(F-219) An Evaluation of Photointiators and Viability Assays on Panc-1 Pancreatic Cancer Cells

Introduction:: Pancreatic tumor models are important and necessary because of the low overall survival rate of pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), and limited treatment options for patients. PDAC tumors experience stiffening of the pancreatic extracellular matrix (ECM) as the disease progresses due to increased collagen deposits and collagen crosslinking. To design in vitro pancreatic tumor models and represent increasing tissue stiffness around pancreatic tumors, we chose methacrylated collagen (ColMA) with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator, which stiffens when exposed to 405nm light (photo-crosslinking). However, it is important to know if LAP is cytotoxic to pancreatic cancer cell lines (i.e., Panc-1) we use in our in vitro tumor models, because current research literature has mixed results on whether LAP is cytotoxic to mammalian cells. Outcomes also appear dependent on individual cell populations, and there is limited information for pancreatic cells specifically. Prior work from our research group investigated metabolic activity of Panc-1 within ColMA+LAP  using alamarBlueTM to evaluate cytotoxicity. While results showed LAP was not toxic, artificially high fluorescence signals in absence of cells suggested that free radicals produced from LAP exposure to light seemed to interfere with alamarBlue™ reduction. Therefore, the current project aimed to: 1) use a free radical scavenger with alamarBlue™ to assess metabolic activity and potential free radical interference, 2) investigate alternative viability assays to assess cytotoxicity, and 3) compare LAP to another photoinitator, ruthenium and sodium persulfate (RUT). 

Materials and Methods:: We evaluated the cytotoxic potential of LAP in two independent studies. First, we compared alamarBlue™ and CyQUANT MTT™ assays to evaluate the reliability of both assays and their ability to give the more accurate assessment of metabolic activity in Panc-1 cells exposed to LAP and photo-crosslinking. Experimental groups included samples with standard culture media (DMEM, 10% FBS, 1x Pen-Strep), samples with and without 2% LAP in media, and samples with and without 2% ascorbic acid in media. Samples were also prepared to be evaluated with and without 405 nm light exposure (5 minutes) at each time point. The experiment ran for 48 hours in a humidified incubator (37°C, 5% CO2) with measurements taken at 4hrs, 24hrs, and 48hrs after light exposure. A NIVO plate reader was used to evaluate changes in metabolic activity (fluorescence) for each assay. The second study evaluated Panc-1 cells with LIVE/DEAD Viability assay which uses Calcein AM (live) and Ethidium homodimer-1 (dead) fluorescent stain. This study ran for 6 days with all measurements taken on Day 6. Experimental samples were like the first study, except none of the groups contained ascorbic acid and light was applied only on Day 0 and Day 3. The second study also adds Ruthenium Sodium Persulfate (RUT) as a photoinitiator, to also compare its effects on the Panc-1 cells. For this study, the LIVE/DEAD assay result (% live) was calculated from readings from using a NIVO plate reader in fluorescence to compare to the initial alamarBlue™ study. 

Results, Conclusions, and Discussions:: For the first study, two figures were generated to represent the data collected. Figure 1., shows the comparison of the alamarBlue™ assay with and without ascorbic acid based on results from prior work. Figure 1 shows how the addition of the ascorbic acid decreased the fluorescence and therefore decreased the viability of the cells. The results also showed us that ascorbic acid concentration was too high and may have been made incorrectly. Figure 2 is the 24hr time point of the second portion of the first study that compares the alamarBlue™ and CyQUANT MTT™ assays. The data collected from the MTT™ assay had consistently higher fluorescence than the alamarBlue™ at 24hrs and appeared to be less influenced by the presence of ascorbic acid. We can infer that the MTT™ assay was more effective in analyzing the metabolic activity in cell culture, albeit the differences in signal were small. The results also give us more confidence that free radicals produced in photo-crosslinking cause interference for the alamarBlue™ reading, as both assays evaluate the metabolic activity of cell culture and should not have consistently different results. This result also helped us decide to stop using the ascorbic acid in tandem with the alamarBlue™ assay and moving forward. Table 1 shows the LIVE/DEAD spectrometry result of Panc-1 cells after a 6-day culture period. The LAP groups and the positive control had a low live percentage of cells between 15-25%. While the RUT had a live percentage that varied around 0-2% which was much lower than its counterparts. This experiment led us to no longer use the RUT as our photointiator and decided that microscopy would be a better option for visual confirmation of viability. As an outcome from this study, we decided not to use alamarBlue™ and have decided to focus on LIVE/DEAD viability assay with image-based assessment. Other work from our group (not shown) has confirmed that imaging is a more accurate representation of the cells we are assessing in 2D and 3D, despite being a higher throughput method.  

Acknowledgements (Optional): : Acknowledge Athenia Jones, Jessica Netto, Hannah Borges, and Madison Sanborn for their assistance. We would like to acknowledge the Pancreatic Cancer Action Network Career Development Award (#831461) for funding this work.

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