Preparation of thermoreversible polymeric gel (TPG)
The delivery system proposed for intravesical inoculation together with MB49 cells consists of a binary polymeric system (Fig. 1) composed of poloxamer 407 (Sigma-Aldrich, St. Louis, USA) and 0.5% chitosan (medium molecular weight, 75–85% deacetylated, Sigma-Aldrich) in a 0.5% acetic acid solution. Chitosan (0.5–1.5% w/w) was initially dissolved in a solution of acetic acid (0.5% v/w), which improved mechanical and mucoadhesive properties that could provide prolonged retention time . The chitosan solution was refrigerated and used as a solvent for the poloxamer (14–20% w/w) dispersion. All formulations had pH levels between 6.0 and 6.5 .
MB49 tumor cell culture
Murine transitional carcinoma cell line (MB49-NCI Thesaurus Code: C25823), donated by Dr. Yi Lou (University of Iowa), were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and 2 mmol/L of L-glutamine (Cultilab, Campinas, Brazil), supplemented with 10% fetal bovine serum (FBS, Cultilab) and 1% penicillin/streptomycin antibiotics (Vitrocell Embriolife, Campinas, Brazil) (complete DMEM). The cell culture was maintained in an incubator at 37 °C in an atmosphere of > 95% humidity and 5% carbon dioxide, in 75 cm2 bottles. Cell growth and adherence were monitored daily, under an inverted microscope, until 90% confluence of the cell monolayer was observed . Adherent cells were then removed from the surface with the addition of a 2.5% trypsin solution (Life Technologies, Carlsbad, CA, USA) and direct cell quantification, in a Neubauer chamber, was performed with the aid of a Trypan blue solution. at 0.4% (Life Technologies), where cellular integrity was confirmed by the exclusion method.
Effect of TPG concentration on the MB49 cells viability and clonogenicity
Resazurin viability assay
The biocompatibility of TPG with MB49 cells was evaluated by culturing, in quadruplicate and overnight, MB49 cells (1 × 105 cells/well in 96-well microplates) and exposing them to 200 μl of a solution of complete DMEM and TPG at different concentrations (0 [negative control], 6.25 [TPG 6.25], 12.5 [TPG 12.5], 25 [TPG25], and 50% [TPG50]) for 24 h. After the period of incubation, the resulting cells were washed with phosphate-buffered saline (PBS 1X) and the cell viability test was performed by adding 20 µL/well of resazurin (70 µmol/L solution diluted in PBS 1X, Sigma-Aldrich, USA) to fresh DMEM without SFB. The absorbance was measured in a spectrophotometer after 3 h of metabolization of resazurin in resorufin using a spectrophotometer with wavelengths of 570 and 600 nm as reference, following the methodology described by Borra et al. (2009) . The cell without treatment was used as a negative control with 100% viability and the cell treated with 1% triton X for 10 min before the quantification, as a reference to 0% viability.
To eliminate the gel interference in the viability measurement, a new assay with cell removal after TPG exposure was performed. In this new assay, MB49 cells were initially cultured overnight in 6-well microplates, until they reach 90% confluence, and exposed to solutions containing 25 and 50% TPG in complete DMEM for 24 h. Next, the adherent cells were removed from the surface of the wells by trypsinization (2.5%, Life Technologies, Carlsbad, USA), diluted in 5 mL of DMEM with 10% of FBS, centrifuged at 200 g, resuspended in 200 μL and re-plated in 96-well microplates for another 24 h. Following this time period, the viability was quantified using the same resazurin protocols as before.
The influence of TPG on the proliferation capacity of tumor cells was investigated through the colony formation assay. Initially, 5 × 105 MB49 cells were cultured in 6-well microplates with 5 mL of high-glucose DMEM medium complemented with 2 mmol/L of L-glutamine (Cultilab), 10% FCS (Cultilab), and 1% penicillin/streptomycin (Vitrocell Embriolife).The microplate was incubated for 48 h at 37 °C in a 5% carbon dioxide atmosphere. When the cell monolayer reached 90% confluence, the supernatant was discarded, and the cells were exposed for another 24 h to 5 mL of a solution of complete DMEM and TPG at different concentrations (25 and 50%). After that, adherent cells were collected from the surface of the wells using trypsin (2.5%), washed/neutralized with DMEM + 10% FBS, centrifuged at 200 g, and resuspended in 1 mL of DMEM. The cell quantification of the control group (without exposure to TPG) was performed with 0.4% Trypan blue (Life Technologies) using a Neubauer chamber.
