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Amy P N Skubitz - One of the best experts on this subject based on the ideXlab platform.
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abstract a06 synthetic peptides derived from the cell adhesion molecule nectin 4 inhibit the formation of ovarian cancer 3d Spheroids
Clinical Cancer Research, 2020Co-Authors: Kristin L M Boylan, Rory D Manion, Heena Shah, Amy P N SkubitzAbstract:A unique aspect of ovarian cancer progression is the collection of ascites fluid in the abdominal cavity, which contains free-floating tumor cells in the form of single cells and also multicellular aggregates or Spheroids. Spheroids are more resistant to standard chemotherapy due in part to their slow proliferation rate and also to the protection afforded by the tight cell aggregation. Agents that prevent Spheroid formation have the potential to improve chemotherapeutic response. Our previous work on the function of the cell surface adhesion molecule Nectin-4 in ovarian cancer demonstrates that it plays a key role in the formation of ovarian cancer Spheroids. We also showed that synthetic peptides derived from the amino acid sequences of Nectin-4 and its binding partner Nectin-1 could inhibit cell adhesion in an in vitro assay. The purpose of this study was to further examine the role of Nectin-4 in the formation of ovarian cancer Spheroids using live-cell digital microscopy to monitor Spheroid formation over time. Synthetic peptides derived from the amino acid sequences of Nectin-4 and Nectin-1 were tested for their ability to alter the Spheroid formation of two ovarian cancer cell lines. Several peptides disrupted ovarian cancer Spheroid formation, warranting further investigation. For the peptide with the strongest inhibitory effect on Spheroid formation, we tested scrambled versions of the peptide, which did not affect the formation of Spheroids. This peptide also inhibited Spheroid formation in a concentration-dependent manner and was not cytotoxic. These results suggest that Nectin-derived peptides could augment the effect of chemotherapy, as they would maintain the cancer cells as single cells or small aggregates that are more sensitive to chemotherapy. Citation Format: Kristin L.M. Boylan, Rory D. Manion, Heena Shah, Amy P.N. Skubitz. Synthetic peptides derived from the cell adhesion molecule Nectin-4 inhibit the formation of ovarian cancer 3D Spheroids [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A06.
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inhibition of ovarian cancer cell Spheroid formation by synthetic peptides derived from nectin 4
International Journal of Molecular Sciences, 2020Co-Authors: Kristin L M Boylan, Rory D Manion, Heena Shah, Keith M Skubitz, Amy P N SkubitzAbstract:The formation of 3D multicellular Spheroids in the ascites fluid of ovarian cancer patients is an understudied component of the disease progression. Spheroids are less sensitive to chemotherapy, in part due to the protection afforded by their structure, but also due to their slower proliferation rate. Previous studies suggest that the cell adhesion molecule Nectin-4 plays a key role in the formation of ovarian cancer Spheroids. In this study, we further examined the role of Nectin-4 at early time points in Spheroid formation using real-time digital photography. Human NIH:OVCAR5 ovarian cancer cells formed aggregates within 8 h, which further contracted into compact Spheroids over 24 h. In contrast, Nectin-4 knockdown cells did not form tightly compacted Spheroids. Synthetic peptides derived from Nectin-4 were tested for their ability to alter Spheroid formation in two ovarian cancer cell lines. Nectin-4 peptide 10 (N4-P10) had an immediate effect on disrupting ovarian cancer Spheroid formation, which continued for over 24 h, while a scrambled version of the peptide had no effect. N4-P10 inhibited Spheroid formation in a concentration-dependent manner and was not cytotoxic; suggesting that N4-P10 treatment could maintain the cancer cells as single cells which may be more sensitive to chemotherapy.
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ovarian carcinoma Spheroids disaggregate on type i collagen and invade live human mesothelial cell monolayers
Clinical & Experimental Metastasis, 2005Co-Authors: Kathryn M Burleson, Linda K Hansen, Amy P N SkubitzAbstract:Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or Spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites Spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via β1 integrin subunits. Here, we assessed the ability of Spheroids that were generated from the human ovarian carcinoma cell line NIH: OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, Spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the β1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, Spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the Spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the β1 integrin subunit drastically reduced Spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked Spheroid invasion into the mesothelial cell monolayers. ɛ-amino-N-caproic acid, a serine protease inhibitor, partially inhibited Spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, Spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.
