Absence Of Epidermal Growth Factor Research Articles

Cellular Uptake of Silica Particles Influences EGFR Signaling Pathway and is Affected in Response to EGF.

The human epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is involved in several key cellular processes, such as cell proliferation and differentiation, and it has been linked to the development and progression of various cancers (e.g., breast and lung). Researchers have attempted to improve current cancer-targeted therapies by conjugating molecules on the surface of (nano)particles to efficiently target and inhibit EGFR. However, very few in vitro studies have investigated the effect of particles per se on EGFR signaling and dynamics. Furthermore, the impact of concomitant exposure of particles and EGFR ligands, such as epidermal growth factor (EGF) on cellular uptake efficiency has received little attention. The purpose of this research was to determine the effects of silica (SiO2) particles on EGFR expression and intracellular signaling pathways in A549 lung epithelial cells, in the presence or absence of epidermal growth factor (EGF). We showed that A549 cells are able to internalize SiO2 particles with core diameters of 130 nm and 1 µm without affecting cell proliferation or migration. However, both SiO2 particles interfere with the EGFR signaling pathway by raising the endogenous levels of extracellular signal-regulated kinase (ERK) 1/2. Furthermore, both in the presence and absence of SiO2 particles, the addition of EGF increased cell migration. EGF also stimulated cellular uptake of 130 nm SiO2 particles but not 1 µm particles. The increased uptake is primarily associated with EGF-stimulated macropinocytosis. This study shows that SiO2 particle uptake interferes with cellular signaling pathways and can be boosted by concurrent exposure to the bioactive molecule EGF. SiO2 particles, both alone and in combination with the ligand EGF, interfere with EGFR signaling pathway in a size-dependent manner.

Abundance of Dual Specificity Phosphatase (DUSP) 1 and DUSP6 mRNA Is Regulated by Hippo Signaling in Bovine Pre-ovulatory Granulosa Cells.

Ovulatory disorders are a major cause of infertility in humans as well as economically important species. In physiological conditions, the LH surge induces the expression of epidermal growth factor (EGF)-like ligands that activate the EGR receptor (EGFR) and subsequently the mitogen-activated protein kinase (MAPK) pathway. The magnitude and duration of MAPK phosphorylation are regulated by dual-specificity phosphatases (DUSPs). Besides this well-known cascade, other signaling pathways such as the Hippo pathway modulate the ovulatory cascade and are reported to crosstalk with MAPK signaling. Here, we tested the hypothesis that LH and the Hippo pathway regulate DUSP expression in bovine pre-ovulatory granulosa cells. The abundance of DUSP6 mRNA but not DUSP1 was decreased by LH (P < 0.05). Cells were then pre-treated (1h) with two inhibitors of Hippo signaling, verteporfin (1µM) or peptide-17 (25µM), before exposure for 6h to LH or to EGF. Treatment with verteporfin increased DUSP1 mRNA levels (P < 0.05) in the presence or absence of EGF or LH and treatment with peptide-17 increased DUSP6 and not DUSP1 mRNA abundance. These data indicate a differential regulation of DUSP1 and DUSP6 mRNA by the Hippo pathway in pre-ovulatory granulosa cells, which suggests a complex control of MAPK signaling around ovulation.

Abstract 3075: EUS-guided biopsy of pancreatic mass lesions for the development of patient-derived organoids in Puerto Rico

