BMSC enhance the survival of paclitaxel treated squamous cell ...

RESEARCH PAPER

RESEARCH PAPER

Cancer Biology & Therapy 11:3, 349-357; February 1, 2011; © 2011 Landes Bioscience

BMSC enhance the survival of paclitaxel treated squamous cell carcinoma cells in vitro Agmal Scherzed, Stephan Hackenberg, Katrin Froelich, Michael Kessler, Christian Koehler, Rudolf Hagen, Andreas Radeloff, Gudrun Friehs and Norbert Kleinsasser* Department of Oto-Rhino-Laryngology; Plastic, Aesthetic and Reconstructive Head and Neck Surgery; Julius-Maximilian-University of Wuerzburg; Wuerzburg, Germany

Key words: bone marrow derived mesenchymal stem cells, squamous carcinoma cell line, paclitaxel, HNSCC, BMSC, cytokines Abbreviations: BMSC, bone marrow derived mesenchymal stem cells; HNSCC, head and neck squamous cell carcinoma

The five-year survival rate of patients suffering from head and neck squamous cell carcinoma (HNSCC) is unsatisfying despite the advances in carcinoma treatment. Recent studies suggest that stem cells can be used as a gene therapy carrier for cancer treatment. Stem cells produce different cytokines such as growth factors in a paracrine manner and cancer cells may show drug resistance in the presence of such growth factors. Reports in the literature concerning treatment of cancer using bone marrow derived stem cells (BMSC) are controversial, which led us to investigate the effects of paclitaxel on human HNSCC cell lines (FaDu and HLaC 78) cultivated simultaneously with BMSC in a transwell system (co-culture). Co-culture and HNSCC cell lines were treated with 10 nM of paclitaxel for 24 h. Morphology, viability and apoptosis were measured by microscopy, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the Annexin V-propidium iodide test. The survival of HNSCC cell lines treated with paclitaxel in co-culture increased significantly compared to control cells. Apoptosis of HNSCC cell lines in co-culture was attenuated significantly. In conclusion, BMSC increase HNSCC resistance to treatment with paclitaxel in vitro. Tumor-stroma interactions are critical components of tumor biology including tumor invasion and metastatic potential. Therefore particular attention must be paid to the complex tumor-stroma interactions to fully understand how tumor cells become chemoresistant.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Introduction

Squamous cell carcinoma represent the sixth most common malignancy and are a major cause of cancer morbidity and mortality. Approximately 500,000 new head and neck squamous cell carcinoma (HNSCC) are diagnosed each year worldwide.1,2 Despite advances in treatment options for patients, survival rates have not significantly improved. HNSCC is most commonly associated with tobacco smoke and increased alcohol consumption.3 Paclitaxel is a mitotic inhibitor used in cancer chemotherapy. It is a product of the Pacific yew bark, Taxus brevifolia. It promotes tubulin polymerization and inhibits microtubule dynamics, which is essential for cell division and vital interphase processes.4 Paclitaxel has been used against a broad range of cancers because of its potent cytotoxic activity.4-6 Moreover, in locally advanced carcinoma it has been used as a radio-sensitizer with promising results.7 Recent studies suggest bone marrow derived mesenchymal stem cells (BMSC) as a gene therapy carrier for cancer treatment.8 But there is also evidence of tumor progression after in vivo application of BMSC.9 Stem cells are found in several tissues like fat, neurons and skin.10 They are characterized as

undifferentiated cells, able to self-renew with a high proliferative capacity.11 BMSC have exhibited the ability to leave the bone marrow, circulate in the blood, and home in on injured tissues and inflammation.12,13 Recently, it has been shown that BMSC are capable of directed migration towards the tumors of various types and origins.14 Indeed, BMSC are a source of circulating stem cells that are recruited from the blood into peripheral solid organs in times of tissue stress or injury.15 Furthermore, chemokine receptors, their ligands and adhesion molecules play an important role in the tissue-specific homing of leukocytes.16 Similar to wound healing, it is thought that tumor expansion also requires BMSC for angiogenesis and growth. Due to the capability of bone marrow derived mesenchymal stem cells to home in on cancer, BMSC are very interesting and it has been suggested that they could be used as vehicles for target drug delivery.17 The influence of BMSC on chemo-sensitivity and apoptosis of HNSCC cells is our primary focus. The objective of the present study was to analyze the in vitro effects of paclitaxel on the human squamous cell carcinoma cell line (HNSCC cell lines) FaDu and HLaC 78 cultivated simultaneously with BMSC in terms of morphological changes, viability and apoptosis.

