1. Introduction
Lung cancer is the second most commonly occurring cancer, with the highest mortality rate worldwide, accounting for 2.2 million cases and 1.8 million deaths in 2020. Incidence and mortality are projected to continue to rise by approximately 60%, to an estimated 3.6 million and 3 million, respectively, in 2040 [
1]. Most lung cancer cases are NSCLC, accounting for 80–85% of all lung cancer cases [
2]. The development of targeted and immunotherapies has revolutionized the treatment of NSCLC. However, side effects, resistance and efficacity in a small therapeutically sensitive group of patients create inequalities in access to such agents [
3,
4,
5]. Therefore, this underscores the need for safer and more efficacious agents.
Metabolic reprogramming is one of the cancer hallmarks that has been a promising target for the development of effective therapeutic approaches [
6]. Compared to the normal cells that rely mainly on mitochondrial oxidative phosphorylation (OXPHOS) under aerobic conditions, cancer cells deviate from this normal metabolic phenotype by relying mainly on cytosolic glycolysis and lactic fermentation, even in the presence of oxygen, to meet the needs of high proliferation [
7]. This phenomenon is known as the Warburg effect, which has been exploited as a therapeutic target to inhibit tumor growth [
8]. PDK is among the essential enzymes controlling glycolysis and OXPHOS [
9]. It shut down the mitochondrial OXPHOS by phosphorylating and inhibiting pyruvate dehydrogenase (PDH), a key enzyme catalyzing the oxidative conversion of pyruvate into acetyl coenzyme A in mitochondria [
10].
DCA is a small-molecular-weight drug that was used in lactic acidosis, congenital mitochondrial defects and diabetes [
11]. Interestingly, DCA showed an ability to shift the tumor metabolism from cytosolic aerobic glycolysis to mitochondrial OXPHOS by inhibiting PDK and enhancing the activity of PDH [
12]. Hence, DCA has been reported to have anti-cancer effects by increasing the efflux of cytochrome c and other apoptotic-inducing factors and the upregulation of ROS levels with consequent cancer cell death [
11,
13,
14,
15]. However, in clinical investigations, the safety profile of DCA was a concern. Even so, Garon et al. (2014), who conducted a clinical trial with DCA on lung cancer patients, concluded that: “in the absence of a larger controlled trial, firm conclusions regarding the association between the patient’s adverse events and DCA are unclear”. They recommended that DCA should be considered with platinum-based chemotherapy in hypoxic tumors rather than as a single agent in advanced non-small-cell lung cancer [
16]. DCA is believed to be a potent molecule that warrants further investigation of its anti-cancer potential on NSCLC.
This study aimed to investigate the impact of DCA on NSCLC cellular viability, colony growth, and cellular migration and invasion in vitro, in addition to tumor growth, metastasis and toxicity in vivo. In addition, DCA’s direct impact on angiogenesis was assessed in vitro. Furthermore, we investigated the effect of DCA in combination therapies with chemotherapy and the first generation of EGFR tyrosine kinase inhibitors (EGFR-TKi).
3. Discussion
Despite the recent advances in the screening, diagnosis and management of lung cancer, in addition to the remarkable progress in understanding its molecular biology, lung cancer is the second most commonly diagnosed cancer with the highest mortality rate worldwide in 2020 [
1]. Therefore, various efforts are being devoted to the development of effective agents and approaches with good safety margins to target lung cancer in an attempt to provide a cure or improve the patient’s overall survival. This study aimed to investigate the impact of the metabolic drug DCA on lung cancer growth, migration, invasion and angiogenesis in vitro and tumor growth and metastasis in vivo as well as the effect of targeting metabolism by DCA on the cytotoxic effect of approved chemotherapy and targeted therapy as a step to achieve a better efficacy and better safety profile.