Abstract Pancreatic cancer is an aggressive cancer that is diagnosed early in only 10% of cases and has a 5-year survival rate of less than 9%. The process in determining the optimal treatment for each patient is both long and tedious - and current strategies do not enable therapy optimization at the individual level. The incidence rate for pancreatic cancer in Puerto Rico between 2013 and 2017 was 8.2%, which is lower than the rate reported for non-Hispanic whites and blacks (16% and 12.9%, respectively). The causes for such health disparity are unknown but it is very likely associated with changes at the genomic and tissue microenvironment level at the sub-population level. To identify pathological markers in a more patient-specific manner, patient-derived models need to be integrated into studies of drug screening and efficacy. Patient-derived organoids (PDO) are becoming a potential model for diagnosis and the study of disease progression due to their ability to recreate the pathology of the tumor in the patients. Although several PDO studies have highlighted their potential for therapeutic applications, the ability to successfully culture and propagate these using standard methods has not been shown in samples collected from the Hispanic population. We assessed the feasibility of establishing PDOs from tissue specimens of a pancreatic mass lesion from Puerto Rican patients using standard methods for pancreatic organoids. A total of 9 samples were collected from endoscopic ultrasound fine-needle biopsies to examine the pathological features of the sample and the retention of the parental tumor characteristics in culture. All tissue specimens were cultured in Matrigel domes within 24 hours of extraction and organoid establishment was monitored for 7-8 days. All samples were positive for pancreatic adenocarcinoma but only around 50% of them were successfully cultured in Matrigel. High ATP levels were observed in tumor organoids after one week in culture which is indicative of a high cell viability. Examination of pathological markers indicated that all samples were negative for epidermal growth factor (EGFR), 5/6 were positive for smooth muscle actin (SMA), 4/6 were positive for Vimentin, and 1/6 were positive for CA 19.9. Absence of EGFR was unexpected as this marker is highly expressed and abundant in 40-70% of pancreatic cancers. Samples with high levels of SMA, a fibroblast marker, resulted in a higher organoid yield than samples with no SMA present. Overall, the data suggests that the presence of fibroblasts is supportive of organoid establishment in culture and that standard culture methods need to be optimized to increase organoid yields. Future work will seek to increase the sample size to test alternative propagation methods and examine drug sensitivity in patient-derived pancreas organoids of the Puerto Rican population. Citation Format: Andrea S. Flores Perez, Janet Mendez Vega, Ana M. Reyes Ramos, Carlos Micames, Madeline Torres Lugo, Maribella Domenech. EUS-guided biopsy of pancreatic mass lesions for the development of patient-derived organoids in Puerto Rico [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3075.

Growth factor dependence of the proliferation and survival of cultured lacrimal gland epithelial cells isolated from late-embryonic mice.

Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) regulate the growth and morphogenesis of various exocrine glands with branched morphologies. Their roles in lacrimal gland (LG) development remain unknown, but fibroblast growth factor (FGF) 10 is crucial for early LG organogenesis. To clarify the roles of EGF, HGF, and FGF10 in LG development, LG epithelial cells were isolated from late-embryonic and neonatal mice; cultured; and treated with EGF, HGF, or FGF10 and their respective receptor tyrosine kinase (RTK) inhibitors AG1478, PHA665752, or SU5402. EGF and HGF increased the number of viable cells by enhancing DNA synthesis, FGF10 and SU5402 showed no such effect, and RTK inhibitors exhibited the opposite effect. EGF and HGF receptors were immunostained in cultured late-embryonic LG epithelial cells and terminal LG acini from late embryos and adult mice. HGF was detected in neonatal LG epithelial cell culture supernatants by western blotting. In the absence of EGF and HGF RTK inhibitors, growth factor addition increased the number of viable cells and suppressed cell death. However, when one RTK was inhibited and a growth factor targeting an intact RTK was added, the number of dead cells increased as the number of viable cells increased. No cells survived when both RTKs were inhibited. In explant cultures of LGs from embryos, AG1478 or PHA665752 decreased the number of Ki67-positive proliferating epithelial cells in terminal acini. Thus, EGF and HGF may function in a cooperative autocrine manner, supporting cell proliferation and survival during LG development in late-embryonic and neonatal mice.