*Correspondence to: Norbert Kleinsasser; Email: [email protected] Submitted: 08/30/10; Revised: 11/01/10; Accepted: 11/10/10 DOI: 10.4161/cbt.11.3.14179 www.landesbioscience.com

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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 1. Multidifferentiation potential of BMSC. (A) Microscopic analysis of BMSC cultured in osteogenic medium (DMEM-EM, 10 -7 M dexamethasone, 10 -3 M β-glycerophosophate and 2-4 M ascorbate-2-phosphate) and analyzed on day 14. The von Kossa staining characterizes the mineralization by staining the calcium mineral component dark brown (magnification: x200). (B) Differentiation into adipogenic lineage was achieved by cultivating with DMEM-EM, 10 -7 M dexamethasone and 10 ng/ml recombinant human insulin. The adipogenic differentiation was confirmed by staining with Oil red O to show the presence of intracellular lipid droplets (magnification: x200). (C) BMSC were cultured in defined chondrogenic differentiation media containing 10 μg/ml TGFβ3. Chondrogenic differentiation was shown using the Alcian blue staining after 21 days of incubation (magnification: x200).

Results Differentiation of BMSC. The potential of BMSC to differentiate into osteocytes, adipocytes and chondrocytes was confirmed by von Kossa, Alcian blue and Oil Red O staining (Fig. 1). Cytokine assay. To evaluate the release of different cytokines, a semi-quantitative cytokine assay was performed. BMSC supernatants contained a variety of cytokines responsible for inflammation (Tumor necrosis factor (TNF)α, TNFβ, interleukine (IL)-6 and Oncostatin-M) and anti-inflammation (IL-10), chemotaxis (Monocyte chemotactic protein (MCP)-1, MCP-2, MCP3, IL-8), angiogenesis (Angiogenin) as well as growth factors

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(Vascular Endothelial Growth Factor [VEGF], Insulin-like growth factor [IGF]-1, IL-7, Growth regulated oncogene [GRO], GRO-α platelet-derived growth factor [PDGF]-BB [Fig. 2]). DMEM as negative control contained EGF. The positive dots in the DMEM group demonstrated, that the assay was performed correctly. Transwell migration assay. The transwell migration assay was performed in order to examine the migration effect of carcinoma cells on bone marrow derived mesenchymal stem cells. BMSC showed a significant enhancement (p < 0.05 FaDu and p < 0.05 HLaC 78) of cell migration to carcinoma cells as compared to medium containing FCS as a chemoattractant (Fig. 3). Paclitaxel treatment of squamous carcinoma cell line. After paclitaxel treatment the cell morphology was analyzed by

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©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Figure 2. Cytokine assay of BMSC cultivated with DMEM without supplements. The dot-blot assay was used to semi-quantitatively analyze the presence of different cytokines in supernatants. (A) To assign the different dots to cytokines, a table was used as a coordinate system. Positive dots are marked grey in the presented table. (B) Bone marrow derived mesenchymal stem cells release various types of cytokines which are responsible for angiogenesis, inflammation and growth. (C) As control, DMEM without supplement was used and no cytokines were available.

microscopy. There were no morphological differences in carcinoma cells between co-culture and control group cells before and after paclitaxel treatment (Fig. 4). Cells treated with paclitaxel were rounded as a sign of apoptosis. The MTT assay revealed a p value of p < 0.001 FaDu cells, a p value of p < 0.01 HLaC 78 cells and a significant attenuation of cell apoptosis in co-culture with BMSC after paclitaxel treatment compared to carcinoma cells incubated with paclitaxel alone. This result was also reproduced in the Annexin V-propidium iodide test (Figs. 5–7).

