Effects of intravesical therapy with platelet-rich plasma (PRP) and Bacillus Calmette-Guérin (BCG) in non-muscle invasive bladder cancer

Tissue and Cell 52 (2018) 17–27

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Effects of intravesical therapy with platelet-rich plasma (PRP) and Bacillus Calmette-Guérin (BCG) in non-muscle invasive bladder cancer

T

Lara Paro Diasa, Ângela C. Malheiros Luzob, Bruno B. Volpeb, Marcela Durána, ⁎ Sofia E.M. Galdamesc, Luiz A.B. Ferreirad, Nelson Duráne,f, Wagner J. Fávaroa,e, a

Laboratory of Urogenital Carcinogenesis and Immunotherapy, Department of Structural and Functional Biology, University of Campinas (UNICAMP), Campinas, SP, Brazil b Public Umbilical Cord Blood Bank, Haematology Hemotherapy Center/INCT do Sangue, University of Campinas (UNICAMP), Campinas, Brazil c Department of Engineering of Materials and Bioprocesses, School of Chemical Engineering, University of Campinas (UNICAMP), Campinas, SP, Brazil d Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, SP, Brazil e NanoBioss, Institute of Chemistry, University of Campinas (UNICAMP), Campinas, SP, Brazil f Nanomedicine Research Unit (Nanomed), Federal University of ABC (UFABC), Santo André, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Platelet-rich plasma Bladder cancer Immunotherapy Bacillus Calmette-Guerin

This study describes the effects of a promising therapeutic alternative for non-muscle invasive bladder cancer (NMIBC) based on Bacillus Calmette-Guerin (BCG) intravesical immunotherapy combined with Platelet-rich plasma (PRP) in an animal model. Furthermore, this study describes the possible mechanisms of this therapeutic combination involving Toll-like Receptors (TLRs) 2 and 4 signaling pathways. NMIBC was induced by treating female Fischer 344 rats with N-methyl-N-nitrosourea (MNU). After treatment with MNU, the animals were distributed into four experimental groups: Control (without MNU) group, MNU (cancer) group, MNU + PRP group, MNU + BCG group and MNU + PRP + BCG group. Our results demonstrated that PRP treatment alone or associated with BCG triggered significant cytotoxicity in bladder carcinoma cells (HTB-9). Animals treated with PRP associated to BCG clearly showed better histopathological recovery from the cancer state and decrease of urothelial neoplastic lesions progression in 70% of animals when compared to groups that received the same therapies administered singly. In addition, this therapeutic association led to distinct activation of immune system TLRs 2 and 4-mediated, resulting in increased MyD88, TRIF, IRF3, IFN-γ immunoreactivities. Taken together, the data obtained suggest that interferon signaling pathway activation by PRP treatment in combination with BCG immunotherapy may provide novel therapeutic approaches for non-muscle invasive bladder cancer.

1. Introduction Bladder cancer (BC) is an important type of cancer worldwide. The European Association of Urology considers BC as the eleventh most common cancer diagnosed worldwide (Babjuk et al., 2016; Witjes et al., 2016). In this year (2017), the American Association for Cancer estimated that approximately 79,030 new cases would be diagnosed in the United States (60,490 and 18,540 cases in men and women, respectively) with deaths in the USA by BC may reach approximately 16,870

(12,240 men and 4630 women) (American Cancer Society, 2017). The initial therapy for superficial bladder cancer (non-muscle invasive bladder cancer – NMIBC) is complete macroscopic eradication by transurethral resection of the bladder tumour (TURBT) (Askeland et al., 2012). Since there is a considerable risk of recurrence and/ or progression of non-muscle invasive bladder tumours after TURBT, intravesical adjuvant therapy with Bacillus Calmette-Guerin (BCG) is recommended for all stages: pTis (flat carcinoma in situ), pTa (papillary carcinoma in situ) and pT1 (high-grade urothelial cancer invading the