Abstract PO-062: EUS-guided biopsy of pancreatic mass lesions for the development of patient-derived organoids in Puerto Rico

Abstract Pancreatic cancer is an aggressive cancer that is diagnosed early in only 10% of cases and has a 5-year survival rate of less than 9%. The process in determining the optimal treatment for each patient is both long and tedious – and current strategies do not enable therapy optimization at the individual level. The incidence rate for pancreatic cancer in Puerto Rico between 2013 and 2017 was 8.2%, which is lower than the rate reported for non-Hispanic whites and blacks (16% and 12.9%, respectively). The causes for such health disparity are unknown but it is very likely associated with changes at the genomic and tissue microenvironment level. To identify patient and sub-population specific markers, patient-derived models need to be integrated into studies of drug screening and efficacy. Patient-derived organoids (PDO) are becoming a potential model for diagnosis and the study of disease progression due to their ability to recreate the pathology of the tumor in patients. Although several PDO studies have highlighted their potential for therapeutic applications, the ability to successfully culture and propagate these using standard methods has not been established in samples collected from the Hispanic population. We assessed the feasibility of establishing PDOs from tissue specimens of a pancreatic mass lesion in Puerto Rican patients using standard methods for pancreatic organoids. A total of 10 samples were collected from endoscopic ultrasound (EUS) fine-needle biopsies to examine the pathological features of the tissue and the retention of the parental tumor characteristics in culture. All tissue specimens were cultured in Matrigel domes within 24 hours of extraction and organoid establishment was monitored for 7-8 days. All samples were positive for pancreatic adenocarcinoma but only 50% of them were successfully cultured in Matrigel. High ATP levels were observed in tumor organoids after one week in culture which is indicative of a high cell viability. Examination of pathological markers indicate that all samples were negative for epidermal growth factor (EGFR), 5/6 were positive for smooth muscle actin (SMA), 4/6 were positive for Vimentin, and 1/6 were positive for CA 19.9. Absence of EGFR was unexpected as this marker is highly expressed and abundant in 40-70% of pancreatic cancers. Samples with high levels of SMA, a fibroblast marker, resulted in a higher organoid yield than samples with no SMA present. Overall, the data suggests that the presence of fibroblasts is supportive of organoid establishment in culture and that standard culture methods need to be optimized to increase organoid yields. Future work will seek to increase the sample size to test alternative propagation methods and examine drug sensitivity in patient-derived pancreas organoids of the Puerto Rican population. Citation Format: Andrea S. Flores Pérez, Janet Mendez Vega, Ana M. Reyes Ramos, Carlos Micames, Madeline Torres-Lugo, Maribella Domenech. EUS-guided biopsy of pancreatic mass lesions for the development of patient-derived organoids in Puerto Rico [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-062.

Epidermal Growth Factor Modulates Palmitic Acid-Induced Inflammatory and Lipid Signaling Pathways in SZ95 Sebocytes.

Epidermal growth factor (EGF) acts as a paracrine and autocrine mediator of cell proliferation and differentiation in various types of epithelial cells, such as sebocytes, which produce the lipid-rich sebum to moisturize the skin. However, sebum lipids via direct contact and by penetrating through the epidermis may have regulatory roles on epidermal and dermal cells as well. As EGF receptor (EGFR) is expressed throughout the proliferating and the lipid-producing layers of sebaceous glands (SGs) in healthy and acne-involved skin, we investigated the effect of EGF on SZ95 sebocytes and how it may alter the changes induced by palmitic acid (PA), a major sebum component with bioactive roles. We found that EGF is not only a potent stimulator of sebocyte proliferation, but also induces the secretion of interleukin (IL)6 and down-regulates the expression of genes involved in steroid and retinoid metabolism. Importantly, when applied in combination with PA, the PA-induced lipid accumulation was decreased and the cells secreted increased IL6 levels. Functional clustering of the differentially regulated genes in SZ95 sebocytes treated with EGF, PA or co-treated with EGF+PA further confirmed that EGF may be a potent inducer of hyperproliferative/inflammatory pathways (IL1 signaling), an effect being more pronounced in the presence of PA. However, while a group of inflammatory genes was up-regulated significantly in EGF+PA co-treated sebocytes, PA treatment in the absence of EGF, regulated genes only related to cell homeostasis. Meta-analysis of the gene expression profiles of whole acne tissue samples and EGF- and EGF+PA -treated SZ95 sebocytes showed that the EGF+PA co-activation of sebocytes may also have implications in disease. Altogether, our results reveal that PA-induced lipid accumulation and inflammation can be modulated by EGF in sebocytes, which also highlights the need for system biological approaches to better understand sebaceous (immuno)biology.

