The synergistic effect of Canady Helios cold atmospheric plasma and a FOLFIRINOX regimen for the treatment of cholangiocarcinoma in vitro

Abstract

Cholangiocarcinoma (CCA) is a rare biliary tract cancer with a low five-year survival rate and high recurrence rate after surgical resection. Currently treatment approaches include systemic chemotherapeutics such as FOLFIRINOX, a chemotherapy regimen is a possible treatment for severe CCA cases. A limitation of this chemotherapy regimen is its toxicity to patients and adverse events. There exists a need for therapies to alleviate the toxicity of a FOLFIRINOX regimen while enhancing or not altering its anticancer properties. Cold atmospheric plasma (CAP) is a technology with a promising future as a selective cancer treatment. It is critical to know the potential interactions between CAP and adjuvant chemotherapeutics. In this study the aim is to characterize the efficacy of FOLFIRINOX and CAP in combination to understand potential synergetic effect on CCA cells. FOLFIRINOX treatment alone at the highest dose tested (53.8 µM fluorouracil, 13.7 µM Leucovorin, 5.1 µM Irinotecan, and 3.7 µM Oxaliplatin) reduced CCA cell viability to below 20% while CAP treatment alone for 7 min reduced viability to 3% (p < 0.05). An analysis of cell viability, proliferation, and cell cycle demonstrated that CAP in combination with FOLFIRINOX is more effective than either treatment alone at a lower FOLFIRINOX dose of 6.7 µM fluorouracil, 1.7 µM leucovorin, 0.6 µM irinotecan, and 0.5 µM oxaliplatin and a shorter CAP treatment of 1, 3, or 5 min. In conclusion, CAP has the potential to reduce the toxicity burden of FOLFIRINOX and warrants further investigation as an adjuvant therapy.

Figure 1

Reduction of KKU-055 cell viability after 48-h exposure to FOLFIRINOX, compared to DMSO treated cells controls (mean ± SEM). Each drug dosage level is labeled by the corresponding concentration of 5-fluorouracil (cohort = 4, 2 /cohort, n = 8, t test). *p < 0.05.

Figure 2

Reduction of KKU-055 cell viability 48 h after CAP treatment for 1–7 min at 120p which corresponds to 28.7 W compared to untreated controls (cohort = 4, 2 /cohort, n = 8 t test). *p < 0.05.

Figure 3

The effect of adjunctive FOLFIRINOX treatment in combination with CAP on cholangiocarcinoma cell viability. Four drug dosages, labeled by their corresponding concentration of 5-fluorouracil (5-FU) from Table 1, were combined with three CAP doses of either 1, 3 or 5 min. FOLFIRINOX treated cells were subject to 24 h pretreatment incubation before CAP, and MTT assays were performed 48 h after CAP treatment. T tests were used to determine synergetic treatment combinations and are indicated as *p < 0.05 or **p < 0.005.

Figure 4

KKU-055 cells were imaged 6, 24, and 48 h after CAP or CAP and FOLFIRINOX treatments with an untreated negative control. (A) The total number of cells in five representative images per treatment condition is plotted (cohort = 3, 2/cohort, n = 6, t test). *p < 0.05. (B) Representative images of Ki67 and DAPI staining of cells after CAP treatment at 120p for 3 min. (C) Representative images of cells subject to 24 h pretreatment with FOLFIRINOX (6.7 µM 5-FU, 1.7 µM leucovorin, 0.6 µM irinotecan, and 0.5 µM oxaliplatin) before CAP at 120p for 3 min.

Figure 5

(A–H) Representative phase contrast images of fluorescently labeled KKU-055 cells 0, 24, and 48 h after no treatment, FOLFIRINOX (6.7 µM 5-FU, 1.7 µM leucovorin, 0.6 µM irinotecan, and 0.5 µM oxaliplatin), CAP at 120p (28.7 W) for 1, 3 or 5 min, and both in combination.

Figure 6

The FOLFIRINOX dosage of 6.7 µM 5-FU, 1.7 µM leucovorin, 0.6 µM irinotecan, and 0.5 µM oxaliplatin was combined with CAP at 1, 3, and 5 min to characterize the cell cycle response. (A-H) The number of cells in either G1 phase, G1-S transition, S/G2/M phase, or M-G1 transition per well in each treatment group from 0 to 24 or 0 to 6 h.