanized on day 14 before administration or day 21 for the evaluation of pulmonary histopathology and hydroxyproline (HYP) content. Inflammatory factors of lung, including transforming growth factor (TGF)-β1, TNF-α, IL-6, MMP2 were analyzed by quantitative real-time PCR (qRT-PCR). Additionally, immunohistochemistry (IHC) and western blotting (WB) were applied to evaluate the expression of α-SMA and activation of TGF-β1-Smad2/3 in lung. Results: USCs highly expressed CD29 and CD90, showing negative expression of haematopoietic stem cell markers (CD45, CD34) and could differentiate into, at least, bone and fat in vitro. In mice challenged with BLM, septal thickening and prominent fibrosis were observed on day 14, with higher HYP content and mRNA levels of TGF-β1, TNF-α and IL-6 exhibited, compared to untreated mice. USCs could migrate to lung and accumulate there in mouse model after intravenous injection. Transplantation of USCs into BLM-induced mice improved their pulmonary histopathology, decreasing Ashcroft score, Szapiel score, HYP content and mRNA levels of TGF-β1 and MMP2 of lung, similar to the effects of BMSCs. IHC and WB further revealed that USCs could inhibit activation of the TGF‑β1-Smad2/3 pathway of lung in vivo. Conclusions: Transplantation of USCs effectively reverses pulmonary fibrotic phenotype in an experimental IPF model, inhibiting the TGF-β1-Smad2/3 pathway, a key driver of fibrosis. These results suggest the therapeutic application of USCs for IPF, instead of other types of MSCs obtained invasively.

Boswellic acid as a potential adjunct for bone healing after endodontic surgery: In vitro study

Abstract Introduction: The role of Acetyl -11-keto-β-boswellic acid (AKBA) in regulating osteoblast differentiation was recently brought to light. Therefore, the current study was designed to explore the osteogenic differentiation capability of AKBA on bone marrow mesenchymal stem cells (BMMSCs) as a potential therapeutic agent to accelerate the healing process in apicoectomy. Materials and Methods: BMMSCs were characterized by flow cytometry. Cellular viability and proliferation assays were used with different concentrations of AKBA. Cells were divided into 5 groups to test osteogenic differentiation: Group I: negative control, Group II: positive control, Group III: BMMSCs were treated with 1 μM AKBA, Group IV: BMMSCs were treated with 0.1 μM AKBA, and Group V: BMMSCs were treated with 0.01 μM AKBA. Mineralization assays and gene expression analysis were assessed, and the significance difference between groups was established at P &lt; 0.05. Results: The flow cytometry analysis demonstrated that BMMSCs had positive expression for mesenchymal stem cell marker and negative expression for hematopoietic markers. The concentration of 0.01 μM gave significantly higher cell density than the untreated cells after 7 days (P &lt; 0.05). Cells treated with 0.1 and 0.01 μM AKBA revealed a significantly higher ALP activity, alizarin red, and von Kossa staining than control groups (P &lt; 0.05). High expression of osteogenic genes was detected in BMMSCs treated with 0.1 μM AKBA (P &lt; 0.05). Conclusions: It was declared that the concentration of 0.1 μM AKBA has no toxicity on BMMSC viability and proliferation with an impact on BMMSC osteogenic differentiation. Therefore, AKBA (0.01 μM) could be used in bone regeneration during periradicular surgery.

Molecular stratification of the human fetal vaginal epithelium by spatial transcriptome analysis.

The human vaginal epithelium is a crucial component of numerous reproductive processes and serves as a vital protective barrier against pathogenic invasion. Despite its significance, a comprehensive exploration of its molecular profiles, including molecular expression and distribution across its multiple layers, has not been performed. In this study, we perform a spatial transcriptomic analysis within the vaginal wall of human fetuses to fill this knowledge gap. We successfully categorize the vaginal epithelium into four distinct zones based on transcriptomic profiles and anatomical features. This approach reveals unique transcriptomic signatures within these regions, allowing us to identify differentially expressed genes and uncover novel markers for distinct regions of the vaginal epithelium. Additionally, our findings highlight the varied expressions of keratin ( KRT) genes across different zones of the vaginal epithelium, with a gradual shift in expression patterns observed from the basal layer to the surface/superficial layer. This suggests a potential differentiation trajectory of the human vaginal epithelium, shedding light on the dynamic nature of this tissue. Furthermore, abundant biological processes are found to be enriched in the basal zone by KEGG pathway analysis, indicating an active state of the basal zone cells. Subsequently, the expressions of latent stem cell markers in the basal zone are identified. In summary, our research provides a crucial understanding of human vaginal epithelial cells and the complex mechanisms of the vaginal mucosa, with potential applications in vaginal reconstruction and drug delivery, making this atlas a valuable tool for future research in women's health and reproductive medicine.