Epidermal growth factor promotes glioblastoma cell death under glucose deprivation via upregulation of xCT (SLC7A11)

The cystine/glutamate antiporter xCT (SLC7A11) is frequently overexpressed in many cancers, including glioblastoma. Cystine taken up by the cells via xCT is reduced to cysteine, which is used to synthesize glutathione for antioxidant cellular defense. However, overexpression of xCT causes cell death under glucose-limited conditions. We found that stimulation of glioblastoma cells with epidermal growth factor (EGF) induces the upregulation of xCT and promotes cell death under glucose deprivation. Treatment with the mTOR inhibitor Torin 1 suppressed the EGF-induced upregulation of xCT and cell death. EGF increased xCT mRNA levels, which was suppressed by Torin 1. The lysosome inhibitor bafilomycin A1 increased xCT protein levels in the absence of EGF or in the presence of EGF and Torin 1. Taken together, our study suggests that EGF promotes glioblastoma cell death under glucose-limited conditions via the upregulation of xCT at transcriptional and protein levels in an mTOR-dependent manner.

Abstract 1498: Fibronectin mediates mutation specific interactions between subclonal populations of mutant PIK3CA and HER2 cells in breast cancer

Abstract Breast cancer is the second most common cancer in the world and the most frequently occurring cancer among women. It is a heterogeneous disease and intratumor heterogeneity is one of the main drivers of disease progression, recurrence, therapeutic resistance and metastasis. Herein we have demonstrated that sub clonal populations containing PIK3CA and HER2 mutants exhibit distinct transformative properties accurately modeling a heterogenous tumor in patients. AAV-mediated gene targeting to was used to heterozygously incorporate a PIK3CA (E545K) or a HER2 (L755S or V777L) mutation, separately, into both the MCF-10A and MCF-7 cell lines. Cocultures were carried out by mixing an equal number of cells containing the PIK3CA mutation and cells containing a HER2 mutation into a single culture. Lentiviral transduction was used to tag the cells with either green or red fluorescent proteins to visualize growth patterns in coculture. Cellular fractions in coculture were quantified using droplet digital PCR. Microarray analysis of cocultures identified fibronectin as a significant mediator of growth and fibronectin knockouts were created using the CRISPR Cas9 gene editing system. MCF-10As carrying the PIK3CA mutation are epidermal growth factor (EGF) independent and those carrying the HER2 mutation are highly EGF dependent. We have identified that interactions between the PIK3CA mutants and the HER2 mutants imparts EGF independence to the HER2 mutants. The effect rendered is so potent that the HER2 mutants dominate the coculture kinetics despite the absence of EGF. This distinct transformation was observed both in vitro and in vivo in the tumorigenic MCF-7 cell line. Interestingly, this phenomenon is mutation specific and was not observed in either the MCF-10A and MCF-7 cells containing wildtype HER2 when co-cultured with the PIK3CA mutants. Microarray analysis demonstrated elevated fibronectin, suggesting it may be a mediator of this observation. Competitive inhibition of fibronectin-integrin interaction using synthetic peptides that mimic the RGD binding domain of fibronectin, reduced the growth of HER2 mutants in coculture. Notably, knocking out fibronectin in the PIK3CA mutants reversed the EGF independence observed in the HER2 mutants in both the MCF-10A and MCF -7 cell lines. Our data suggests that mutation specific interactions between mutant PIK3CA and mutant HER2 cells in a genetically heterozygous environment imparts a selective growth advantage to the HER2 mutants. This effect is mediated by an extracellular matrix protein, fibronectin. These interactions could be used to therapeutically target the complications that arise due to tumor heterogeneity. Citation Format: Swathi Karthikeyan, Ian Waters, Lauren Dennison, David Chu, Joshua Donaldson, Dong Ho Shinn, Sarah Croessmann, Ben Ho Park. Fibronectin mediates mutation specific interactions between subclonal populations of mutant PIK3CA and HER2 cells in breast 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 1498.

Epidermal growth factor and three-dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte-like cells.