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Discussion The present study focused on the effects of bone marrow derived mesenchymal stem cells on the viability of squamous carcinoma cell lines during paclitaxel treatment. The carcinoma cell lines showed enhanced survival and decreased apoptosis in the presence of BMSC (co-culture). The study was performed to elucidate the results of current investigations in the literature with conflicting conclusions concerning the effects of BMSC on tumors. The benefit of BMSC is postulated, for example, in a


Figure 3. To analyze the chemoattractant effect of cancer on bone marrow derived mesenchymal stem cells a transwell system was used. BMSC were coated on the top of the membrane. (A) FaDu and (B) HLaC 78 were cultured on the bottom of the same well plate. As control, RPMI with 10% FCS was used. FCS functioned as a chemoattractant. HNSCC cell lines induced a significant enhancement in BMSC migration (p < 0.05).

reduction of tumor growth in a prostate cancer bone metastasis model. Chanda et al. showed a significant inhibition of tumor growth by implanting BMSC at the tumor site.23 Other in vitro studies suggest BMSC as a carrier of suicide gene therapy in glioma cells with promising results.17 Drug resistance of tumor cells is recognized as the primary cause of failure of chemotherapeutic treatment of most human tumors. Although pharmacological factors including inadequate drug concentration at the tumor site can contribute to clinical resistance, cellular factors play a major role in chemoresistance of several tumors.24 One major mechanism by which tumor cells acquire a chemoresistant phenotype is the protection from drug-induced apoptosis. Resistance towards cytotoxic drugs can be present before the onset of chemotherapy (intrinsic) or develops during treatment course (extrinsic).25,26 The role of tumor-BMSC-interactions in development and manifestation of chemoresistance in HNSCC has been the subject of this study. Cytokines may play a key role in enhancing the carcinoma cell line’s survival during paclitaxel treatment. Tsang et al. were able to show drug resistance of squamous cell carcinoma induced during long-term treatment with epidermal growth factor (EGF), which was possibly due to a downregulation of topoisomerase II.27 Moreover, Dhawan showed the interaction of paclitaxel and topoisomerase II in vitro.28 We used an in vitro transwell model to investigate the effect of BMSC on squamous cell carcinomas during paclitaxel treatment. To examine the migration of BMSC towards cancer cells in vitro, the transwell system was used as well. The apoptosis rate of carcinoma cell lines cultivated simultaneously with stem cells was attenuated under paclitaxel treatment. This drug resistance may be induced by the autocrine and paracrine secretion of cytokines. Several studies point to the ability of BMSC to produce growth factors such as vascular endothelial growth factors (VEGF), insulin-like growth factor (IGF)-1 or transforming growth factor (TGF)β in a paracrine manner.29,30 BMSC migrate towards tumors and participate in the formation of a tumor-associated

stroma.31 This may have an influence on the tumor survival in the presence of anticancer drugs. IL-6 is a cytokine which stimulates tumor growth by activation of Ras/Raf/MEK/Erk1/2.32,33 Moreover, IL-6 is regarded as one of the most important cytokines in multiple myeloma disease progression.34,35 Our cytokine assay represented semi-quantitatively the release of IL-6. Deregulated growth factor signaling pathways might promote cell proliferation and render cancer cells resistant to apoptosis. Since IL-6 is present in wound inflammation, there may be a synergistic effect of cytokines secreted in cancer and BMSC. Bone marrow derived mesenchymal stem cells participate in the formation of tumor-associated stroma.31 Moreover, Antoine discovered that BMSC migrate into breast cancer and “educate” the tumor to metastasize.36 We observed an enhanced migration of BMSC towards carcinoma cell lines HLaC 78 and FaDu. The ability of BMSC to seek out the site of tissue damage has been demonstrated in several studies.37,38 Since cancer represents a wound that never heals, many cytokines are released. It is also suggested that BMSC have an effect on the tumor growth kinetic39 and several studies illustrate the capacity of BMSC to home in on tumor sites endogenously or when injected systemically.40,41 In summary, the present study showed a reduction in apoptosis and an enhancement in the viability of squamous carcinoma cell lines treated with paclitaxel and cultured simultaneously with bone marrow derived mesenchymal stem cells. BMSC could have influenced the viability of HNSCC through paracrine secretion of various cytokines. BMSC increased HNSCC resistance to treatment with paclitaxel in vitro. Tumor-stroma interactions are critical components of tumor biology including tumor invasion and metastatic potential. Therefore particular attention must be paid to the complex tumor-stroma interactions to fully understand how tumor cells become chemoresistant. The current study offers a possible approach for further in vitro and in vivo investigation concerning BMSC effects on squamous cell carcinoma.


