The SIVB’s In Vitro Animal Cell Sciences Section (IVACS) held the virtual Student and Post-Doctoral Oral Presentation Competition on Monday, June 8, 2020. The top 3 abstracts were chosen for the competition based on their scientific merit and the quality of their write-up. Although the competition was virtual, due to COVID-19 pandemic, each presentation was very engaging and at a high scientific level. Our three final contestants delivered professional pre-recorded presentations and continued lively on-line discussions with the audience and judges thought the virtual conference. The panel of IVACS judges that evaluated the presentations included: Dr. John W. Harbell (JHarbell Consulting, LLC), Dr. Barbara Doonan (New York Medical College), Dr. Michael J. Fay (Midwestern University), Dr. Brad L. Upham (Michigan State University), Dr. Mae Ciancio (Midwestern University), Dr. Debora Esposito (North Carolina State University), Dr. Kristina Martinez-Guryn (Midwestern University), as well as the session moderators Dr. Addy Alt-Holland (Tufts University) and Dr. Kolla Kristjansdottir (Midwestern University). Evan M. Hill, from University of Michigan School of Public Health, won the 1st place award for his presentation “Sulforaphane Inhibits Colon Adenoma Organoid Formation and Induces Differentiation in a Dose-dependent Manner.” Sepideh Mohammadhosseinpour, from Arkansas State University, won the 2nd place award for her presentation “Prenylated Stilbenoids as Potential Therapeutic Agents for Triple Negative Breast Cancer.” Connor P. Dyer, from Midwestern University, won the 3rd place award for his presentation “Decellularized Plant Biomaterials Are Adaptable 3D Scaffolds to Study Cellular Growth, Invasion, and Ex Vivo Tissue Regeneration.” Certificates and monetary awards were sent to the three contestants via mail. Congratulations to these three contestants for their fantastic virtual presentations!

Submitted by Addy Alt-Holland and Kolla Kristjansdottir

First Place

Sulforaphane Inhibits Colon Adenoma Organoid Formation and Induces Differentiation in a Dose-dependent Manner

Colorectal cancer ranks as the 2nd leading cause of cancer mortality in the United States. Preventive strategies for individuals with familial colon cancer syndromes remain limited. Sulforaphane (SFN), an isothiocyanate in cruciferous vegetables and potent activator of the Nrf2 transcription factor, has chemopreventive effects in vitro and in vivo, including inducing growth arrest and/or apoptosis. We have previously demonstrated that SFN inhibits breast cancer stem cells, the hypothesized cells of origin for carcinogenesis. Whether SFN can act as a cancer preventive agent through targeting colon stem cells is unknown. To investigate this, we treated APC-mutant patient-derived human colon adenoma organoids with 4 doses of SFN (1.5, 3.1, 6.25, and 12.5 µM) or DMSO-control for 6 days and quantified the number and size of organoids formed. Additionally, we performed RNAseq on each treatment group and calculated benchmark doses for pathway alterations using BMDExpress2. Organoid formation decreased with SFN treatment in a dose-dependent manner and the highest dose decreased organoid formation by 80% compared to control. The number of differentially expressed genes (DEG) also increased in a dose-dependent manner. Organoids treated with the lowest dose had 254 DEG (83 down-regulated and 171 up-regulated) compared to control while the highest dose had 7099 DEGs (3478 down and 3621 up). Increasing doses of SFN increased transcription of many Nrf2 target genes, including NQO1, HMOX1, and ALDH3A1. Increasing doses of SFN also increased the expression of colon differentiation genes, including KRT20, AQP3, and ALPP, and decreased the expression of colon stem cell genes LGR5, OLFM4, and EPHB2. BMDExpress2 analysis estimated the median benchmark dose for the gene ontology “stem cell proliferation” pathway at less than 1µM.  Ongoing work is characterizing the stem cell specific effects of SFN in a mouse model of colorectal carcinogenesis. These results provide evidence that SFN induces a differentiation-associated phenotype in colon adenoma cells at physiologically relevant doses.