Three-dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold-based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte-like cells using embryoid body protocol in the two-dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte-like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or -EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate-based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene-expression patterns, we can conclude that alginate-based 3D coculture system provided a highly efficient protocol for oocyte-like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte-like cell differentiation.

MiR-125a-5p post-transcriptionally suppresses GALNT7 to inhibit proliferation and invasion in cervical cancer cells via the EGFR/PI3K/AKT pathway

BackgroundThe carcinogenesis and progression of cervical cancer is a complex process in which numerous microRNAs are involved. The purpose of this study is to investigate the role of miR-125a-5p in progression of cervical cancer.MethodsRT-qPCR was used to detect the expression of miR-125a-5p and GALNT7 in cervical cancer tissues and cell lines. Then, the miR-125a-5p mimic, miR-125a-5p inhibitor, GALNT7 siRNA, or/and pcDNA-GALNT7 were respectively transfected into HeLa and Caski cervical cancer cells, and Cell Counting kit-8 assay, Transwell assay and flow cytometry analysis were respectively used to observe cell proliferation, invasion and apoptosis. Subsequently, luciferase reporter gene assay was employed in confirming the target relationship between miR-125a-5p and GALNT7. MiR-125a-5p mimic or/and pcDNA-GALNT7 were transfected into the cervical cancer cells at the absence of epidermal growth factor (EGF) or not, and the pcDNA-GALNT7 was transfected into the cervical cancer cells at the absence of inhibitors of multiple kinases or not. Furthermore, the effect of miR-125a-5p on tumor growth was also studied using a xenograft model of nude mice.ResultsMiR-125a-5p was down-regulated in both cervical cancer tissues and cell lines and it inhibited cell proliferation and invasion of cervical cancer cells. MiR-125a-5p directly targeted and post-transcriptionally downregulated GALNT7 that was strongly upregulated in cervical cancer tissues and cell lines. Similar to the effect of miR-125a-5p mimic, silencing GALNT7 inhibited proliferation and invasion of cervical cancer cells. In addition, miR-125a-5p overexpression could counteract both GALNT7- and EGF-induced cell proliferation and invasion. GALNT7 promoted cell proliferation and invasion by activating the EGFR/PI3K/AKT kinase pathway, which could be abated by the inhibitors of the kinases. Moreover, the role of miR-125a-5p inhibited tumor formation in cervical cancer by suppressing the expression of GALNT7 in vivo.ConclusionIn conclusion, miR-125a-5p suppressed cervical cancer progression by post-transcriptionally downregulating GALNT7 and inactivating the EGFR/PI3K/AKT pathway.

GLP-2, EGF, and the Intestinal Epithelial IGF-1 Receptor Interactions in the Regulation of Crypt Cell Proliferation.

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that promotes intestinal growth and proliferation through downstream mediators, including epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1). EGF synergistically enhances the proliferative actions of IGF-1 in intestinal cell lines, and both of these factors are known to be essential for the trophic effects of GLP-2 in vivo. However, whether EGF and IGF-1 interact to mediate the proliferative actions of GLP-2 in vivo remains unknown. Normal and knockout (KO) mice lacking the intestinal epithelial IGF-1 receptor (IE-IGF-1R) were therefore treated chronically with EGF and/or long-acting human hGly2GLP-2, followed by determination of intestinal growth parameters. Intestines from control and IE-IGF-1R KO mice were also used to generate organoids (which lack the GLP-2 receptor) and were treated with EGF and/or IGF-1. Combination treatment with EGF and hGly2GLP-2 increased small intestinal weight and crypt-villus height in C57Bl/6 mice in an additive manner, whereas only hGly2GLP-2 treatment increased crypt cell proliferation. However, although combination treatment also increased small intestinal weight and crypt-villus height in IE-IGF-1R KO mice, the proliferative responses to hGly2GLP-2 alone or with EGF were diminished in these animals. Finally, IGF-1 treatment of organoids undergoing EGF withdrawal was not additive to the effect of EGF replacement on proliferation, but could restore normal proliferation in the absence of EGF. Together, these findings demonstrate that the intestinal proliferative effects of hGly2GLP-2 are augmented by exogenous EGF in a manner that is partially dependent upon IE-IGF-1R signaling.