Sandeep Jain - One of the best experts on this subject based on the ideXlab platform.
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Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes
PLOS ONE, 2014Co-Authors: Yong-soo Byun, Sapna Tibrewal, Yair Ivanir, Cecile M. Sano, Joy Sarkar, Sandeep JainAbstract:Purpose Corneal stromal cells transform to precursor cells in Spheroid culture. We determined whether keratocytes derived from Spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes. Methods Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes. Results Stromal cells formed Spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in Spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in Spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the Spheroids. Conclusion Stemness transcription factor genes are upregulated in Spheroids. Keratocytes derived from Spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.
Yong-soo Byun - One of the best experts on this subject based on the ideXlab platform.
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Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes
2016Co-Authors: Yong-soo Byun, Sapna Tibrewal, Yair Ivanir, Cecile M. Sano, Joy Sarkar, Chia-yang Liu, Eunjae Kim, Lisette Yco, Eep JainAbstract:Purpose: Corneal stromal cells transform to precursor cells in Spheroid culture. We determined whether keratocytes derived from Spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes. Methods: Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes. Results: Stromal cells formed Spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in Spheroids as compared to adherent cells whereas alpha-smooth muscle actin (a-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and a-SMA in Spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the Spheroids. Conclusion: Stemness transcription factor genes are upregulated in Spheroids. Keratocytes derived from Spheroid
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Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes
PLOS ONE, 2014Co-Authors: Yong-soo Byun, Sapna Tibrewal, Yair Ivanir, Cecile M. Sano, Joy Sarkar, Sandeep JainAbstract:Purpose Corneal stromal cells transform to precursor cells in Spheroid culture. We determined whether keratocytes derived from Spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes. Methods Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes. Results Stromal cells formed Spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in Spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in Spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the Spheroids. Conclusion Stemness transcription factor genes are upregulated in Spheroids. Keratocytes derived from Spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.
Boyang Zhang - One of the best experts on this subject based on the ideXlab platform.
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correction deep lumen assay human lung epithelial Spheroid classification from brightfield images using deep learning
Lab on a Chip, 2020Co-Authors: Lyan Abdul, Shravanthi Rajasekar, Dawn S Y Lin, Alexander Sotra, Yuhang Feng, Amy Liu, Sibi Venkatasubramania Raja, Boyang ZhangAbstract:Three-dimensional (3D) tissue models such as epithelial Spheroids or organoids have become popular for pre-clinical drug studies. In contrast to 2D monolayer culture, the characterization of 3D tissue models from non-invasive brightfield images is a significant challenge. To address this issue, here we report a deep-learning uncovered measurement of epithelial networks (Deep-LUMEN) assay. Deep-LUMEN is an object detection algorithm that has been fine-tuned to automatically uncover subtle differences in epithelial Spheroid morphology from brightfield images. This algorithm can track changes in the luminal structure of tissue Spheroids and distinguish between polarized and non-polarized lung epithelial Spheroids. The Deep-LUMEN assay was validated by screening for changes in Spheroid epithelial architecture in response to different extracellular matrices and drug treatments. Specifically, we found the dose-dependent toxicity of cyclosporin can be underestimated if the effect of the drug on tissue morphology is not considered. Hence, Deep-LUMEN could be used to assess drug effects and capture morphological changes in 3D Spheroid models in a non-invasive manner.
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deep lumen assay human lung epithelial Spheroid classification from brightfield images using deep learning
bioRxiv, 2020Co-Authors: Lyan Abdul, Shravanthi Rajasekar, Dawn S Y Lin, Alexander Sotra, Yuhang Feng, Amy Liu, Sibi Venkatasubramania Raja, Boyang ZhangAbstract:Abstract Three-dimensional (3D) tissue models such as epithelial Spheroids or organoids have become popular for pre-clinical drug studies. However, different from 2D monolayer culture, the characterization of 3D tissue models from non-invasive brightfield images is a significant challenge. To address this issue, here we report a Deep-Learning Uncovered Measurement of Epithelial Networks (Deep-LUMEN) assay. Deep-LUMEN is an object detection algorithm that has been fine-tuned to automatically uncover subtle differences in epithelial Spheroid morphology from brightfield images. This algorithm can track changes in the luminal structure of tissue Spheroids and distinguish between polarized and non-polarized lung epithelial Spheroids. The Deep-LUMEN assay was validated by screening for changes in Spheroid epithelial architecture in response to different extracellular matrices and drug treatments. Specifically, we found the dose-dependent toxicity of Cyclosporin can be underestimated if the effect of the drug on tissue morphology is not considered. Hence, Deep-LUMEN could be used to assess drug effects and capture morphological changes in 3D Spheroid models in a non-invasive manner. Significance of the work Deep learning has been applied for the first time to autonomously detect subtle morphological changes in 3D multi-cellular Spheroids, such as Spheroid polarity, from brightfield images in a label-free manner. The technique has been validated by detecting changes in Spheroid morphology in response to changes in extracellular matrices and drug treatments.