The cells from the control group (0 percent TPG) were diluted in a volume of complete DMEM determined to be able to culture 1 × 103 viable MB49 cells/well. Using 48-well microplates, the same volume was utilized to plate cells from the other TPG groups in quintuplicate. The microplates were kept in an incubator for 48 h until the colony-forming units were observed.
TPG interference in the development of the intravesical tumor model
Based on the absence of viability of MB49 cells, the concentration of 50% TPG (TPG50) was chosen to evaluate tumor induction interference. To compare the rate of tumor development, 3 groups of C57BL/6 female mice from Biotério Central of the University of São Paulo-Ribeirão Preto (USP-RP) were used: Group A1 (n = 20), submitted to intravesical inoculation of a solution containing 50 μL of cell suspension with 5 × 105 MB49 cells in DMEM and 50 μL of TPG (TPG50); Group A2 (n = 20), exposed to 100 μl of a solution containing 5 × 105 MB49 cells in DMEM, without any delivery system; Group A3 (n = 10) submitted to intravesical inoculation of a solution containing 50 μL of DMEM and 50 μL of TPG (TPG50) without MB49 cells in order to observe possible side effects promoted by the gel to the animals. All procedures were approved by the Ethics Committee on Animal Use of The Federal University of São Carlos (CEUA/UFSCar, n◦ 9234221018 and 4556170619). Before induction procedures, mice were anesthetized intraperitoneally with ketamine (90 mg/kg, Dopalen®, Ceva, Paulínia, Brazil) and xylazine (10 mg/kg, Anasedan®, Ceva). Transurethral catheterization was performed with a needleless 24G polyethylene intravenous catheter (0.7 × 19 mm) lubricated with petroleum jelly (Vaseline®, Unilever, London, England). After emptying the bladder, a traumatic injury to the bladder epithelium was performed with a catheter needle with a curved tip (5 to 7 degrees), inserted into the catheter and rotated 5 times in order to promote tumor induction . Catheterized animals were kept in the dorsal decubitus positioning for 45 min with the syringe attached to the catheter. Intravesical inoculations were performed randomly among the animals and, subsequently to the inoculation experiment, they were kept in separate boxes according to their group. Lesions were blindly induced in the animals by an operator who had no idea which group the animals belonged to. The tumor induction rate was determined by following the evolution of the lesions for up to 50 days. During this period, the appearance of indicative signs of tumor presence, such as pain behavior, hematuria, palpable abdominal mass or recurrent weight loss were evaluated. After 50 days, all remaining animals were euthanized by an overdose of chemical anesthetics (ketamine, 270 mg/kg, and xylazine, 30 mg/kg) and the bladders were examined for signs of tumor damage.
Immunogenic effect evaluation of the TPG50:MB49
Based on the absence of tumor induction, the 50% concentration of TPG (TPG50) was chosen to serve as an adjuvant in the immunization with non-viable MB49 cells. For this, 54 mice were randomly divided (www.random.org) into three groups: Group B1 (n = 18), submitted to intravesical inoculation of a solution containing 50 μL of cell suspension with 1 × 105 MB49 cells in DMEM and 50 μL of TPG (TPG50); Group B2 (n = 18), submitted to intravesical inoculation of a solution containing 50 μL of DMEM and 50 μL of TPG (TPG50), without MB49 cells; Group B3 (n = 18), exposed to 100 μl of a solution containing only DMEM without any delivery system or tumor cells. Before exposure, the animals were anesthetized and a traumatic injury to the bladder epithelium was performed according to the methodology described above.
