Study On Paclitaxel Combined With Ultrasound Irradiation On Three-dimentional Spheroids

Background and objective:Currently, anticancer drugs are assessed by detecting drug sensitivity in monolayer cells in vitro. But scholars often found that drug sensitivity of tumor cells are not equal to efficacy of most clinical assessment in patients which results in limited application for monolayer cells. The reason for this discrepancy is that the structure of monolayer cells is simple. And it doesn’t reflect the three-dimensional structure of solid tumors and the status of the pathophysiology of tumor cells in vivo. Therefore scholars intend to find a new model which could make up for the deficiency of two-dimentional (2D) monolayer model.In recent years, a number of scholars have established a three-dimensional (3D) spheroid model. Compared to monolayer cells, it is generally considered spheroids have a similar biological characteristics of solid tumors in vivo, including the development of an ECM, tight junctions between epithelial cells and gradients of oxygen, nutrient, pH concentration and cell proliferation from the exterior to the centre and so on. It may be more clinical reference value of doing research on 3D spheroid model.There are several methods to get a spheroid model such as spinner flask culture, serum-free suspension culture, hanging drops method and so on. All of this spheroids show the characteristics of drug-resistant, having a hypoxic microenvironment similar to solid tumours. Serum-free suspension culture method is widely recognized as one of the screening methods to get cancer stem cells. Given a few cancer stem cells is one of the reasons for the presence of drug resistance in solid tumors, we chose serum-free suspension culture to get our 3D spheroid model which simulate the hypoxic microenvironment along with drug-resistant properties.Ultrasound targeted microbubble destruction (UTMD) technology is a potential targeted drug delivery method using different acoustic emission intensity in a specific area, causing rupture microbubbles generated effects of cavitation, microfluidic, shock waves, microjets etc. Then, it can transiently perforate cell membranes and therefore enhance the intracellular uptake of drugs, which is called sonoporation. The fluid flow effect induced by ultrasonic exposure could also increase the transport of any drugs, and then enhance the accumulation of drugs in cells. It have been widely reported that UTMD technology can enhance cellular uptake of drug, gene, protein in monolayer cells. We have previously carried out UTMD technology to drug-resistant monolayer cells and proved its effect of reverse multidrug resistance. But we know little about the synergistic effect of UTMD technology combined with chemotherapy drugs on 3D spheroid model.As one of the most widely used chemotherapeutics drug in clinical, Paclitaxel apply to many different types of cancer such as breast cancer and ovarian cancer. Paclitaxel makes cells cycle arrest which can effectively kill rapidly proliferating tumor cells. Its mechanism is stabilizing microtubules at the time of cell division, interfering with microtubule depolymerization and leaving the cell cycle arrest in mitosis. Due to the lack of mitosis, cells can not proliferate, and eventually induces cells to die. Studies have shown that UTMD technology can promote tumor cells to uptake of a variety of chemotherapeutic agents, thereby increasing the cytotoxicity on tumor cells.In summary, we take into account the different sensitivity of various types of tumor cells to ultrasound and the limitations of monolayer culture model in the application. In order to further investigate the applicability of ultrasound irradiation on different types of spheroids, this study intends to build 3D spheroid model of human breast cancer cell line MCF-7 and human nasopharyngeal carcinoma cell line 5-8F, following by investigating the effects of ultrasound irradiation combined with paclitaxel on this model. Then the related mechanisms are also explored.Materials and methods:1. Established a 3D spheroid modelHuman breast cancer cell MCF-7 and human nasopharyngeal carcinoma cell 5-8F were cultured in DMEM and RPMI 1640 medium each containing 10% fetal bovine serum, respectively. MCF-7,5-8F monolayer cells were digested and resuspended in 6-well plates filled with DMEM/F12 medium and growth factors, then incubated at 37℃,5% CO2 incubator.2. Difference of biological properties between 3D spheroids and 2D monolayer cells2.1 Cell cycle determined by flow cytometerSpheroids and monolayer cells of MCF-7 and 5-8F in the logarithmic phase were collected and digested into single cells. After adding RNase A and PI for 15 min, differences of cell cycle were detected by flow cytometry in each group.2.2 Q-PCR analysis of related gene expression levels between 3D spheroids and 2D monolayer cellsRNA samples were respectively extracted from spheroids and monolayer cells of MCF-7 and 5-8F. After reversely transcribed, their expression levels of ABCB1, ABCG2, ABCC1, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) were detected by Q-PCR.2.3 Cell viability between 3D spheroids and 2D monolayer cells to