Evan Michael Hill, University of Michigan Medical School, Ann Arbor, MI. In Vitro Cellular and Developmental Biology, 56:S20-21, 2020

First Place

Sulforaphane Inhibits Colon Adenoma Organoid Formation and Induces Differentiation in a Dose-dependent Manner

Colorectal cancer ranks as the 2nd leading cause of cancer mortality in the United States. Preventive strategies for individuals with familial colon cancer syndromes remain limited. Sulforaphane (SFN), an isothiocyanate in cruciferous vegetables and potent activator of the Nrf2 transcription factor, has chemopreventive effects in vitro and in vivo, including inducing growth arrest and/or apoptosis. We have previously demonstrated that SFN inhibits breast cancer stem cells, the hypothesized cells of origin for carcinogenesis. Whether SFN can act as a cancer preventive agent through targeting colon stem cells is unknown. To investigate this, we treated APC-mutant patient-derived human colon adenoma organoids with 4 doses of SFN (1.5, 3.1, 6.25, and 12.5 µM) or DMSO-control for 6 days and quantified the number and size of organoids formed. Additionally, we performed RNAseq on each treatment group and calculated benchmark doses for pathway alterations using BMDExpress2. Organoid formation decreased with SFN treatment in a dose-dependent manner and the highest dose decreased organoid formation by 80% compared to control. The number of differentially expressed genes (DEG) also increased in a dose-dependent manner. Organoids treated with the lowest dose had 254 DEG (83 down-regulated and 171 up-regulated) compared to control while the highest dose had 7099 DEGs (3478 down and 3621 up). Increasing doses of SFN increased transcription of many Nrf2 target genes, including NQO1, HMOX1, and ALDH3A1. Increasing doses of SFN also increased the expression of colon differentiation genes, including KRT20, AQP3, and ALPP, and decreased the expression of colon stem cell genes LGR5, OLFM4, and EPHB2. BMDExpress2 analysis estimated the median benchmark dose for the gene ontology “stem cell proliferation” pathway at less than 1µM.  Ongoing work is characterizing the stem cell specific effects of SFN in a mouse model of colorectal carcinogenesis. These results provide evidence that SFN induces a differentiation-associated phenotype in colon adenoma cells at physiologically relevant doses.

 

Evan Michael Hill, University of Michigan Medical School, Ann Arbor, MI. In Vitro Cellular and Developmental Biology, 56:S20-21, 2020

Second Place

Prenylated Stilbenoids as Potential Therapeutic Agents for Triple Negative Breast Cancer

Breast cancer is the most prevalent type of cancer in women worldwide. Triple negative breast cancer (TNBC) is known to be one of the deadliest types since it does not respond to hormonal treatments. Therefore, there is an ongoing search for new treatments to increase survival rates for this disease. The goal of this study is to assess the efficacy of prenylated stilbenoids from peanut as natural products for the prevention and treatment of TNBC. To induce the production of these compounds, peanut hairy root cultures were co-treated with elicitors and then the prenylated stilbenoids were purified via semi-preparative high-performance liquid chromatography from extracts of the culture medium. The cytotoxicity of the prenylated stilbenoids arachidin-1 and arachidin-3 and the non-prenylated stilbenoid resveratrol was studied in TNBC cell lines MDA-MB-231 and MDA-MB-436. Epithelial breast cell line MCF-10A was used as a control. Cytotoxicity and apoptosis were measured by the MTS cell proliferation and Apo-ONE Homogeneous Caspase-3/7 assays, respectively. To further investigate the apoptosis and cell cycle stage, cells were studied using flow cytometry after treatment with each stilbenoid. These studies showed that the prenylated stilbenoids exhibited higher cytotoxicity to the cancer cells than non-prenylated stilbenoid resveratrol and arachidin-3 induces higher level of apoptosis when compared with resveratrol. Furthermore, the increased cytotoxicity correlated with increased levels of the apoptosis markers caspase-3 and caspase-7. This highlights the significance to continue research with prenylated stilbenoids in TNBC. Current studies focus on elucidating the signaling pathways affected by these compounds in TNBC cells in order to advance our understanding of the anticancer mechanisms of these natural products.

Sepideh Mohammadhosseinpour, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S21, 2020

Second Place

Prenylated Stilbenoids as Potential Therapeutic Agents for Triple Negative Breast Cancer

Breast cancer is the most prevalent type of cancer in women worldwide. Triple negative breast cancer (TNBC) is known to be one of the deadliest types since it does not respond to hormonal treatments. Therefore, there is an ongoing search for new treatments to increase survival rates for this disease. The goal of this study is to assess the efficacy of prenylated stilbenoids from peanut as natural products for the prevention and treatment of TNBC. To induce the production of these compounds, peanut hairy root cultures were co-treated with elicitors and then the prenylated stilbenoids were purified via semi-preparative high-performance liquid chromatography from extracts of the culture medium. The cytotoxicity of the prenylated stilbenoids arachidin-1 and arachidin-3 and the non-prenylated stilbenoid resveratrol was studied in TNBC cell lines MDA-MB-231 and MDA-MB-436. Epithelial breast cell line MCF-10A was used as a control. Cytotoxicity and apoptosis were measured by the MTS cell proliferation and Apo-ONE Homogeneous Caspase-3/7 assays, respectively. To further investigate the apoptosis and cell cycle stage, cells were studied using flow cytometry after treatment with each stilbenoid. These studies showed that the prenylated stilbenoids exhibited higher cytotoxicity to the cancer cells than non-prenylated stilbenoid resveratrol and arachidin-3 induces higher level of apoptosis when compared with resveratrol. Furthermore, the increased cytotoxicity correlated with increased levels of the apoptosis markers caspase-3 and caspase-7. This highlights the significance to continue research with prenylated stilbenoids in TNBC. Current studies focus on elucidating the signaling pathways affected by these compounds in TNBC cells in order to advance our understanding of the anticancer mechanisms of these natural products.