The present study showed that DCA (3.125–100 mM) produced a concentration- and time-dependent reduction in the cellular viability and growth of pre-formed colonies of A549 and LNM35 cell lines. The IC50 of DCA at 48 h was approximately 25 mM in both cell lines. Our results are in agreement with other reports in which DCA (10–90 mM) inhibited the cellular viability of colorectal cancer (CRC) cell lines, namely, SW620, LS174t, LoVo and HT-29 in a concentration-dependent manner at 48 h with an IC50 range of 30–50 mM according to the cell line type [
18]. Similarly, DCA (20 mM) significantly decreased the viability of CRC cells, namely, SW480, LoVo and HT-29 at 48 h, with a greater effect on the poorly differentiated SW480 cells and metastatic LoVo cells compared to the well-differentiated HT-29 cells [
19]. On the other hand, a higher IC50 was reported in cervical cancer cells, Hela and SiHa cells [
20], while DCA (20 mM) failed to inhibit the cellular viability of the breast cancer MCF-7 cell line [
21].
Our in vitro data were validated by testing the effect of DCA on tumor progression in vivo using a chick embryo CAM and athymic mice models. Firstly, we demonstrated that a significant growth reduction was achieved in the A549 and LNM35 xenografted on chick embryo CAM by using DCA doses of 50 mM and 100 mM, respectively. During the writing of this manuscript, a study was published that investigated the effect of sodium DCA on U87 MG and PBT24 glioblastoma cell lines xenografted on a chick embryo CAM [
22]. The authors reported a variation in U87 MG and PBT24 tumor growth in response to the different concentrations of sodium DCA. It was reported that 10 mM of sodium DCA was effective in reducing the PBT24 tumor growth but not U87 tumor growth, reflecting some differences in the biology of the two cell lines [
22]. Secondly, we demonstrated that treatment with DCA at doses of 200 mg/kg everyday (5 days/week) caused a significant 40% reduction in xenografted A549 tumor growth, while a higher dose of DCA (500 mg/kg) was required to produce a significant decrease in xenografted LNM35 tumor growth. In this context, it has been previously reported that DCA (100 mg/kg) increased the tumor doubling time of A549 and H1975 NSCLC from approximately 3 to 6.5 days [
15], but failed to produce a significant inhibitory effect in MDA-MB-231 tumor-bearing mice [
23]. On the other hand, a significant growth delay was also observed in HT-29 xenografts treated with oral DCA (200 mg/kg) daily for four days [
24].
Investigating the toxicity of the potential anti-cancer drugs is as important as investigating their efficacy since severe toxicity can prevent their use in the clinic. DCA showed no cytotoxicity to the chick embryos and athymic mice. The percentage of alive embryos was the same in the DCA-treated and control groups. Additionally, DCA did not affect mice behavior, weight, complete blood count, liver and kidney function parameters compared to the control group. These findings are consistent with previous preclinical and clinical reports that showed no evidence of severe hematologic, hepatic, renal, or cardiac toxicity with DCA treatment [
13,
14]. Few patients treated with DCA complained of common gastrointestinal effects. Additionally, the most common limitation to DCA administration is reversible peripheral neuropathy, which can be minimized by dose reduction or the complementary administration of antioxidants [
11]. Incorporating DCA into drug delivery systems (DDS) such as nanoparticles is a promising approach to retain the anti-cancer activity of DCA with minimal side effects [
25,
26,
27].