Extracellular vesicles transmit epithelial growth factor activity in the intestinal stem cell niche.

Extracellular vesicles (EV) are membrane-surrounded vesicles that represent a novel way of intercellular communication by carrying biologically important molecules in a concentrated and protected form. The intestinal epithelium is continuously renewed by a small proliferating intestinal stem cell (ISC) population, residing at the bottom of the intestinal crypts in a specific microenvironment, the stem cell niche. By using 3D mouse and human intestinal organoids, we show that intestinal fibroblast-derived EVs are involved in forming the ISC niche by transmitting Wnt and epidermal growth factor (EGF) activity. With a mouse model that expresses EGFP in the Lgr5+ ISCs, we prove that loss in ISC number in the absence of EGF is prevented by fibroblast-derived EVs. Furthermore, we demonstrate that intestinal fibroblast-derived EVs carry EGF family members, such as amphiregulin. Mechanistically, blocking EV-bound amphiregulin inhibited the EV-induced survival of organoids. In contrast, EVs have no role in transporting R-Spondin, a critical niche factor amplifying Wnt signaling. Collectively, we prove the important role of fibroblast-derived EVs as a novel transmission mechanism of factors in the normal ISC niche.

Deciphering Hydrodynamic and Drug-Resistant Behaviors of Metastatic EMT Breast Cancer Cells Moving in a Constricted Microcapillary.

Epithelial to mesenchymal transition (EMT) induces cell migration, invasion, and drug resistance, and consequently, contributes to cancer metastasis and disease aggressiveness. This study attempted to address crucial biological parameters to correlate EMT and drug-treated cancer cells traversing through microcapillaries, reminiscent of metastatic conditions. MDA-MB-468 breast cancer cells induced to undergo EMT by treatment with 20 ng/mL of epidermal growth factor (EGF) were initially passed through several blockages and then through a constricted microchannel, mimicking the flow of invasive metastatic cells through constricted blood microcapillaries. EMT cells acquired enhanced migratory properties and retained 50% viability, even after migration through wells 10–15 μm in size and a constricted passage of 7 μm and 150 μm in length at a constant flow rate of 50 μL/h. The hydrodynamic properties revealed cellular deformation with a deformation index, average transit velocity, and entry time of 2.45, 12.3 mm/s, and 31,000 μs, respectively for a cell of average diameter 19 μm passing through one of the 7 μm constricted sections. Interestingly, cells collected at the channel outlet regained epithelial character, undergoing reverse transition (mesenchymal to epithelial transition, MET) in the absence of EGF. Remarkably, real-time polymerase chain reaction (PCR) analysis confirmed increases of 2- and 2.7-fold in the vimentin and fibronectin expression in EMT cells, respectively; however, their expression reduced to basal level in the MET cells. A scratch assay revealed the pronounced migratory nature of EMT cells compared with MET cells. Furthermore, the number of colonies formed from EMT cells and paclitaxel-treated EMT cells after passing through a constriction were found to be 95 ± 10 and 79 ± 4, respectively, confirming that the EMT cells were more drug resistant with a concomitant two-fold higher expression of the multi-drug resistance (MDR1) gene. Our results highlight the hydrodynamic and drug-evading properties of cells that have undergone an EMT, when passed through a constricted microcapillary that mimics their journey in blood circulation.

Morphology and dynamics of tumor cell colonies propagating in epidermal growth factor supplemented media