Associations of early-life and adult anthropometric measures with the expression of stem cell markers CD44, CD24, and ALDH1A1 in women with benign breast biopsies.

According to the stem cell hypothesis, breast carcinogenesis may be related to the breast stem cell pool size. However, little is known about associations of breast cancer risk factors, such as anthropometric measures, with the expression of stem cell markers in non-cancerous breast tissue. The analysis included 414 women with biopsy-confirmed benign breast disease (BBD) in the Nurses' Health Study (NHS) and NHSII. Birthweight, weight at age 18, current weight, and current height were reported via self-administered questionnaire. Immunohistochemical staining of stem cell markers (CD44, CD24, ALDH1A1) in histopathologically normal epithelial and stromal breast tissue was quantified with an automated computational image analysis system. Linear regression was used to examine the associations of early-life and adult anthropometric measures with log-transformed stem cell marker expression, adjusting for potential confounders. Birthweight (≥10.0 vs. <5.5 lbs: β [95% CI]=4.29 [1.02, 7.56]; p-trend=0.001 in stroma) and adult height (≥67.0 vs. <63.0 inch: 0.86 [0.14, 1.58]; p-trend=0.02 in epithelium and stroma combined) were positively associated with CD44 expression. Childhood body fatness was inversely (p-trend=0.03) and adult height was positively associated with CD24 expression in combined stroma and epithelium (p-trend=0.03). Our data suggest that anthropometric measures, such as birthweight, adult height, and childhood body fatness, may be associated with the stem cell expression among women with BBD. Anthropometric measures, such as birthweight, height, and childhood body fatness, may have long-term impacts on stem cell population in the breast.

Age-related reduction in the functional properties of adult stem cells located in the peripheral region of human retinal pigment epithelium.

Adult stem cells (SCs) with self-renewal and multilineage potential have been reported upon culturing human retinal pigment epithelial (RPE) cells. The current study aimed to identify the location of SCs in human RPE and to elucidate the age-related changes. Peripheral, equatorial, and central RPE cells from donors of three age groups were analyzed for their sphere-forming, clonal, and label-retaining cell properties. Furthermore, native human RPE flatmounts were immunostained for SC and proliferating cell markers. Cells with higher sphere-forming and clonal ability were identified only in young donors (<30 years) and were restricted to the periphery. Upon culturing, cells from peripheral and equatorial regions had the label-retaining cell (LRC) property. With aging, the LRCs were restricted to the periphery and were reduced. In young donors, Ki67+ proliferating cells were not observed in native RPE. However, such cells were observed in the peripheral RPE of older donors correlating with the need for regeneration. The native RPE cells were negative for SC marker expression. The above findings highlighted the presence of SCs with the ability to proliferate in the peripheral RPE and a reduction in these functional properties of SCs with aging.

Generation of a human induced pluripotent stem cell line (SHUPLi002-A) from PBMCs of a healthy female donor

Human induced pluripotent stem cells (iPSCs) have good multi-lineage differentiation potential, which is an ideal model for studying the pathogenesis of diseases and drug screening.Here, we generated an iPSC line, SHUPLi002-A, from peripheral blood mononuclear cells (PBMCs) of a healthy 28-year-old female individual using episomal vectors. SHUPLi002-A is characterized by expression of classical pluripotent stem cell markers as well as teratoma formation assays to evaluate their differentiation capacity in vivo.