The anti-cancer effect of DCA was reported to be partly due to the induction of apoptosis, as was observed in colorectal cancer cells [
19] and NSCLC cells [
15] or due to the inhibition of angiogenesis. Angiogenesis inhibitors such as the anti-VEGF antibody Bevacizumab and VEGF receptor blocker Ramucirumab have been approved clinically for the management of lung cancer [
28]. Despite their approved efficacy, their modest overall therapeutical effects with the associated side effects highlight a clear need for a more effective approach targeting angiogenesis [
28]. Our study demonstrated that DCA (25 mM) is a promising anti-angiogenic agent by being able to significantly inhibit endothelial cell tube formation and sprouting in vitro. In addition, lower concentrations of DCA (6.25 and 12.5 mM) did not affect the HUVECs tube formation. These findings are consistent with a report by Schoonjans and coworkers, who demonstrated that 5 mM and 10 mM DCA did not affect HUVECs’ tube formation in vitro [
29]. In agreement with our data, DCA caused a reduction in the tumor microvessel density in treated rats, in which HIF1α suppression was also reported within the tumor cells [
30]. On the other hand, Zhao and coworkers recently reported that DCA stimulates angiogenesis in a vascular dementia rat model by improving endothelial precursor cell function [
31].
Approximately 30–40% of NSCLC patients presented with metastatic disease at the time of diagnosis. Distant metastases negatively affect the treatment options, response and survival [
32] and are the main cause of lung cancer deaths [
33]. Metastasis is a multistep process involving the detachment of cancer cells, migration, invasion and colonization at distant sites. Therefore, therapeutic agents and regimens reducing such a hallmark in cancer are of high importance in cancer therapy. Despite the demonstrated anti-angiogenic activity of DCA, this study showed no impact of DCA on the metastasis of LNM35 cells xenografted in athymic mice treated orally with an effective dose. In this study, LNM35 cells xenografted by subcutaneous inoculation in athymic mice caused a 90% incidence of axillary lymph node metastases, and DCA failed to reduce the incidence and the growth of these lymph node metastases. The LNM35 cell line was established in 2000 as the first human lung cancer cell line having lymphogenous metastatic properties with 100% incidence following a subcutaneous injection into the lateral flank of nude mice [
34]. Additionally, DCA did not show any inhibitory effects on the migratory and invasive properties of LNM35 and A549 cells in vitro. Similarly, it was reported that DCA monotherapy was not effective in reducing lung metastases from metastatic breast cancer cells xenografted in nude mice [
23].
Combination therapy has been a fundamental approach in cancer management. Combining different anti-cancer drugs allows the targeting of different essential signaling pathways to enhance therapeutic benefits, avoid the acquired resistance and decrease the severity of side effects [
35]. Chemotherapy plays an integral part in the management of NSCLC patients. A regimen of platinum (cisplatin or carboplatin) plus paclitaxel, gemcitabine, docetaxel, vinorelbine, irinotecan, or pemetrexed is usually used [
36]. The nonselective characteristics of chemotherapeutic agents results in a modest increase in survival with significant toxicity to the patient [
37]. This underscores the need for better strategies to improve patients’ outcomes with minimal side effects. In the present study, DCA failed to enhance the anti-cancer effect of camptothecin and gemcitabine in both NSCLC cell lines. Additionally, DCA failed to significantly enhance the anti-cancer effects of cisplatin in the A549 cell line in vitro, but it enhanced the cytotoxic effect of cisplatin in the LNM35 cell line, reflecting the role of the genetic background of cancer cells in determining the cell death pathway induced by the drugs. Kim et al. reported that A549 cells have a lower rate of aerobic glycolysis compared to H460 cells due to differential expression in some metabolic enzymes [
38]. Aerobic glycolysis in cancer has been linked to chemoresistance, and the inhibition of related pathways has been suggested as a mechanism for overcoming such resistance. For instance, the overexpression of PDK4 in high-grade bladder cancer makes the co-administration of DCA with cisplatin cause a dramatic reduction in tumor growth compared to DCA or cisplatin alone [
39]. Similarly, the administration of DCA with paclitaxel was reported as a successful approach to overcome the paclitaxel-resistant NSCLC cells due to PDK2 overexpression [
40]. Furthermore, Galgamuwa et al. stated that pre-treatment with DCA significantly attenuated the nephrotoxicity induced by cisplatin in mice, retaining the cisplatin anti-cancer effects [
41].
