FOLFIRINOX regimen reduced cholangiocarcinoma cell viability
To determine the possible synergistic effects of FOLFIRINOX on KKU-055 cells, an optimal dosage of the four drugs in combination must be able to reduce cell viability significantly. A serial dilution of 6 doses of FOLFIRINOX was done to establish a baseline toxicity measurement for each dose (Table 1 and Figure 1). Control cells treated with DMSO remained viable 99% (± 6) suggesting that DMSO had no significant effects on cell growth, and all reduction was due to FOLFIRINOX (cohort = 4, 2 /cohort, n = 8, t test p > 0.05). Cell viability decreased significantly at doses equal to or higher than the 3.4 nM 5-FU level (cohort = 4, 2/cohort, n = 8, t test p < 0.05). Exposure to the lowest FOLFIRINOX dose decreased viability to 94% (± 3) and was not statistically significant compared to the control DMSO which reached 103% (± 3). When KKU-055 cells were treated with the highest dose of FOLFIRINOX (53.8nM Fluorouracil, 13.1nM Leucovorin, 5.1nM Irinotecan, and 3.7nM Oxaliplatin), cell viability was reduced to 19% (± 1.9).
Assessment of the combined treatment of CAP and FOLFIRINOX
A dose dependence experiment was performed on KKU-055 cells to establish CAP efficacy. MTT assays were conducted 48 hours post CAP treatment. Cell viability was significantly reduced by CAP for all durations, and the highest treatment of 7 minutes reduced viability to 3% (p < 0.005, Figure 2).
KKU-055 cells were exposed to 24 hours of FOLFIRINOX pretreatment at 6.7 – 53.8 nM [5-FU] (Table 1) and CAP at 120p for 1, 3, or 5 minutes. Viability reduction was measured 48 hours after treatment (Figure 3). Cells without either treatment were negative controls. Complete cell death was observed with a combination of FOLFIRINOX (53.8nM 5-FU dose) and CAP for 5minutes where viability was reduced to 1%.
A two-way ANOVA test followed by post hoc Fisher exact tests (with Bonferroni correction) was conducted on this combination treatment experiment. Sources of variation were a change in either CAP dose or FOLFIRINOX dose. Then the variance between the two was tested to determine if one treatment had an effect of the other. There were three hypotheses for this test; H1: The observed viability between drug dosage groups is equal, H2: the observed viability between CAP dosage groups is equal, and H3: there is no interaction between the two treatments. For all three hypotheses p < 0.05, so we can reject each one. Student paired t-tests and two-way ANOVA test followed by post hoc Fisher exact tests (with Bonferroni correction) were then conducted to compare each combination treatment with every other experiment group (Table 2).
Dosage combinations were considered synergetic when combination treatment reduced viability significantly more than the corresponding CAP or FOLFIRINOX dosage alone. In cases when the FOLFIRINOX dose was 13.5nM [5-FU] or higher the drug alone was strong enough to reduce KKU-055 viability to below 30%, and this made drug treatment significantly more effective than 1 or 3 minutes of CAP (cohort = 4, 2/cohort, n = 8 t test p < 0.05, Figure 3, Table 2). With these high doses of drug enhanced efficacy then could not be determined. Statistically significant for all the drug combinations are indicated in Table 2.
The FOLFIRINOX dose (6.73nM fluorouracil, 1.71nM leucovorin, 0.63nM irinotecan, and 0.47nM oxaliplatin) in combination with 5 minutes of CAP achieved a 91% reduction in cell viability (Figure 3). This FOLFIRINOX dose in combination with 5 minutes of CAP was more effective in reducing cell viability than the drug alone (p < 0.001). Also, when this dose was combined with 1 minute of CAP the combination treatment was statistically more effective than 1 minute of CAP alone (p < 1 × 10-5). The efficacy of this FOLFIRINOX dose in combination treatments was observed and the drug alone did not statistically reduce cell viability more than CAP alone so this dosage was selected for following confocal microscopy and cell cycle analysis.
Decrease in Cell Proliferation
Cell proliferation was examined by Ki-67/DAPI co-staining at 6, 24, or 48 hours post CAP, FOLFIRINOX, or combination treatment. The 6.7nM 5-FU dose of drug (Table 1) was combined with 1, 3, and 5 minutes of CAP. In five images, nuclei that were in focus were outlined and each mean fluorescence intensity (MFI) of Ki-67 channel was recorded. The mean of Ki-67 MFI was calculated for each treatment group including for No Treatment and Isotype control. A Ki-67+ cell threshold was determined as a cell with a MFI greater than the lowest mean of MFI of all groups other than Isotype control. There was a significant (cohort = 3, 2/cohort, n = 6, t test p < 0.05) decrease in cell count with FOLFIRINOX and 3 minutes of CAP treatment combined at 6 hours compared to no treatment controls (Figure 4A). In cells treated with combination CAP 3min and FOLFIRINOX, less cells were observed (Figure 4B-C). All cells were then graded as Ki-67+ or Ki-67- on this scale. Representative images at the 3-min CAP timepoint and total cell counts of all timepoints are shown in Figure 4. Ki-67 can be seen co-localized within the outlined nucleoli in cells regardless of treatment group (Figure 4B-C).
Induction of cell cycle arrest with combination treatment
Experiments were designed to measure cell confluence and cell cycle distribution after combining the 6.7nM 5-FU dose of FOLFIRINOX (Table 1) and CAP at 3 and 5 minutes. Cells were placed in the IncuCyte Live Cell imaging system immediately after CAP where confluence was monitored. Representative images of 0 hour, 24 hours, and 48 hours timepoints are shown to demonstrate cell confluence within treatment wells (Figure 5A-H). In Figure 5, morphological differences can be seen between experiment conditions. No treatment and drug only treated cells are confluent at 48 hours with most cells visibly fluorescent. In combination treatment wells, cells are not confluent and large clusters of cellular debris are visible 48 hours after treatment.
The number of cells in different phases of the cell cycle was quantified through fluorescence measurements. The quantifications during the first 48 hours after treatment are shown at the CAP 1, 3 and 5 minute doses (Figure 6A-H). In the no treatment and FOLFIRINOX only treated groups, most cells are in labeled grey in the mitotic phase, and this line increases over time (Figure 6A-B). Also, the number of cells in S/G2/M increases in these wells. Conversely, cells treated with FOLFIRINOX and CAP were not proliferating. At a CAP dosage of 1 minute, cells were moving through the cell cycle, as shown in the grey line (Figure 6C). With a combination of FOLFIRINOX and CAP 1 minute this progression is reduced, and the grey line plateaued 24 hours after treatment (Figure 6D). At CAP dosages of 3 and 5 minutes, the grey line of cells in M-G1 phase trended down after treatment. CAP and FOLFIRINOX combination treatment hindered the cell cycle, and the number of cells in the mitotic phase was reduced compared to FOLFIRINOX or a low dose (1 minute) of CAP alone. Figure 6 needs to be compared with Figure 5 since the presence of dead cells were counted in Figure 6. For example, the yellow line in Figure 6G-H is not true G1-S phase signal but residual fluorescence of oxidized protein by CAP treatment as shown in representative phase images Figure 5E-H cells started to die at the 24 hours timepoint.