Abbreviation: BC, bladder cancer; BCG, Bacillus Calmette-Guerin; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; EGF, epidermal growth factor; GFs, growth factors; HGF, hepatocyte growth factor; HRP, horseradish peroxidase; IFN-γ, interferon-gamma; IGF, insulin growth factor; IL, interleukin; IRF-3, interferon regulatory factor 3; MNU, n-methyl-Nnitrosourea; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor-κB; NMIBC, non-muscle invasive bladder cancer; PDGF, platelet-derived growth factor; PRP, platelet-rich plasma; pT1, high-grade urothelial cancer invading the lamina propria; pTa, papillary tumor; pTis, flat carcinoma in situ; TGF-β, transforming growth factor; TLR, toll-like receptor; TNF-α, tumor necrosis factor α; TRIF, TIR-domain-containing adapter-inducing interferon-β; VEGF, vascular endothelial growth factor ⁎ Corresponding author at: Laboratory of Urogenital Carcinogenesis and Immunotherapy, Institute of Biology, University of Campinas (UNICAMP), Avenida Bertrand Russell, s/n., 13083-865, Campinas, SP, Brazil. E-mail address: [email protected] (W.J. Fávaro). https://doi.org/10.1016/j.tice.2018.03.011 Received 12 October 2017; Received in revised form 21 March 2018; Accepted 22 March 2018 Available online 23 March 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved.

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proliferation. Thus, considering the numerous attempts to associate BCG intravesical immunotherapy with chemotherapeutic and immunomodulatory agents, as well as the promising results of PRP use in tissue repair, the present study describes the effects of a promising therapeutic alternative for NMIBC based on BCG intravesical immunotherapy combined with PRP in an animal model for NMIBC. Furthermore, this study describes the possible mechanisms of this therapeutic combination involving TLRs 2 and 4 signaling pathways.

lamina propria) (Askeland et al., 2012). BCG acts as a stimulant of the reticuloendothelial system, causing a local inflammatory process with the infiltration of granulocytes, macrophages and lymphocytes. As a result, there is an enhancement of both phagocytosis and ratio of T helper/suppressor cells (Ratliff et al., 1993). Followed by the mentioned events, many cytokines are produced, among them interleukins (IL-1, IL-2, IL-6, IL-8, IL-10, IL-12), Tumor Necrosis Factor α (TNF-α), interferon (IFN), the intercellular adhesion molecule, etc (Jackson et al., 1995). The response triggered by Th1 is the predominant event, being responsible for tumor ablation (Gandhi et al., 2013). However, BCG treatment is associated with therapeutic failure in up to 50% of patients, and several side effects occur in up to 90% of patients (Douglass and Schoenberg, 2016; Chou et al., 2017; Packiam et al., 2017). The most frequent local side effects of the BCG therapy include dysuria, cystitis, urinary frequency and macroscopic haematuria, while systemic side effects include malaise and fever, as well as major complications such as sepsis and death can occur in some cases (Douglass and Schoenberg, 2016; Chou et al., 2017; Packiam et al., 2017). The treatment of NMIBC remains a challenge in the pharmaceutical field due the recurrence and progression of the disease, as well as the pronounced side effects still associated to the available therapeutic modalities. Although important strategies have been investigated in different preclinical studies and clinical trial phases, efficient and welltolerated approaches need to be developed in order to improve therapeutic efficacy and also the life quality of patients suffering from NMIBC. A reasonable outcome for NMIBC’ patients depends on many factors, including besides the characteristics of the cancer, also the technology and the anaesthetic approach, the ability of the surgeon and strong multidisciplinary aids. It was suggested that the future of NMIBC care will be integration of all these factors, in order to reduce healthcare costs and upgrade patients’ quality of life and willingness. All of these meaning that patients with the poorest prognosis will receive the most aggressive treatment, while low-risk patients will not be subjected to unnecessary procedures (Malmström et al., 2017). Immunotherapy is one of the approaches to cancer treatment (Laheru and Jaffee, 2005; Ott et al., 2017). In this context, compounds that modulate immune system, through Toll-like receptors (TLRs), could be a valuable strategy for the cancer treatment, whether used alone or in combination with existing therapies (Laheru and Jaffee, 2005; Garay et al., 2007). TLRs play key roles in innate immunity and their activation can trigger two different responses in tumors: they stimulate immune system to attack tumor cells and/or eliminate the inhibitory machinery to the immune system (Akira and Takeda, 2004; Takeda and Akira, 2004; Pradere et al., 2014; Zhao et al., 2014). Considering the importance of TLRs modulation in the treatment of tumors, including NMIBC, our research group investigated whether platelet-rich plasma (PRP) could exert some effect on modulation of these receptors. PRP is a platelet lysate, concentrated in a small volume of plasma, with the presence of several growth factors (GFs), such as: platelet-derived growth factor (PDGF); transforming growth factor (TGF-β); vascular endothelial growth factor (VEGF); epidermal growth factor (EGF), basic fibroblast growth factor (bFGF); insulin growth factor (IGF); hepatocyte growth factor (HGF); and cytokines (Sommeling et al., 2013; Lana et al., 2014; Cugat et al., 2015). It plays important actions in various healing/ tissue repair stages (Okuda et al., 2003; Cole et al., 2010; Lana et al., 2017). Hua et al. (2012) compared the conventional treatment for cervical ectopy (laser therapy) with a new treatment based on activated PRP gel in humans. The results showed same therapeutic efficacy for both treatments, but the degree of side effects was significantly lower in the PRP treatment. The exact mechanism of PRP in squamous reepithelialization of cervical ectopy is unclear. However, these same authors hypothesized that reepithelialization of cervical ectopy was attributed to GFs presence in the PRP, such as PDGF, TGF-β, IGF-1, FGF, VEGF, which act as regulatory agents, stimulating chemotaxis and cellular differentiation and