Generation of a human iPSC line from a Parkinson’s disease patient with a novel CHCHD2 mutation (p.R145Q)

Mutations in CHCHD2 have been reported to be associated with familial Parkinson’s disease (PD). We generated a human induced pluripotent stem cell (hiPSC) line by reprogramming dermal fibroblasts from a PD patient harboring a novel CHCHD2 mutation (c.434G > A, p.R145Q). This line exhibited human embryonic stem cell (hESC)-like clonal morphology, expression of undifferentiated stem cell markers, a normal karyotype and trilineage differentiation capacity and thus the potential to serve as a model for further investigating the underlying molecular mechanisms of CHCHD2 function in PD.

Short-chain fatty acids induced lung tumor cell death and increased peripheral blood CD4+ T cells in NSCLC and control patients ex vivo.

Despite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth. This study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer. In this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations. To investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects. Overall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

Enhancing the anticancer effect of androgen deprivation therapy by monocarboxylate transporter 1 inhibitor in prostate cancer cells.

Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets. PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor. Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions. Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT.

Hsa_circTCF25 Facilitates Oncogenesis in Gastric Cancer Cells by Modulating miR-149 Expression.

Circular RNA (circRNA) is related to gastric carcinogenesis and progression. This study explored the effects of circTCF25 on gastric cancer cell proliferation, migration, invasion, and cancer stem cell markers, as well as the potential network of circTCF25-miR and miR-149. circTCF25 expression was detected in tissue specimens and cells by real-time quantitative reverse transcription polymerase chain reaction. Cell Counting Kit-8 and transwell assays were used to measure the effects of circTCF25 knockdown on proliferation, migration and invasion. The potential network of circTCF25 was analyzed using bioinformatic analysis. circTCF25 was overexpressed in human gastric cancer tissues, and a series of cancer cell lines, and was associated with shorter overall survival. Interfering with circTCF25 reduced gastric cancer cell proliferation, migration, invasion and expression of cancer stem cell markers. CircTCF25 reduced expression of miR-149, apparently by acting as a miR-149 sponge. A new circTCF25-miR-149 competitive endogenous RNA network in gastric cancer was constructed, and most core genes were associated with the malignant growth and metastatic behavior of gastric cancer. circTCF25 may have prognostic value and an oncogenic role in gastric cancer. A circTCF25-miR-149 RNA regulatory network was established which may provide novel biomarkers or potential therapeutic targets for treating gastric cancer.

Osteopontin promotes tumor growth and metastasis and GPX4-mediated anti-lipid peroxidation in triple-negative breast cancer by activating the PI3k/Akt/mTOR pathway

PurposeTriple-negative breast cancer (TNBC) features high aggressiveness, metastasis rate, drug resistance as well as poor prognosis. Osteopontin (OPN) is a key protein in the process of osteogenesis and has emerged as a new tumor marker in recent years.MethodsCell viability was tested with the CCK-8 kit. Transwell and wound healing were adopted to test cell invasive and migratory abilities. Tumor sphere formation was detected by tumor sphere formation assay. Human umbilical vein endothelial cell (HUVEC) tube formation assay was used to measure the angiogenesis of tumor cells. Western blot was applied for the estimation of the expression of cancer stem cell markers, angiogenesis-, signaling pathway-related proteins as well as OPN. Bioinformatics tools predicted OPN expression in breast cancer tissues. The levels of oxidative stress-related markers were assessed with ELISA. Following the overexpression of OPN in MD-MB-436 cells and the addition of the PI3K/AKT/mTOR pathway inhibitor LY294002, the aforementioned functional experiments were implemented again to investigate the mechanism. Finally, in vivo experiments of tumor-bearing mice were performed for further verification. ResultsThe proliferative, invasive, migratory and tumor sphere formation capabilities as well as angiogenesis of TNBC cells were conspicuously increased in contrast to non-TNBC cell lines. OPN expression in TNBC tissues and cells was dramatically enhanced. OPN upregulation significantly elevated cell proliferative, invasive and migratory capabilities as well as tumor sphere formation and angiogenesis. The mechanism might be achieved by activating PI3K/AKT/mTOR signaling to regulate glutathione peroxidase 4 (GPX4)-mediated anti-lipid peroxidation.ConclusionOPN promoted tumor sphere formation and angiogenesis in TNBC by activating the PI3K/AKT/mTOR pathway to regulate GPX4-mediated anti-lipid peroxidation levels.

