From left to right: D.E. Daniels, S.A. Rizvi, and M.A. Brandt

The SIVB’s In Vitro Animal Cell Sciences Section (IVACS) held the Student and Post-Doctoral Oral Presentation Competition on Monday, June 12, 2023. The top 3 abstracts were chosen for the competition based on their scientific merit and the quality of their write-up. Each presentation was very engaging and was followed by questions from the audience and the competition judges. Our three final contestants delivered professional presentations at a high scientific level. The panel of IVACS judges that evaluated the presentations included: John W. Harbell (JHarbell Consulting, LLC), Barbara Doonan (New York Medical College), Michael J. Fay (Midwestern University), Brad L. Upham (Michigan State University), Michael K. Dame (University of Michigan), Mae Ciancio (Midwestern University), Joshua Gasiorowski (Midwestern University), Anissa Belfetmi-stone (Harvard Medical School), Zoe Xiaofang Zhu (Tufts University), as well as the session moderators Addy Alt-Holland (Tufts University) and Kolla Kristjansdottir (Midwestern University). Shuaa A. Rizvi, from Midwestern University, won the 1st place award for her presentation “Identification of Stratifin as a Potential Protein Biomarker Indicative of Oral Squamous Cell Carcinoma Progression.” Dominique E. Daniels, from Wilfrid Laurier University, won the 2nd place award for her presentation “Encapsulation of Long dsRNA in Extracellular Vesicles, SARS-CoV2 Virus-like Particles, and Liposomes for the Stimulation of Antiviral RNA Interference Against Human Coronaviruses.” Mari Austad Brandt, from Norwegian University of Life Sciences (NMBU), won the 3rd place award for her presentation “Developing Cell Lines from Young and Adult Coho Salmon (Oncorhynchus kisutch).” Certificates and monetary awards were presented to the three contestants at the IVACS Business Meeting. Congratulations to these three contestants for their informative and engaging presentations!

Submitted by Addy Alt-Holland and Kolla Kristjansdottir

First Place

Identification of Stratifin as a Potential Protein Biomarker Indicative of Oral Squamous Cell Carcinoma Progression

Eleanor Jane Brant
Shuaa Rizvi

It has been shown that the expression of a squamous epithelium keratinocyte protein, Stratifin, is downregulated in cancerous tissues. This study aims to examine Stratifin expression at the cellular level in four cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. Thus, examining the utility of Stratifin as a diagnostic and prognostic indicator for the disease.This study evaluated the differential expression of Stratifin across four cell lines representing successive progression steps of OSCC. Western blotting was completed to quantify the expression of Stratifin in primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells that represent normal oral keratinocytes, oral premalignant lesions, locally invasive OSCC cells, and metastatic OSCC cells, respectively. Stratifin expression was found to be downregulated alongside the progression from normal oral keratinocytes to premalignant, and from premalignant to metastatic cancer cells. Quantitative analysis revealed that SFN was downregulated significantly in the metastatic cells compared to the normal and dysplastic cells (p< 0.05). There was also a significant downregulation in the expression of SFN in the dysplastic cells compared to the normal (p< 0.05). However, there was no significant difference in SFN expression between the dysplastic and the locally invasive cancer cells. Stratifin proved to be differentially expressed at the cellular level in cell lines. Particularly, we documented a significant decrease of Stratifin expression with the progression from normal to premalignant to metastatic cells. Ultimately, the results showed that the expression of SFN was downregulated as OSCC progressed in a step-wise manner. Thus, Stratifin can potentially serve as a diagnostic and prognostic biomarker for patients suffering from oral cancer.

Shuaa Rizvi, Masters of Biomedical Science, College of Graduate Studies, Midwestern University, Downers Grove, IL 60515. Abstract presentation A-1002.