2. Materials and methods 2.1. PRP preparation The peripheral blood of 4 healthy human volunteer, aged above 18 years, was collected to obtain PRP according to protocol described by Perez et al. (2013). Each healthy human volunteer did not use drugs within 72 h prior to collection in order to avoid the influence of drugs on the individual’s platelet production. The peripheral blood from each human volunteer was collected using tubes of 3.5 mL (Vacuette - REF 454327, Greiner Bio-One GmbH, Austria) containing 0.5 mL of 3.2% sodium citrate (w/v) as anticoagulant (blood/sodium citrate 9:1). Immediately after collection, the peripheral blood was centrifuged at 100 × g for 10 min at 25 °C (Routine 380R, Hettich Zentrifugen, Munich, Germany). After centrifugation, the blood was separated into three layers: supernatant, intermediate layer and pellet (Fig. 1). The supernatant, which has the highest concentration of platelets, was collected using a 200 μL micropipette, transferred in a dry collection tube (Fig. 1) and kept on ice until administration to the animals. Samples of whole blood and PRP (obtained after centrifugation) were characterized by counting white and red blood cells in haematological counter (ABX Micron ES 60, Horiba Medical, Montpellier, France) (Fig. 1). The use of human volunteers in this study was approved by the Human Research Ethics Committee – UUNICAMP (CAAE number: 51774515.0.0000.5404). 2.2. In vitro cytotoxicity analysis using the MTT assay In order to evaluate cell viability in presence of PRP, BCG and PRP + BCG treatments, MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazoline bromide) method was performed (Mosmann, 1983). Due to test being carried out, a cell line of Homo sapiens urinary bladder grade II carcinoma 5637 (HBT-9) were obtained from the Rio de Janeiro cell bank and incubated at 37 °C, 5% CO2 in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg L sodium bicarbonate and fetal bovine serum to a final concentration of 10%. Then, the bladder grade II cells (HBT-9) were seeded in 96-well microplates at the density of 1.5 × 104 cells per well. Thereafter, PRP, BCG and PRP + BCG treatments were added to the wells at 5% concentration (50 μl per treatment). Subsequently, the microplates were incubated at 37 °C for 24 h in order to determine cell viability as a function of dose and timedependent effects from PRP, BCG and PRP + BCG treatments. After exposure of different treatments to the cells, the wells were washed with Phosphate Buffered Saline pH 7.4 (PBS) and a 0.5 mg/mL MTT solution (Sigma-Aldrich, USA) diluted in serum-free Roswell Park Memorial Institute medium (RPMI) was added. Thus, the cells were incubated for an additional 2 h at 37 °C. Finally, the culture medium was removed from the plate and 100 μl of dimethyl sulphoxide (DMSO) was added for dissolution of the formazan crystals. The plates were shaken for 10 min and the absorbance measured on microplate reader (Cytation 5, BioTek Instruments, Inc., USA) at λ = 570 nm. The values were expressed as percentages of reduction of MTT in relation to the control. 18