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Figure 4. Microscopic analysis of HNSCC cell line under different conditions. Microscopic analysis of (A) FaDu and (B) HLaC 78 (cultivation with RPMI-EM), no rounded cells were visible. Co-culture consisting of (C) FaDu and BMSC, (D) HLaC 78 and BMSC cultivated with RPMI-EM and 10 nM of paclitaxel. (E) FaDu and (F) HLaC 78 cultivated with RPMI-EM and 10 nM paclitaxel.

Material and Methods Culture of human carcinoma cell line FaDu. The head and neck squamous carcinoma cell line HLaC 78 has been established from a lymph node metastasis of a laryngeal squamous cell carcinoma.18 Furthermore, we used FaDu cells, a cell line derived from


a human hypopharyngeal squamous cell carcinoma.19 Cells were grown in RPMI-1640 medium (Biochrom AG, Berlin, Germany) with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ ml streptomycin, 1% sodium pyruvate (100 mM, Biochrom AG) and 1% non-essential amino acids (100-fold concentration, Biochrom AG) (RPMI-expansion medium [RPMI-EM]).


Figure 5. MTT assay of HNSCC cell lines under different conditions. After one week in co-culture cells were treated with 10 nM of paclitaxel for 24 h. After 24 h the medium was changed. Following another 24 h period, an MTT test was done. There was significant enhancement in cell viability in coculture compared to the control group. (A) FaDu, p < 0.001 and (B) HLaC 78, p < 0.01.


Figure 6. Representative flow cytogramme of an Annexin V binding (abscissa) versus PI uptake (ordinate) in the HNSCC cell line FaDu. The numbers in the upper left quadrant (Q1 = annexin V-/PI+; represent the percentage of damaged cells), upper right quadrant (Q2 = annexin V+/PI+; represent the percentage of necrotic cells), lower left quadrant (Q3 = annexin V-/PI-; represent the percentage of viable cells) and lower right quadrant (Q4 = annexin V+/PI-; represent the percentage of apoptotic cells). (A) Representative flow cytogramme of co-culture of control cells. (B) Representative flow cytogramme of co-culture of FaDu and BMSC after 24 h of paclitaxel treatment. (C) Representative flow cytogramme of FaDu after 24 h of paclitaxel treatment. (D) Columns representing apoptosis of control, co-culture of BMSC and FaDu and FaDu alone. Graphs represent five repetitions of tests. There was a significant attenuation of cell apoptosis after treatment with 10 nM of paclitaxel in co-cultures compared to the control group, p < 0.001.

The cells were cultured at 37°C with 5% CO2 in culture flasks. Medium was replaced every other day and passaging was performed after reaching 70–80% confluence by trypsinization (0.25% trypsin; Gibco Invitrogen, Karlsruhe, Germany), washing and seeding in new flasks or treatment wells. Experiments were performed using cells in the exponential growth phase.

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Bone marrow derived mesenchymal stem cells. Bone marrow was obtained from voluntary trauma patients undergoing surgery in the Department of Orthopedics. The study was approved by the Ethics Committee of the Medical Faculty, University of Wuerzburg (12/06) and informed consent was obtained from all of the individuals included. BMSC were isolated as described


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Figure 7. Representative flow cytogramme of an Annexin V binding (abscissa) versus PI uptake (ordinate) in the HNSCC cell line HLaC 78 (see Fig. 6). Graphs represent five repetitions of tests. There was a significant attenuation of cell apoptosis after treatment with 10 nM of paclitaxel in co-cultures compared to the control group, p < 0.05.