Second Place

Encapsulation of Long dsRNA in Extracellular Vesicles, SARS-CoV2 Virus-like Particles, and Liposomes for the Stimulation of Antiviral RNA Interference Against Human Coronaviruses

Eleanor Jane Brant
Dominique Daniels

Viruses produce long dsRNA (LdsRNA) during replication, and its presence in the cell triggers the innate antiviral immune responses. In the RNA interference response to LdsRNA (dsRNAi), LdsRNA is used as a template to bind complementary viral mRNA and halt viral protein replication. While in vitro delivery of 700 bp LdsRNA can stimulate the dsRNAi response in human cells against Human coronaviruses, we hypothesize that in vivo antiviral treatments will require carriers for tissue targeting and protection from LdsRNA degradation. We evaluated extracellular vesicles (EVs) and virus-like particles (VLPs) as carriers for 700 bp HCoV-229E N gene LdsRNA in treatments against HCoV-229E. LdsRNA-EVs were produced by treating U937 (human monocytes) with LdsRNA and extraction of EVs with ExoQuickÒ. To encapsulate LdsRNA, SARS-CoV2 VLPs were disassembled in 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM EGTA, 20mM DTT, then reassembled with 5mM CaCl2 after LdsRNA addition. A green fluorescent protein (GFP) plasmid was encapsulated in VLPs and delivered to HepG2 (human hepatoblastoma) cells. LdsRNA carriers were treated with RNase III and GFP-VLPs were treated with DNase I. LdsRNA Immunoblot confirmed LdsRNA packaging in EVs and protection against RNase III degradation. HEL-299 were treated with LdsRNA-EVs and dsRNA delivery was confirmed by immunocytochemistry. Agarose gel electrophoresis (AGE) of LdsRNA-VLPs showed successful LdsRNA encapsulation and protection from RNAse III degradation. LdsRNA Immunoblot confirmed LdsRNA packaging in EVs and protection against RNase III degradation. AGE of GFP-VLPs before and after DNase I treatment confirmed plasmid encapsulation and fluorescence microscopy of GFP-VLP treated HepG2 cells showed GFP expression suggesting an intact delivery mechanism. EVs and VLPs can encapsulate and protect LdsRNA from degradation and are suitable LdsRNA carriers for antiviral experiments. This is the first study to explore EVs or VLPs as LdsRNA carriers for antiviral dsRNAi in vertebrates, and if effective this strategy could be applied to other vertebrate viruses.

Dominique Daniels, Department of Biology, Faculty of Science, Wilfrid Laurier University, Waterloo, ON, CANADA. Abstract presentation A-1001.

Third Place

Developing Cell Lines from Young and Adult Coho Salmon (Oncorhynchus kisutch)

Eleanor Jane Brant
Mari Austad Brandt

Coho (Oncorhynchus kisutch) or Silver salmon is one of 5 native salmon species found in North America’s west coast. Coho are economically important as fishery and aquaculture species and are the preferred salmon species for recreational fishermen. In the Fraser Valley Canada, angling for Coho is restricted to hatchery reared salmon which are easily recognized by the missing adipose fin that are clipped before they are released to the wild. A call for donations of fresh Coho salmon scraps (heads, tails, entrails) from local anglers led to several donated specimens from which cell cultures were made. We were successful in establishing primary cultures from each specimen received and some of these have now been passaged leading to the establishment of adult Coho salmon cell lines. Additionally, culled fingerling Coho were secured from a local hatchery and primary cultures were also obtained from all processed specimens. Cellosaurus, a database cataloguing reported cell lines in the scientific literature, indicates the existence of only 4 Coho salmon cell lines, all of which were derived from embryos (CSE-119 and CoE-45, 115 & 345). We report here the ease of establishing primary cultures from young and adult Coho salmon tissues including skin, fins, gills, muscle, brain, liver, heart, intestine, spleen, gonads, fat, tongue, teeth, and olfactory rosettes. Although many were lost to contamination, some have been passaged and continue growing vigorously. Our aim was to develop an epidermal cell line from Coho skin as these appear to have interesting features in vivo, such as ability to hypertrophy in response to sea lice infestation. It is well known that among salmonids, Coho is most resistant to sea lice, thus our interest in developing an epidermal cell line from Coho that could be useful to the aquaculture industry. Coho cells from muscle and mesenchymal tissues are also of interest to the emerging cellular agriculture industry. Thus, characterization of epidermal and mesenchymal cells is underway, and we describe here the initiation of some of these novel adult Coho salmon cell lines.

Mari Austad Brandt, Faculty of Biosciences, Norwegian University of Life Sciences (NMBU), NORWAY.. Abstract presentation A-1000.

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