The discovery of targeted therapy has helped physicians to tailor the treatment options for NSCLC patients. Many targeted drugs have been developed and become part of the first-line treatment of NSCLC, such as gefitinib and erlotinib, which are considered the first generation of EGFR-TKi [
42]. Gefitinib and erlotinib were approved more than 10 years ago for the treatment of chemotherapy-naive patients with advanced EGFR-mutant NSCLC as the first-line treatment. They are also used as a second-line therapy after chemotherapy failure [
43]. Some reports showed that erlotinib has good efficacy in patients with EGFR-wild-type NSCLC [
44]. A maintenance dose can benefit these patients after platinum-based chemotherapy, considered the backbone therapy in wild-type EGFR NSCLC [
45]. Despite the remarkable benefits, many patients acquired therapeutic resistance after 10–14 months of treatment due to a secondary mutation in the EGFR gene [
46].
In this study, we were seeking to investigate the ability of DCA to sensitize the EGFR wild-type NSCLC cell lines when combined with gefitinib or erlotinib in vitro. DCA significantly enhanced the inhibitory effect of gefitinib and erlotinib on the cellular viability of A549 and LNM35. This study also showed additive effects on LNM35 colony growth upon combining DCA with gefitinib or erlotinib for seven days of treatment. Furthermore, this combination produced synergistic effects on A549 colony growth after fourteen days of treatment. In addition, all these combination protocols lead to a substantial decrease in the cellular density of individual colonies of both A549 and LNM35. In this context, it has been reported that DCA with gefitinib or erlotinib synergistically inhibits the viability and colony formation capacity of EGFR-mutant cells (NCI-H1975 and NCI-H1650) due to synergistic effect in promoting apoptosis. In EGFR wild-type cells (A549 and NCI-H460), they showed, in comparison to the individual treatments, that combination caused an elevated fraction affected (Fa) value in cellular viability without reaching the level of synergism in EGFR wild-type cells (A549 and NCI-H460), and this combination did not significantly repress the colony formation of these cell lines [
47]. The differences in the experimental conditions between the aforementioned report and our study could explain such variable results. In their clonogenic assay, the investigators treated the individual cells for three successive days, followed by incubation with a drug-free medium for 15 days to form colonies; however, in our experiments, the cells were firstly incubated for ten days to form colonies followed by seven and fourteen days of treatment.
In summary, this study demonstrated that DCA is a promising anti-cancer agent for NSCLC by inhibiting the cellular viability and colony growth of NSCLC cells in vitro and tumor growth in the chick embryo CAM and nude mice, in which the safety of this agent was also assessed. DCA inhibits the ability of endothelial cells to form capillary-like structures and sprouting in vitro, suggesting the inhibition of angiogenesis as a potential mechanism behind the anti-cancer effect. This study also revealed the potential value of DCA when combined with gefitinib or erlotinib in vitro. The findings of this study pave the way for validating the impact of the combination of DCA with gefitinib or erlotinib on tumor growth in vivo, in addition to investigating the impact of DCA when combined with the second- and third-generation EGFR-TKi.
4. Materials and Methods
4.1. Cell Culture and Reagents
NSCLC cells, A549 and LNM35, were maintained in RPMI-1640 medium (Gibco, Paisley, UK) in a humidified incubator at 37 °C and 5% CO2. The medium was supplemented with 1% of penicillin–streptomycin solution (Hyclone, Cramlington, UK) and 10% of fetal bovine serum (Hyclone, Cramlington, UK). Human umbilical vein endothelial cells (HUVECs) were maintained in an EndoGROTM-VEGF complete media kit (Merck Millipore, Massachusetts, USA) in a humidified incubator at 37 °C and 5% CO2 in flasks coated with 0.2% gelatin. The culture medium of all cells was changed every 3 days, and cells were passed once a week when the culture reached 95% confluency for cancer cells and 80% for HUVECs.