Abstract 2776: Exploring the differences between brain tumor initiating cell extracellular vesicles and secreted factors that influence the tumor microenvironment

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Within the tumor, there is a subtype of cells deemed brain tumor initiating cells (BTICs), which contribute to tumor progression, therapy resistance, and tumoral heterogeneity. The tumor microenvironment, surrounding tumors, contains an ecosystem of cells in which BTICs can interact with and further promote tumor progression. In the lateral wall of the lateral ventricles, the neurogenic niche of the subventricular zone (SVZ), contains neural progenitor cells (NPCs) that self-renew and generate highly migratory neuroblasts. Interestingly, GBM tumors proximal to the SVZ are more proliferative, migratory, and negatively impact the survival of patients. We have previously observed that the presence of GBM induces reciprocal changes to the SVZ altering its neurogenic capacity and resulting in a more proliferative tumor. BTICs share similar self-renewal capacity to NPCs and may exert intercellular communication resulting in increased malignancy features. The mechanisms of intercellular communication, present in the SVZ include paracrine, autocrine, direct cell contact, gap junctions, nanotubes, and more recently extracellular vesicles (EVs). However, the exact mechanisms of how BTICs communicate with NPCs is not well understood. EVs are a heterogenous group of cell-derived membranous structures that serve as means of intercellular communication, allowing for cells to exchange proteins, lipids, RNA, and other genetic material. Cancer cells release higher amounts of EVs than non-malignant cells, and these EVs help communicate with other nearby cells, leading to promotion of tumorigenesis. We have observed that BTICs-derived EVs increase the proliferation of human NPCs, while altering their morphology and expression of stem cell markers. These effects are not observed when NPCs are treated with vesicle-free BTIC-derived secreted factors. To investigate the distinctions between BTIC secreted factors and protein cargo contained in BTIC-EVs, we employ the proximity protein labelling system, TurboID, and express this system in BTIC-EVs. The overarching goal of this project is to characterize BTIC-derived EV cargo to identify proteins that modify the GBM tumor microenvironment. We hypothesize that the phenotype of non-cancer NPCs is altered by distinct protein cargo contained in BTIC-derived EVs. The results of these experiments will help us to elucidate what BTIC-specific EV cargo impacts the tumor microenvironment, with the potential of revealing therapeutic targets for GBM. Citation Format: Marissa Russo, Maria Jose Ulloa Navas, Emily Norton-Ramos, Alfredo Quiñones Hinojosa, Hugo Guerrero Cazares. Exploring the differences between brain tumor initiating cell extracellular vesicles and secreted factors that influence the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2776.

Abstract 2875: Resistance to conventional chemo and radiotherapy associated with increased DNA repair and stem cell-like phenotype in head and neck squamous cell carcinoma expressing HPV E5