by Lee et al.20 from fresh bone marrow aspirate using Ficoll density gradient centrifugation (30 min, 1,300 rpm, density = 1,077 g/ml, Biochrom AG). The cells in the interphase were collected and washed twice using phosphate buffered saline (PBS) (Roche Diagnostics GmbH, Mannheim, Germany) containing 2% FCS. The pellet was resuspended in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Invitrogen), 10% FCS, 100 U/ ml penicillin, 100 μg/ml of streptomycin (DMEM-expansion medium [DMEM-EM]) and were counted using a Neubauer chamber. The cells were incubated overnight at 37°C and 5% CO2 in DMEM-EM. Following incubation, the tissue culture plates were washed to remove residual non-adherent cells. Medium was changed every 2nd day. When cells reached >70% confluence (P0), they were trypsinized with 0.25% trypsin, resuspended in DMEM-EM and subcultered at a concentration of 2,000 cells/cm2. The morphology was analyzed by inverted microscopy (Leica DMI 4000B Inverted Microscope, Leica Microsystems, Wetzlar, Germany). In vitro differentiation. Differentiation procedures were performed to analyze the multidifferentiation potential of the cells. BMSC were cultivated with DMEM-EM. The differentiation into mesenchymal tissues was performed according to the following protocols: Osteogenic differentiation. Osteogenic differentiation was carried out in a 24-well plate (BD Falcon) with 1 x 104/well cells until 70% confluence was reached. The osteogenic induction medium was prepared as described by Pittenger et al.11 and was composed of DMEM-EM, 10 -7 M dexamethasone,


10-3 M β-glycerophosophate and 2-4 M ascorbate-2-phosphate (all Sigma-Aldrich, Schnelldorf, Germany). Every 3rd day the medium was changed. As negative control, cells were maintained in DMEM-EM. The von Kossa staining detected an osteogenic differentiation stage and was performed according to the protocol as previously described in reference 21. Cells were washed with PBS without Ca++ and Mg++ three times and fixed with 70% Ethanol for 20 min at room temperature (RT). After washing, the cells were stained according to the protocol for 60 min with 5% silver nitrate (Sigma-Aldrich) under ultraviolet light (UV-light) (Desaga Lightbox UVIS 254/366 nm, Burladingen, Germany) at RT. Next, cells were fixed with 5% sodium thiosulfate (Applichem, Darmstadt, Germany) for 5 min. Finally, cell nuclei were stained with Nuclear Fast Red. Von Kossa staining is routinely used to characterize mineralization by staining the calcium mineral component dark brown. Adipogenic differentiation. Adipogenic differentiation was performed in monolayer culture as described by Pittenger et al.11 Cells were plated at a density of 1–2 x 104 cells/cm2. Adipogenic differentiation was induced by DMEM-EM, 10 -7 M dexamethasone (Sigma Aldrich) and 10 ng/ml recombinant human insulin. It was confirmed by staining with Oil Red O to show the presence of intracellular lipid droplets. Chondrogenic differentiation. For chondrogenic differentiation the pellet culture system was used. The cell pellets were cultured in a defined chondrogenic differentiation medium (Lonza, Basel, Switzerland) supplemented with 10 ng/ml transforming growth factor-beta 3 (TGFβ3). The medium was