Twenty one days after the initial inoculation, three animals from each group were separated to collect the splenocytes and the rest (n = 15) were submitted to the induction procedures of the orthotopic syngeneic tumor model of urothelial bladder cancer, in which a solution containing 1 × 105 MB49 cells in DMEM were inoculated into the bladder after anesthesia and traumatic injury to the bladder epithelium.
Effect of TPG50:MB49 immunization on tumor development
To determine post-challenge tumor development, the animals were kept under daily observation for up to 35 days. Tumor development was assessed through the appearance of indicative signs of tumor presence such as pain behaviors, hematuria, increase in abdominal volume, or recurrent weight loss. As soon as the animals showed any of the indicative signs, they were euthanized and the bladders were removed to quantify the tumor mass, thus avoiding unnecessary suffering to the animals. After euthanasia, the abdominal cavity, liver, spleen, and kidneys were examined looking for metastases. Before the bladder was removed, it was examined for signs of a tear in the muscle layer. The difference between tumor tissue and normal bladder tissue is often easy to identify since the tumor mass is well vascularized. The collected bladder tissue samples were washed in 0.9% saline solution, dried and weighed. Tumor growth rate values were calculated by dividing the bladder weight by the value in days from induction to euthanasia of the animals.
TPG50:MB49 immunization effect on the antitumor response of splenocytes
Isolation of murine splenocytes
The splenic tissues of three animals from groups B1, B2 and B3 (n = 9), which did not undergo tumor induction, were collected. Splenic tissues were compressed using the rough surface of two frosted microscope slides and the splenic homogenate obtained was washed with RPMI 1640 medium (LGC Biotecnologia, Cotia, Brazil) and resuspended with syringes and needles of decreasing size (18 G, 22 G and 26 G, respectively), until a homogeneous solution was obtained. Splenic homogenates were centrifuged at 200 g for 10 min and resuspended in 360 μL of filtered distilled water for 10 s in order to promote hemolysis, followed by the addition of 40 μL of 10 × filtered PBS. The centrifugation procedure, followed by the hemolysis procedure, was repeated three times. Splenic samples from each group were plated in pools using 6-well microplates containing 5 ml of RPMI 1640 culture medium (LGC Biotecnologia), supplemented with 0.05 mM β-mercaptoethanol (Sigma-Aldrich), 10% FBS (Cultilab) and 1% penicillin/streptomycin antibiotics (Vitrocell Embriolife). After 2 h of cultivation, the supernatant from each well was collected and transferred to a new plate. The viability of the splenocytes were evaluated using 0.4% trypan blue (Life Technologies) and the splenic samples were kept in an incubator overnight until the co-culture assay.
Co-culture assay of tumor cells and splenocytes
For the co-culture assay, MB49 tumor cells labeled with green fluorescent protein (MB49:GFP+) were plated in 96-well microplates (5 × 104 cells/well) containing 200 μl of complete DMEM, according to Andrade et al. (2010) . The MB49:GFP+ cell culture was kept in an incubator overnight and then used for co-culture with the splenocytes in the proportions of 1:10 and 1:50. This co-cultivation was carried out in quadruplicate and using complete 200 μl of DMEM without phenol red (Thermo Fisher Scientific, Waltham, USA). To determine the co-cultivation cell viability, the co-culture was kept in an incubator and, after 24 h, it was analyzed using the fluorescence system ImageXpress® Micro XLS (Molecular Devices, San Jose, USA), . The cell viability of the co-culture was also analyzed using the resazurin assay as described above.
Quantitative data were analyzed using the one-way or two-way ANOVA statistical and Tukey`s tests using the Prism® software (version 5.0, GraphPad Software, San Diego, USA). The results were expressed as mean ± standard error of the mean (SEM) and, in all analyses, 5% was adopted as the statistical significance limit (p < 0.05). In cases where the results did not follow normality, the data were analyzed using the Kruskal–Wallis and Dunn’s multiple comparison tests. The survival analysis was performed by the Kaplan Mier method associated with the Gehan-Breslow-Wilcoxon test.
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