Sodium DCA, cisplatin, camptothecin, gemcitabine HCl, erlotinib HCl and gefitinib were purchased from Sigma-Aldrich (Saint Louis, MO, USA). DCA was freshly dissolved in HyPure water (Hyclone, Cramlington, UK) before starting any experiment to make a stock solution of 1M, which was then diluted to the required concentrations for treatment.
4.2. Cellular Viability
A549 and LNM35 cells were seeded at a density of 5000 cells/well into a 96-well plate. After 24 h, cells were treated with an increasing concentration of DCA (3.125–100 mM) in duplicate for 24, 48 and 72 h, whereas control cells were treated with a drug vehicle (Hypure water) mixed with medium. At the indicated time points, a CellTiter-Glo® Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA) was used to determine the effect of DCA on cellular viability by quantifying the ATP that will be proportional to the number of the metabolically active cells. The luminescent signal was measured by a GloMax® Luminometer (Promega Corporation, Madison, WI, USA). Cellular viability was presented as a percentage (%) by comparing the viability of DCA-treated cells to the control cells, the viability of which is assumed to be 100%.
In the second set of experiments, cells were treated for 48 h with an increasing concentration of gefitinib and erlotinib (5–80 µM). Additionally, cells were treated for 48 h with a combination of DCA and other anti-cancer agents, namely, cisplatin, camptothecin, gemcitabine, gefitinib and erlotinib. Cellular viability was determined using a CellTiter-Glo® Luminescent Cell Viability assay and the GloMax® Luminometer (Promega Corporation, Madison, WI, USA). The viability was presented as a percentage (%) by comparing the viability of drug-treated cells with the control cells.
4.3. Clonogenic Assay
Into a 6-well plate, A549 and LNM35 cells were seeded, respectively, at 50 and 100 cells/well. Cells were kept to grow into colonies for 7–10 days in a humidified atmosphere at 37 °C and 5% CO2, with the medium being changed every three days. Formed colonies were treated every 3 days for 7 days with increasing concentrations of DCA (6.25–50 mM). Afterwards, colonies were washed three times with 1× PBS, fixed and stained for 2 h with 0.5% crystal violet dissolved in 50% methanol (v/v). Finally, colonies were washed with 1× PBS and photographed, and colonies with more than 50 cells were counted. Data are presented as the colony percentage (%) by comparing the DCA-treated colonies with the control colonies. Colony cell density was assessed by photographing the colonies in each group using an inverted phase-contrast microscope (4×).
In the second set of experiments, formed colonies were treated every 3 days for 7 or 14 days with a combination of DCA and gefitinib or DCA and erlotinib. Data are presented as colonies percentage (%) by comparing the drug-treated colonies with the control colonies.
4.4. In Ovo Tumor Growth Assay
Fertilized Leghorn eggs were incubated in the egg incubator set at a temperature of 37.5 °C and humidity of 50% for the first 3 days after fertilization. At embryonic day 3 (E3), the CAM was dropped by drilling a small hole into the eggshell opposite to the round, wide end followed by aspirating ~1.5–2 mL of albumin using a 5 mL syringe with 18G needle. Then, a small window was cut into the eggshell above the CAM using a delicate scissor and sealed with a semipermeable adhesive film (Suprasorb® F). The eggs were kept again in the egg incubator until embryonic day 9 (E9), in which cancer cells were trypsinized, centrifuged and suspended in an 80% Matrigel® Matrix (Corning, Bedford, UK) to have 1 × 106 cells/100 µL for A549 and 0.3 × 106 cells/100 µL for LNM35. A 100 µL inoculum was added onto the CAM of each egg for a total of 10–13 eggs per condition. At embryonic day 11 (E11), formed tumors were treated topically by dropping 100 µL of the DCA prepared in 0.9% NaCl for the first group or the drug vehicle for the control group. Treatment was repeated at E13 and E15. All the described steps were performed under aseptic conditions. Finally, at embryonic day 17 (E17), embryos were humanely euthanized by a topical addition of 10–30 µL pentobarbitone sodium (300 mg/mL, Jurox, Auckland, New Zealand). Tumors were carefully extracted from the normal upper CAM tissues, washed with 1× PBS and weighted to determine the effect of DCA on tumor growth. Data are presented as comparisons of the average weight of tumors in the control group and DCA-treated group. Drug toxicity was assessed by comparing the percentage of alive embryos in the control and DCA-treated groups at the end of the experiment. Alive embryos were determined by checking the voluntary movements of the embryos in addition to the integrity and pulsation of the blood vessels.