replaced every 2–3 days for 3 weeks. After 21 days the pellets were embedded in Tissue-Tek® O.C.T TM Compound (Optimal Cutting Temperature Paraffin, Tissue-Tek®). The cryo sections were stained with Alcian blue to show the presence of glycosamineglycane. Treatment with paclitaxel. To analyze the effects of BMSC on HNSCC cell lines, a transwell system (Corning Incorporating Costar, Wiesbaden, Germany) was used. First we generated a coculture: 5 x 104 FaDu cells and HLaC 78 cells were coated with 1 ml RPMI-EM on the bottom of a 12 round bottom plate. 1 x 105 BMSC with 0.5 ml RPMI-EM were coated in transwells and transferred to the well plates containing HNSCC cell lines. Every other day the medium was changed. When cells reached >70% confluence, they were trypsinized with 0.25% trypsin (Gibco Invitrogen), counted and subcultured. Simultaneously, HNSCC cell lines were cultivated in parallel during the entire investigation in RPMI-EM as the control group. After one week, cells were treated with 10 nM paclitaxel for 24 h. After 24 h the medium was then changed. Following another 24 h period, the viability was measured by the MTT assay and apoptosis was evaluated using the Annexin V-propidium iodide test. Analytical assays. The following assays were performed to analyze secretion, viability, migration and apoptosis of the cells. Cytokine assay. The dot blot assay (RayBiotec Inc., Norcross, GA) was used as a semi-quantitative method to obtain cytokine secretion of BMSC after cultivation in DMEM without supplements. After 2 days, the supernatants were investigated for the presence of cytokines. The detection procedure followed the manufacturer’s protocols. Briefly, the supernatants were added to the membrane, which was spotted with antibodies against various cytokines. The membrane was blocked with blocking buffer for 30 min. After several washing steps, each of the samples reacted for 2 h at RT, followed by an incubation period with a mixture of biotin-conjugated antibodies against several cytokines and horseradish peroxidase-conjugated streptavidin. The labeled proteins were observed by enhanced chemiluminescence using detection buffer and exposure to an X-ray film. As negative control DMEM without supplements was used. The cytokines represented as dots with different intensity and growth size. MTT assay. The viability of cells was studied using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich) colorimetric staining method according to Mosmann et al.22 All plates were incubated with 1 ml of MTT

(1 mg/ml) followed by 5 h incubation at 37°C with 5% CO2. After removal of MTT, 1 ml of isopropanol was added following incubation for 1 h at 37°C with 5% CO2. The color conversion of the blue formazan dye was measured with the multi-plate reader (Titertek Multiskan PLUS MK II, Labsystems, Helsinki, Finland) at a wavelength of 570 nm. Migration assay. Transwells (Corning Incorporating Costar, Wiesbaden, Germany) were coated with 50% extra cellular matrix (EMC) gel (Sigma-Aldrich) for 30 min. 1 x 105 BMSC were coated on the top surface of the membrane and incubated with DMEM-EM for 24 h at 37°C with 5% CO2. Subsequently, 4.5 x 104 HNSCC cell lines were coated on the bottom of the well plate chamber, followed by incubation for 24 h. Non migratory cells on the upper membrane were removed with a cotton swab; cells that migrated onto the lower surface of the membrane were stained for 25 min with 1% crystal violet (Sigma-Aldrich). After one washing step with aqua destillata, cells were incubated with 500 μl 10% acetic acid for 20 min. The measurement was performed using a multi-plate reader (Titertek Multiskan PLUS MKII, Labsystems) at 570 nm. Annexin V-propidium iodide test. Apoptosis was evaluated using the Annexin V-APC kit of BD Pharmingen (BD Biosience, Heidelberg, Germany) according to the kit manual. In brief, cells in suspension and adherent cells were harvested and washed twice with cold PBS. Cells were resuspended in 1:10 binding buffer (0.1 M Hepes, pH 7.4, 1.4 M NaCl, 25 mM CaCl2) at a concentration of 1 x 106 cells/mL and 100 μL aliquots of this cell suspension (1 x 105 cells) were then transferred to a 5 ml culture tube. 5 μl of Annexin V-APC and 5 μl of propidium iodide (PI) were added to each aliquot containing 1 x 105 cells. After incubation for 15 min in the dark at RT, cells were resuspended with 400 μl 1:10 binding buffer. We used a FACScanto flow cytometer (Beckton Dickinson, Heidelberg, Germany) to analyze the samples. PI only stains cells that present damaged membranes, as observed during necrosis. Statistical analysis. All Data were transferred to standard spreadsheets and analyzed by statistical analysis (GraphPad Prism 4.0 Software). First column analysis was performed to test whether the distribution was Gaussian. In the case of Gaussian distribution unpaired t-test, in the case of not Gaussian distribution Kruskal-Wallis test was performed. Statistical significance (p < 0.05) is indicated in the figures as follows: ***p < 0.0001; **p < 0.0001–0.001; *p < 0.01–0.05.


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