The epidermal growth factor (EGF) plays a key role in physiological and pathological processes. This work reports on the influence of EGF concentration (cEGF) on the modulation of individual cell phenotype and cell colony kinetics with the aim of perturbing the colony front roughness fluctuations. For this purpose, HeLa cell colonies that remain confluent along the whole expansion process with initial quasi-radial geometry and different initial cell populations, as well as colonies with initial quasi-linear geometry and large cell population, are employed. Cell size and morphology as well as its adhesive characteristics depend on cEGF. Quasi-radial colonies (QRC) expansion kinetics in EGF-containing medium exhibits a complex behavior. Namely, at the first stages of growth, the average QRC radius evolution can be described by a t1/2 diffusion term coupled with exponential growth kinetics up to a critical time, and afterwards a growth regime approaching constant velocity. The extension of each regime depends on cEGF and colony history. In the presence of EGF, the initial expansion of quasi-linear colonies (QLCs) also exhibits morphological changes at both the cell and the colony levels. In these cases, the cell density at the colony border region becomes smaller than in the absence of EGF and consequently, the extension of the effective rim where cell duplication and motility contribute to the colony expansion increases. QLC front displacement velocity increases with cEGF up to a maximum value in the 2–10 ng ml−1 range. Individual cell velocity is increased by EGF, and an enhancement in both the persistence and the ballistic characteristics of cell trajectories can be distinguished. For an intermediate cEGF, collective cell displacements contribute to the roughening of the colony contours. This global dynamics becomes compatible with the standard Kardar–Parisi–Zhang growth model, although a faster colony roughness saturation in EGF-containing medium than in the control medium is observed.

Epidermal Growth Factor Represses Constitutive Androstane Receptor Expression in Primary Human Hepatocytes and Favors Regulation by Pregnane X Receptor.

Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF–EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O2 ) versus hypoxic (3% O2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc.

Label-Free Raman Imaging to Monitor Breast Tumor Signatures.

Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for further label-free optical tools to diagnose the disease.

Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression.

We derived adult neural stem/progenitor cells (NSPCs) from the sub-ventricular zone of male and female mice to examine direct responses to principal sex hormones. In the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) NSPCs of both sexes expressed nestin and sox2, and could be maintained as neurospheres without addition of any sex hormones. The reverse was not observed; neither testosterone (T), 17β-estradiol (E2) nor progesterone (P4) was able to support neurosphere growth in the absence of EGF and FGF2. Ten nanomolar T, E2 or P4 induced nestin(+) cell proliferation within 20 min and enhanced neurosphere growth over 7 days irrespective of sex, which was abolished by Erk inhibition with 20 μM U0126. Maintaining neurospheres with each sex hormone did not affect subsequent neuronal differentiation. However, 10 nM T, E2 or P4 added during differentiation increased βIII tubulin(+) neuron production with E2 being more potent compared to T and P4 in both sexes. Androgen receptor (AR) inhibition with 20 μM flutamide but not aromatase inhibition with 10 μM letrozole reduced basal and T-induced neurosphere growth in females, while only concurrent inhibition of AR and aromatase produced the same effect in males. This sex-specific effect was supported by higher aromatase expression in male neurospheres compared to females measured by Western blot and green fluorescent protein (GFP) reporter. Ten micromolar menadione induced oxidative stress, impaired neurosphere growth and up-regulated aromatase expression in both sexes. However, under oxidative stress letrozole significantly exacerbated impaired neurosphere growth in males only. While both E2 and T could prevent oxidative stress-induced growth reduction in both sexes, the effects of T were dependent on innate aromatase activity. We show for the first time that intrinsic androgen and estrogen signaling may impact the capacity of NSPCs to produce neural progenitors under pathological conditions of oxidative stress.

An Aminopropyl Carbazole Derivative Induces Neurogenesis by Increasing Final Cell Division in Neural Stem Cells.

P7C3 and its derivatives, 1-(3,6-dibromo-9H-carbazol-9-yl)-3-(p-tolylamino)propan-2-ol (1) and N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-(3-methoxyphenyl)-4-methylbenzenesulfonamide (2), were previously reported to increase neurogenesis in rat neural stem cells (NSCs). Although P7C3 is known to increase neurogenesis by protecting newborn neurons, it is not known whether its derivatives also have protective effects to increase neurogenesis. In the current study, we examined how 1 induces neurogenesis. The treatment of 1 in NSCs increased numbers of cells in the absence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), while not affecting those in the presence of growth factors. Compound 1 did not induce astrocytogenesis during NSC differentiation. 5-Bromo-2′-deoxyuridine (BrdU) pulsing experiments showed that 1 significantly enhanced BrdU-positive neurons. Taken together, our data suggest that 1 promotes neurogenesis by the induction of final cell division during NSC differentiation.