This assay is a randomly assigned unblinded assay that was carried out according to the protocol approved by the animal ethics committee at the United Arab Emirates University. According to the European Directive 2010/63/EU on the protection of animals used for scientific purposes, experiments involved using chicken embryos on and before E18, do not require approvals from the Institutional Animal Care and Use Committee (IACUC).
4.5. Tumor Growth and Metastasis Assay
The animal experiments were performed according to the protocol approved by the UAE university animal ethics committee in March 2019 (protocol code ERA_2019_5872). Six- to eight-week-old athymic NMRI male nude mice (nu/nu, Charles River, Germany) were housed in filtered-air laminar flow cabinets and handled under aseptic conditions. A549 cells (5 × 106 cells/200 µL PBS) and LNM35 cells (0.4 × 106 cells/200 µL PBS) were injected subcutaneously into the lateral flank of the nude mice. Ten days later, when tumors had reached the volume of approximately 50 mm3, animals with A549 xenografts were divided randomly into three groups of 9–10 mice each. These groups were treated orally every day (5 days/week) with DCA 50 mg/kg or 200 mg/kg or drug vehicle for 38 days. On the other hand, animals with LNM35 xenografts were treated orally every day (5 day/week) with DCA 200 mg/kg or drug vehicle for 10 days and DCA 500 mg/kg or drug vehicle for 24 days. Tumor dimensions and animal weights were checked every three or four days. In addition, the physical signs and behavior were checked every day. Tumor volume was calculated using the formula V = L × W2 × 0.5, with L representing the length and W the width of the tumor. At the end of the experiments, animals were anesthetized and sacrificed by cervical dislocation, and tumors were removed and weighted. The effect of DCA on tumor growth was presented by comparing the average tumor weight at the end of the experiment between the control group and the DCA-treated group. It was also assessed by comparing the tumor volume between the control and DCA-treated groups throughout the experiment. Blood samples were collected from each mouse and analyzed using the SCIL VET ABC™ Animal Blood Counter for a complete blood count. In addition, blood plasma was separated by centrifugation for biochemical analysis. To study the impact of DCA on metastasis, axillary lymph nodes were excised and weighted from the animals with LNM35 xenografts at the end of the experiment.
4.6. Vascular Tube Formation Assay
Matrigel
® Matrix (Corning, Bedford, UK) was thawed, and 40–50 µL was added to the wells of a 96-well plate for coating. In order for the Matrigel to solidify, the plate was kept in a humidified incubator at 37 °C and 5% CO
2 for 1 h. HUVECs were trypsinized and seeded on the coated plate at a density of 2.5 × 10
4 cells/100 µL/well in the presence and absence of different concentrations of DCA. After 8 h of incubation, the tube networks at the different wells were photographed using an inverted phase-contrast microscope. The impact of DCA on the ability of HUVECs to form capillary-like structures was assessed by measuring the total lengths of the formed tubes in the control and DCA-treated wells. Total tube lengths were measured manually and through an online image analysis software developed by Wimasis (
https://www.wimasis.com/en/products/13/WimTube - access date 1 March 2019). The effect of the different concentrations of DCA on the viability of HUVECs was determined using a CellTiter-Glo
® Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA), as previously described in the cellular viability section.