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Fig. 1. a–d: Preparation of PRP. (A) Peripheral blood was collected and transferred in specific collection tubes containing 0,5 mL of 3.2% sodium citrate as anticoagulant. (B) After centrifugation, the peripheral blood was separated into three layers: supernatant composed mainly of platelets and few white blood cells dispersed in the plasma; intermediate layer or buff coat composed of white blood cells; and pellet composed of red blood cells and other blood cells. (C) Supernatant was collected using a 200 μL micropipette, transferred in a dry collection tube and kept on ice until administration to the animals. (D) Samples of whole blood and PRP obtained after centrifugation were characterized by counting white and red blood cells in haematological counter.

2.3. NMIBC induction and treatment

2.4. Histopathological analysis

Sixty female Fischer 344 rats seven weeks old were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at the University of Campinas (UNICAMP). The animal experiments were approved by an institutional Committee for Ethics in Animal Use (CEUA/UNICAMP, protocol no. 3901-1). Prior to intravesical catheterisation with a 22-gauge angiocatheter, the rats were anesthetized with 10% ketamine (60 mg/kg, i.m.; Vibra®, Roseira, SP, Brazil) and 2% xylazine (5 mg/kg, i.m.; Vibra®, Roseira, SP, Brazil). The animals remained anesthetized for approximately 45 min after catheterization to prevent spontaneous micturition. Twenty animals were divided into two control groups (n = 10 animals per group): the Control group received 0.30 ml of 0.9% physiological saline, intravesically (I.V.), every other week for 14 weeks; and the Control + PRP group received 0.30 ml of 0.9% physiological saline, I.V., every other week for 8 weeks; after this group received 0.2 mL of PRP (corresponding to 328 × 103–549 × 103 platelets/mm3), I.V., for 4 consecutive weeks (Hua et al., 2012; Costanzo et al., 2015). NMIBC induction was carried out in 40 rats that received n-methyl-N-nitrosourea (MNU; 1.5 mg/kg, dissolved in 0.30 ml of 1 M sodium citrate, pH 6.0) intravesically every other week for eight weeks (Garcia et al., 2016). Two weeks after the last dose of MNU, all rats were submitted to ultrasonography to evaluate the occurrence of tumors. The ultrasounds were evaluated using a portable, software-controlled ultrasound system with a 10–5 MHz 38mm linear array transducer. MNU-treated rats were further divided into four groups (n = 10 per group): the MNU group received 0.30 ml of 0.9% physiological saline, I.V., for 4 consecutive weeks, the MNU + PRP group received 0.2 mL of PRP (corresponding to 328 × 103–549 × 103 platelets/mm3), I.V., for 4 consecutive weeks (Hua et al., 2012; Costanzo et al., 2015); the MNU + BCG group received 106 CFU (40 mg) of BCG, I.V., for 6 consecutive weeks (Garcia et al., 2016); and the MNU + PRP + BCG group received 0.2 mL of PRP (corresponding to 328 × 103–549 × 103 platelets/mm3), I.V., for 4 consecutive weeks (Hua et al., 2012; Costanzo et al., 2015); after PRP treatment, this group received 106 CFU (40 mg) of BCG, I.V., for 6 consecutive weeks, totalizing 10 consecutive weeks of treatment (Garcia et al., 2016). At the end of the treatments, the rats were killed and the urinary bladders were collected and processed for histopathological and immunohistochemistry analyses.