paclitaxel were determined by CCK-8MCF-7 spheroids and monolayer cells were incubated with paclitaxel of different concentrations for 72 h. CCK-8 assay was used to measure the relative cell viability in different groups. The same experiment was done in 5-8F as above.3. Synthesis and characterization of microbubblesThe ultrasound contrast microbubbles (MBs) used in this study were prepared according to the method as follows. Briefly, certain quantities of DSPC、DPPE and PEG4000 were well hydrated with ultrapure water in a 70 ℃ thermostatic water bath for 30min. Subcequently, mixed lipid suspension were added in 50 mL polypropylene tube, and put the shear cutter below liquid level. Afterward, perfluorobutane gas (C3F8) was dispersed by sonication in the admixture with a certain shear rate for 2 min. Then lipid microbubbles were stored in schering bottles filled with whole fluorine propane gas. Particle size, size distribution and concentration of MBs were determined using a Coulter counter. Morphologic characteristics of MBs were determined under a light microscope.4. The optimization of ultrasonic irradiation conditions6-well plates were used to seed MCF-7 and 5-8F spheroids. Ultrasonic irradiation was performed through the bottom of culture plates for each area separately at room temperature. According to papers, four influencing factors were selected to join our orthogonal test. Every factor has 3 levels (duty cycle:10%,20%, 50%; microbubble concentration:10%,20%,30%; ultrasound intensity:0.21 W/cm2, 0.65 W/cm2,1.09 W/cm2; irradiation time:30 s,60 s,90 s). Then a orthogonal table L27(313) was designed. After US, we detected cell viability and cellular uptake of Dil by flow cytometry for each group, combining this two indicators to obtain optimal ultrasonic parameters.5. Cell viability of 3D spheroids treated with paclitaxel with or without ultrasound irradiation was determined by CCK-8MCF-7 spheroids were incubated with paclitaxel of different concentrations (1 ng/mL、10ng/mL、100ng/mL、1 ug/mL、2ug/mL、3 ug/mL、4ug/mL、5 ug/mL) combined with or without ultrasound irradiation for 72 h. Each group was made in quartic and CCK-8 assay was used to test its cell viability. The same experiment was done in 5-8F spheroids as above.6. Cellular uptake of DiI in 3D spheroids after US exposure.MCF-7 spheroids were set in three groups of (1) control, (2) DiI, (3) US+DiI. The third group was irradiated by optimized parameters, and then the second and third group were added the same amount of Dil. All samples were incubated for 24 h. Cellular uptake of DiI in each group was analyzed by flow cytometry. The same experiment was done in 5-8F spheroids as above.7. Difference of intercellular space and hypoxia of each spheroids.7.1 Hematoxylin and eosin staining experiment (paraffin section):the MCF-7 and 5-8F spheroids in logarithmic growth phase were washed twice with PBS, and fixed with 4% paraformaldehyde for 1.5 h. The following steps were done:dehydrated, embedded in paraffin, sliced, dewaxed, stained, and mounted. Finally the sections were observed by optical microscope.7.2 Q-PCR analysis of the mRNA expression level of HIF-1α between MCF-7 and 5-8F spheroids by the method mentioned above.8. Western blot analysis of related proteins expression levels in 3D spheroids after US exposure.We extracted proteins samples of MCF-7 and 5-8F tumor spheroids before and after ultrasound exposure for 24 h,48 h,72 h later. Western blot analyed the expression of P-glycoprotein (P-gp/ABCB1), breast cancer resistence protein (BCRP/ABCG2), multidrug resistence-associated protein (MRP1/ABCC1), HIF-la, VEGF, y-H2AX proteins in MCF-7 and 5-8F spheroids, respectively.9. Statistical AnalysisStatistical analyses were performed using the SPSS 13.0 software package. All data were presented as a mean value with its standard deviation indicated (mean ± SD). The protocol was performed by orthogonal design with 4 factors and 3 levels. And statistical analysis was performed for the obtained data by variance analysis of orthogonal design. The other datas were all analyzed with two-sample t-test. P< 0.05 was considered statistically significant. Defined * as P< 0.05,** as P< 0.01,*** as P< 0.001.Results:1. Established a 3D spheroid modelBoth of MCF-7 and 5-8F monolayer cells can form spheroids in serum-free medium. According to the difference of their growth rate, we chose spheroids of MCF-7 incubated for 15 days and 5-8F incubated for 7 days as experimental subjects.2. Difference of biological properties between 3D spheroids and 2D monolayer cells2.1 Cell cycle determined by flow cytometerCompared to monolayer cells, the cell proportion of MCF-7 and 5-8F tumor spheroids were all increased in Gl phase, reduced in S phase and G2/M phase. The differences were all statistically significant (P< 0.05). It indicated that this two kinds of spheroids were in a relatively quiescent period.2.2 Q-PCR analysis of related gene expression levels between 3D spheroids and 2D monolayer cellsThe mRNA expression levels of ABCB1, ABCG2, HIF-la, VEGF in MCF-7 spheroids were 4.87 times,2.53 times,2.08 times,2.56 times than its monolayer cells, the differences were all statistically significant (P< 0.05). The mRNA expression