4.7. HUVEC Spheroids Sprouting Assay
HUVEC spheroids were prepared by firstly staining the cells by incubating 190,000 cells with 2 µM solution of CellTracker
TM Green CMFDA Dye (Invitrogen Molecular probes, Paisley, UK) for 30 min in a humidified incubator set at 37 °C and 5% CO
2, followed by centrifugation for 5 min and the removal of the supernatant. HUVEC pellet was suspended with supplemented HUVEC medium (5 mL) mixed with methocel solution (1.25 mL), which should be prepared earlier [
48]. Then, 25 µL of the cell suspension was pipetted onto the cover of the Petri dish. Approximately 50 drops were pipetted in each Petri dish. Finally, drops were kept upside down for 24 h in a humidified incubator set at 37 °C and 5% CO
2.
Formed spheroids in each dish (~50 spheroids) were collected separately with 1× PBS and centrifuged at 150× g for 5 min. In the meantime, collagen I working solution was prepared on ice by gentle mixing of rat tail collagen I stock (1500 µL) (Millipore, MA, USA) with 10× medium 199 (150 µL) (Sigma-Aldrich, Saint Louis, MO, USA) and ice-cold sterile 1N NaOH (34 µL), which turned a red color. Each spheroid pellet was layered with methocel solution, having 4% FBS (0.25 mL), collagen I working solution (0.25 mL) and 60 µL of basal medium or VEGF 30 ng/mL or DCA 25 mM or a combination of both. Immediately after gentle mixing, the mixture was added to a pre-warmed 24-well plate and incubated in a humidified incubator set at 37 °C and 5% CO2 for 24 h, allowing for collagen polymerization and spheroid sprouting. After 24 h, spheroids were captured using an inverted microscope with 20× magnification. The sprout length in 12 spheroids in each condition was measured using ImageJ.
4.8. Wound Healing Motility Assay
A549 and LNM35 cells were seeded at a density of 1 × 106 cells/well into a 6-well plate. After 24 h, a scratch was made through the confluent monolayer by using a 200 µL tip. After that, the cells were washed twice with 1× PBS followed by the addition of supplemented fresh medium having a drug vehicle or DCA. At the top of the plate, two places were marked for monitoring the decrease in the wound size over time, using an inverted microscope at objective 4× (Olympus 1X71, Tokyo, Japan). The plates were incubated in a humidified atmosphere at 37 °C, and 5% CO2 and the wound width was measured at 0, 2, 6 and 24 h after incubation. The migration distance was expressed as the average of the difference between the measurements at time zero and the 2, 6 and 24 h time periods.
4.9. Matrigel Invasion Chamber Assay
Following the manufacturer’s protocol (Corning, Bedford, MA, USA), 0.5 mL RPMI-1640 medium, supplemented with 10% FBS, was added to the bottom chambers. After that, cancer cells were seeded at a density of 1 × 105 cells/0.5 mL into the upper chambers in a medium lacking FBS in the presence and absence of DCA. The plate was kept in a humidified incubator at 37 °C and 5% CO2 for 24 h. Invasive cells degrade the Matrigel and pass through the 8 µm pores of the insert. The upper chambers’ non-penetrating cells were removed by gently rubbing the area with a cotton swab. Then, the semipermeable membrane was removed using a very fine scissor. The invasive cells were detected using a CellTiter-Glo® Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA) previously described in the cellular viability section. The effect of DCA on cellular invasion was presented as a percentage (%) by comparing the invading cells in the presence of DCA with the control.
4.10. Statistical Analysis
Apart from the in ovo assay and experiments on nude mice, each experiment was carried out at least three times. Data are expressed as mean ± S.E.M. The statistical analysis was performed using GraphPad Prism version 8.3.1 for Windows (GraphPad Software, San Diego, CA, USA). An unpaired t-test was used to assess the difference between two groups. One-way ANOVA followed by Dunnett’s multiple comparison test were used to compare 3 or more groups to the control group. Additionally, a one-way ANOVA followed by Tukey’s multiple comparison test was used for the combination experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate significant differences.