Kristin L M Boylan - One of the best experts on this subject based on the ideXlab platform.
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abstract a06 synthetic peptides derived from the cell adhesion molecule nectin 4 inhibit the formation of ovarian cancer 3d Spheroids
Clinical Cancer Research, 2020Co-Authors: Kristin L M Boylan, Rory D Manion, Heena Shah, Amy P N SkubitzAbstract:A unique aspect of ovarian cancer progression is the collection of ascites fluid in the abdominal cavity, which contains free-floating tumor cells in the form of single cells and also multicellular aggregates or Spheroids. Spheroids are more resistant to standard chemotherapy due in part to their slow proliferation rate and also to the protection afforded by the tight cell aggregation. Agents that prevent Spheroid formation have the potential to improve chemotherapeutic response. Our previous work on the function of the cell surface adhesion molecule Nectin-4 in ovarian cancer demonstrates that it plays a key role in the formation of ovarian cancer Spheroids. We also showed that synthetic peptides derived from the amino acid sequences of Nectin-4 and its binding partner Nectin-1 could inhibit cell adhesion in an in vitro assay. The purpose of this study was to further examine the role of Nectin-4 in the formation of ovarian cancer Spheroids using live-cell digital microscopy to monitor Spheroid formation over time. Synthetic peptides derived from the amino acid sequences of Nectin-4 and Nectin-1 were tested for their ability to alter the Spheroid formation of two ovarian cancer cell lines. Several peptides disrupted ovarian cancer Spheroid formation, warranting further investigation. For the peptide with the strongest inhibitory effect on Spheroid formation, we tested scrambled versions of the peptide, which did not affect the formation of Spheroids. This peptide also inhibited Spheroid formation in a concentration-dependent manner and was not cytotoxic. These results suggest that Nectin-derived peptides could augment the effect of chemotherapy, as they would maintain the cancer cells as single cells or small aggregates that are more sensitive to chemotherapy. Citation Format: Kristin L.M. Boylan, Rory D. Manion, Heena Shah, Amy P.N. Skubitz. Synthetic peptides derived from the cell adhesion molecule Nectin-4 inhibit the formation of ovarian cancer 3D Spheroids [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A06.
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inhibition of ovarian cancer cell Spheroid formation by synthetic peptides derived from nectin 4
International Journal of Molecular Sciences, 2020Co-Authors: Kristin L M Boylan, Rory D Manion, Heena Shah, Keith M Skubitz, Amy P N SkubitzAbstract:The formation of 3D multicellular Spheroids in the ascites fluid of ovarian cancer patients is an understudied component of the disease progression. Spheroids are less sensitive to chemotherapy, in part due to the protection afforded by their structure, but also due to their slower proliferation rate. Previous studies suggest that the cell adhesion molecule Nectin-4 plays a key role in the formation of ovarian cancer Spheroids. In this study, we further examined the role of Nectin-4 at early time points in Spheroid formation using real-time digital photography. Human NIH:OVCAR5 ovarian cancer cells formed aggregates within 8 h, which further contracted into compact Spheroids over 24 h. In contrast, Nectin-4 knockdown cells did not form tightly compacted Spheroids. Synthetic peptides derived from Nectin-4 were tested for their ability to alter Spheroid formation in two ovarian cancer cell lines. Nectin-4 peptide 10 (N4-P10) had an immediate effect on disrupting ovarian cancer Spheroid formation, which continued for over 24 h, while a scrambled version of the peptide had no effect. N4-P10 inhibited Spheroid formation in a concentration-dependent manner and was not cytotoxic; suggesting that N4-P10 treatment could maintain the cancer cells as single cells which may be more sensitive to chemotherapy.