Sepideh Mohammadhosseinpour, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S21, 2020

 

Third Place

Decellularized Plant Biomaterials Are Adaptable 3D Scaffolds to Study Cellular Growth, Invasion, and Ex Vivo Tissue Regeneration

Traditional cell culturing techniques often employ flat, plastic dishes as substrates, which are obviously very different from the natural 3D extracellular matrices (ECMs) found in the body. While some biomaterials, such as decellularized mammalian donor tissue and synthetic hydrogels, are useful 3D tissue engineering scaffolds, their effectiveness is still limited by cost, fabrication methods, availability, and adaptability. For some scenarios, decellularized plant materials provide a means to readily available and cost effective scaffolds that can also be manipulated into macroscale structures (mm-cm) while maintaining the plant’s native nano to microscale topography that can support mammalian cell adherence and growth. Previous studies have shown that plant material can be decellularized and coated with mammalian ECM proteins to support mammalian cell growth. Our current research has shown that dilute ECM protein coatings can be directly applied to decellularized plant scaffolds without crosslinking while still improving mammalian cell growth. We found that decellularized apple wafers coated with both collagen I and fibronectin provide 2.5X greater mouse mesenchymal stem (C3H10 T1/2) cell growth compared to no protein or single protein coating controls over 3 days. On decellularized potato wafers, collagen I alone supported 1.4x greater C3H10 growth than wafers coated with fibronectin. On decellularized sweet potato wafers, all combinations of fibronectin and/or collagen I coatings lead to similar C3H10 cell growth. Additionally, we have used fluorescence microscopy to show that mouse dorsal root ganglion tissue explants can regenerate on decellularized apple wafers 1-6 days post plating, with axons growing over millimeters in length, comparable to their growth on a traditional plastic dish. Together, this data highlights the cost effectiveness and simple, adaptable methods that can be used to decellularize and process plant materials into scaffolds that reproducibly support mammalian cells and ex vivo tissue cultures.

Connor Patrick Dyer, Midwestern University, Downers Grove, IL. In Vitro Cellular and Developmental Biology, 56:S20, 2020

Third Place

Decellularized Plant Biomaterials Are Adaptable 3D Scaffolds to Study Cellular Growth, Invasion, and Ex Vivo Tissue Regeneration

Traditional cell culturing techniques often employ flat, plastic dishes as substrates, which are obviously very different from the natural 3D extracellular matrices (ECMs) found in the body. While some biomaterials, such as decellularized mammalian donor tissue and synthetic hydrogels, are useful 3D tissue engineering scaffolds, their effectiveness is still limited by cost, fabrication methods, availability, and adaptability. For some scenarios, decellularized plant materials provide a means to readily available and cost effective scaffolds that can also be manipulated into macroscale structures (mm-cm) while maintaining the plant’s native nano to microscale topography that can support mammalian cell adherence and growth. Previous studies have shown that plant material can be decellularized and coated with mammalian ECM proteins to support mammalian cell growth. Our current research has shown that dilute ECM protein coatings can be directly applied to decellularized plant scaffolds without crosslinking while still improving mammalian cell growth. We found that decellularized apple wafers coated with both collagen I and fibronectin provide 2.5X greater mouse mesenchymal stem (C3H10 T1/2) cell growth compared to no protein or single protein coating controls over 3 days. On decellularized potato wafers, collagen I alone supported 1.4x greater C3H10 growth than wafers coated with fibronectin. On decellularized sweet potato wafers, all combinations of fibronectin and/or collagen I coatings lead to similar C3H10 cell growth. Additionally, we have used fluorescence microscopy to show that mouse dorsal root ganglion tissue explants can regenerate on decellularized apple wafers 1-6 days post plating, with axons growing over millimeters in length, comparable to their growth on a traditional plastic dish. Together, this data highlights the cost effectiveness and simple, adaptable methods that can be used to decellularize and process plant materials into scaffolds that reproducibly support mammalian cells and ex vivo tissue cultures.

Connor Patrick Dyer, Midwestern University, Downers Grove, IL. In Vitro Cellular and Developmental Biology, 56:S20, 2020