levels of ABCG2, ABCC1, HIF-1α, VEGF expression were 3.46 times,1.49 times, 4.05 times and 2.22 times in 5-8F tumor spheroids compared to monolayer cells, and the differences were all statistically significant (P< 0.05) except the gene of ABCC1. It indicated that it is successful to establish a relatively drug-resistant models along with hypoxic microenvironment.2.3 Cell viability between 3D spheroids and 2D monolayer cells to paclitaxel were determined by CCK-8Compared to monolayer cells respectively, both of MCF-7 and 5-8F improved resistance to paclitaxel after being spheroids, and significantly reduced the cell killing effect of paclitaxel. The differences were statistically significant (P< 0.05). This result was consistent with the result of Q-PCR mentioned above.3. Synthesis and characterization of microbubblesThe prepared microbubbles had a good shape and uniform distribution without aggregation under optical microscope. The mean diameter of MBs was 2.01 ± 0.93 μm and the concentration was (3.93±1.0)×108/mL which were all determined by the Coulter counter.4. The optimization of ultrasonic irradiation conditionsTo obtain optimized US condition, cell viability and cellular uptake of MCF-7 and 5-8F spheroids were respectively detected under different ultrasonic parameters which were designed by orthogonal experiment. The optimal US conditions for MCF-7 spheroids was duty cycle 10%, MBs concentration 10%, irradiation time 60 s and irradiation intensity 0.65 W/cm2 and for 5-8F spheroids was duty cycle 20%, MBs concentration 10%, irradiation time 90 s and irradiation intensity 0.65 W/cm2.5. Cell viability of 3D spheroids treated with paclitaxel with or without ultrasound irradiation determined by CCK-8At low-dose paclitaxel (< 1 ng/mL for MCF-7 spheroids and< 2 ng/mL for 5-8F spheroids), the cytotoxicity of paclitaxel did not significantly increase at this two kinds of spheroids combined with ultrasonic irradiation. Compared to the group without US, the cytotoxicity of paclitaxel significantly increased when combined with ultrasonic irradiation at high-dose paclitaxel (> 1 ng/mL for MCF-7 spheroids and> 2 ng/mL for 5-8F spheroids), and the differences were statistically significant of this two (P< 0.05). It indicated that optimized ultrasonic irradiation conditions can enhance the cytotoxicity of paclitaxel to MCF-7 and 5-8F spheroids.6. Cellular uptake of DiI in 3D spheroids after US exposure.Optimized ultrasounic irradiation conditions could promote cellular uptake of DiI in MCF-7 and 5-8F spheroids, and the increasing extent of MCF-7 spheroids was significantly higher than 5-8F which was consistent with the results of CCK-8. It indicated that ultrasonic irradiation can increase drug accumulation within the spheroids.7. Difference of intercellular space and hypoxia of each spheroids..7.1 HE staining showed that intercellular space of MCF-7 spheroids was wider than 5-8F spheroids. Cell arrangement was relatively looser in MCF-7 spheroids compared to 5-8F. And the last one was compact with tight structure.7.2 Q-PCR showed that mRNA expression level of HIF-la in 5-8F spheroids was 1.48 times taller than MCF-7 spheroids, with a statistically significant difference (P< 0.001). This difference may be due to that two kinds of spheroids have different intercellular spaces. More oxygen went into MCF-7 spheroids which had larger intercellular space under US exposure. The microenvironment with relative high oxygen concentration caused low expression level of HIF-la in MCF-7 spheroids.8. Western blot analysis of related proteins expression in 3D spheroids after US exposure.Compared to control group, P-gp, BCRP, MRP1, VEGF expression levels of 5-8F spheroids were down-regulated after ultrasonic irradiation. Expression level of HIF-1α protein did not change. P-gp expression level of MCF-7 spheroids increased slightly after US, and BCRP, MRP1, HIF-la, VEGF expression levels did not change significantly. It indicated that protein expression levels were different in various types of spheroids in response to US exposure, and US can decrease the expression levels of some proteins in 5-8F spheroids.9. Western blot analysis of DNA damage.γ-H2AX protein expression level was all significantly increased at 72 h after ultrasound exposure for MCF-7 and 5-8F spheroids, which meant DNA damage. It indicated that ultrasonic irradiation can cause DNA damage of spheroids and may further affect cell proliferation and the expression levels of related genes and proteins.Conclusion:1. The optimal ultrasonic irradiation conditions to promote cellular uptake of paclitaxel are different for various types of spheroids.2. US could enhance cytotoxity of paclitaxel for MCF-7 and 5-8F spheroids.3. The related mechanisms of enhancing cytotoxity induced by US combined with paclitaxel are:sonoporation enhance the cell membrane permeability, fluid flow generated by US and the difference of intracellular space increase the convective transport of paclitaxel toward the inner areas of the spheroids, downregulation the expression of drug-resistant and hypoxic proteins, induction of DNA damage.4. The influence mechanisms of ultrasonic irradiation are different in various types of spheroids.