Abstract Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is one of the fastest-rising cancers in the US. While HPV+ HNSCC patients exhibit better responses to chemoradiation compared to HPV- HNSCC patients, a subset of HPV+ patients do not respond as well and develop recurrent/refractory disease. In this study, we investigated the potential role of HPV E5 in promoting resistance to conventional chemo- and radiotherapy, and the underlying molecular mechanisms. HPV E5 is a small hydrophobic viral oncoprotein with multiple functions including regulation of tumor cell differentiation and apoptosis, modulation of H+ ATPase responsible for acidification of late endosomes, and immune modulation. To test the impact of HPV E5 on therapeutic resistance, we generated HNSCC cell lines and normal keratinocytes expressing HPV16 E5. Upon treatment with radiation (2Gy, 4Gy, 8Gy) and/or cisplatin, HNSCC cells and keratinocytes expressing HPV 16 E5 had increased cell viability, decreased apoptosis, and increased colony-forming capacity compared to empty vector (EV) control lines. Specifically, treatment with 4Gy radiation, 4µM cisplatin, or the combination led to a ~2-fold decrease in % of apoptotic cells in HPV16 E5-expressing HNSCC cells compared to EV and HPV16 E6/E7 HNSCC cells (p &amp;lt;0.05). Examination of DNA damage response and repair pathways indicated that while HPV16 E5 HNSCC cells initially had increased phosphorylation of H2AX 6 hours post treatment, at 24 hours post treatment H2AX phosphorylation in HPV16 E5 cells was significantly decreased compared to EV or HPV16 E6/E7 HNSCC cells. These findings indicate increased DNA repair capacity of HPV16 E5 HNSCC cells. To identify mechanisms underlying this phenotype, we performed RNA-seq and observed enhanced signaling pathways associated with epithelial development and increased expression of stem cell markers in HPV16 E5 HNSCC cells compared to EV control lines. Functionally, we observed a ~2.5-fold increase in sphere-forming capacity and increased expression of markers associated with stem cell phenotype in HPV16 E5 HNSCC cells compared to EV and HPV E6/E7 HNSCC cells. To determine whether these findings translated clinically, 18 HPV+ HNSCC patients from the GSE74927 dataset were grouped according to median expression level of HPV E5 (High vs Low). In support of our hypothesis, HPV E5-High patients exhibited enrichment of pathways associated with extrinsic apoptotic signaling, pluripotency of stem cells, and positive regulation of DNA repair. Taken together these results identify HPV E5 as a potential mediator of resistance to conventional radiation and chemotherapy in HNSCC and suggest that patient tumors expressing HPV E5 may have increased DNA repair capacity and cell survival after cytotoxic therapies. Citation Format: Souvick Roy, Sayuri Miyauchi, Riley Jones, Sangwoo Kim, Andrew B. Sharabi. Resistance to conventional chemo and radiotherapy associated with increased DNA repair and stem cell-like phenotype in head and neck squamous cell carcinoma expressing HPV E5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2875.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6956.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4250.

Abstract 2784: Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion

Abstract Introduction: Overexpression of lipogenic enzymes has been associated with a poor clinical outcome in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein, prevents the downstream activation of Wnt signaling by de-palmitoylating Wnt ligands. Notum is also a CRC stem cell marker, and its expression correlates with poor prognosis in CRC. We found that upregulation of FASN is associated with an increase in Notum expression in mouse adenoma tissues. Therefore, the purpose of this study is to elucidate the mechanisms and contribution of FASN/Notum axis in CRC. Methods: Adenoma and normal intestinal organoids, established from ApcMin/VillinCre-ERT2 and Apc/VillinCre-ERT2 mouse models with hetero- and homozygous deletion of FASN. 4-hydroxytamoxifen (4-OHT) and TVB-3664 (FASN inhibitor) treatments were used to assess the effect of FASN deletion/inhibition on organoid growth and viability. LIVE/DEAD™ Viability/Cytotoxicity and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth. Human NOTUM/Protein notum homolog ELISA Kit was used to analyze Notum secretion. CRC cells, NTC and FASN shRNA, and control and FASN overexpression, were used for Notum analysis. Results: The TCGA data analysis shows that Notum is significantly upregulated in CRC and its expression correlates with expression of FASN in tumor tissue. Utilizing qRT-PCR and Western blot analyses, we show that downregulation of FASN leads to a decrease in expressions of active β-catenin, Notum, and other stem cell markers in transgenic mouse models. Moreover, 4-OHT-mediated inhibition of FASN results in decrease viability and size in ApcMin/FASN/VillinCre-ERT2 and Apc/FASN/VillinCre-ERT2 organoids. Consistently, shRNA-mediated deletion of FASN decreases expression of Notum in CRC cells. In contrast, overexpression of FASN increases the expression levels of active and total β-catenin, Notum, and other stem cell markers in SW480 cells. We show that normal epithelial cells exposed to CRC cell medium exhibit stem-like phenotype which is reduced when cells are exposed to medium collected from Notum shRNA knockdown cells. Moreover, the addition of recombinant Notum restores this phenotype. Conclusion: In summary, downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stem-like properties via upregulation of β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis in CRC. Citation Format: Courtney Olivia Kelson, Josiane Weber Tessmann, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2784.