Samples of urinary bladders (n = 10 per group) were fixed in Bouin solution for 12 h and then washed in 70% ethanol, dehydrated in an increasing series of alcohols, cleared in xylene for 2 h and embedded in plastic polymer (Paraplast Plus, St. Louis, MO, USA). Subsequently, 5μm thick sections were cut on a rotary microtome (Slee CUT5062 RM 2165; Slee Mainz, Mainz, Germany), stained with hematoxylin-eosin and photographed with a Leica DM2500 photomicroscope (Leica, Munich, Germany). A senior uropathologist analyzed the urinary bladder lesions based on the criteria of the Health/World International Society of Urological Pathology Organization (Epstein et al., 1998). 2.5. Immunohistochemistry of toll-like receptor signaling pathway: (TLR2, TLR4, MyD88, TRIF, IRF-3 and IFN-γ) in NMIBC The same tissue samples (n = 10 per group) used for histopathological analysis were used for immunolabelings. The samples were cut into 5-μm thick sections and antigen was retrieved by boiling the sections in a 10 mM citrate buffer, pH 6.0, three times for 5 min each in a conventional microwave oven. The sections were subsequently incubated in 0.3% H2O2 to block endogenous peroxidase activity and nonspecific binding sites were blocked by incubating the sections in blocking solution at room temperature. The primary rabbit polyclonal anti-TLR2 (RRID:AB_2303458; 1:100), mouse monoclonal anti-TLR4 (RRID:AB_10611320; 1:100), rabbit polyclonal anti-MyD88 (RRID:AB_2146724; 1:100), rabbit polyclonal anti-TRIF (RRID:AB_2255834; 1:50), rabbit polyclonal anti-IRF-3 (RRID:AB_218160; 1:50) and mouse monoclonal anti-IFN-γ (RRID:AB_315493; 1:50) were diluted in 1% BSA and applied to the sections overnight at 4 °C. Bound antibody was detected with an Advance™ HRP kit (Dako Cytomation Inc., USA). The sections were lightly counterstained with Harris’ hematoxylin and photographed with a photomicroscope (DM2500 Leica). In order to evaluate the intensity of antigen immunoreactivity, the percentage of positive-staining urothelial cells was examined in ten fields for each antibody under high magnification (400×). The Image J software (https://imagej.nih.gov/ij/) was employed for this analysis. The staining intensity was graded on a scale of 0–3, and expressed as 0 (no immunoreactivity), 0% positive urothelial cells; 1(weak immunoreactivity), 1–35% positive urothelial cells; 2 (moderate immunoreactivity), 36–70% positive urothelial cells; 3 (intense immunoreactivity), > 70% positive urothelial cells (Garcia et al., 2015). 19

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3.2. PRP treatment associated with BCG immunotherapy decreased neoplastic lesions progression induced by MNU carcinogen The Control and Control + PRP groups showed no histological changes in bladder tissue (Fig. 3a–d ; Table 1). Three different cell types composed the normal bladder urothelium: basal cell layer, intermediate cell layer and surface cell layer (umbrella cells) (Fig. 3a–d). In addition, moderate infiltrate of inflammatory cells was observed in the urothelium and lamina propria of the rats from Control + PRP group (Fig. 3c and d). In contrast, MNU group showed deep microscopic changes in the urinary bladder tissue such as pT1 (Fig. 3e, f), pTa (Fig. 3g) and pTis (Fig. 3h) in 40%, 40% and 20% of the rats, respectively (Table 1). The pT1 carcinoma was characterized by tumor cells invading the lamina propria, numerous mitotic figures and pleomorphic cells with enlarged nuclei (Figs. 3e, f, 4 f). The pTa tumor was characterized by cancer cells showing slender papillae with frequent branching, minimal fusion, and variations in nuclear polarity, size, shape, and chromatin pattern and with the presence of nucleoli (Figs. 3g, 4b, c). The pTis carcinoma was characterized by flat lesion in the urothelium surface, showing large and pleomorphic cells, severe nuclear atypia and loss of cellular polarity (Figs. 3h, 4a). The most frequent neoplastic lesions in the MNU + BCG group were pTis (Fig. 4a) and pTa (Fig. 4b) in 50% and 30% of the animals, respectively (Table 1). Furthermore, flat hyperplasia (benign lesion) and low-grade intraurothelial neoplasia (preneoplastic lesion) were found in 10% and 10% of the rats, respectively (Table 1), indicating that this immunotherapy promoted inhibition of tumor progression in 20% of the animals (Table 1). The intravesical treatment with PRP alone showed decrease of bladder neoplastic lesions progression in 30% of the animals (Table 1). Normal bladder tissue morphology and flat hyperplasia were found in 10% and 10% of the animals, respectively (Table 1). Preneoplastic lesions, such as low-grade intraurothelial neoplasia (Fig. 4d), were found in 10% of the rats (Table 1). The most frequent neoplastic lesions found in this group were pTa (Fig. 4c) and pTis in 50% and 20% of the animals, respectively (Table 1). Low-grade intraurothelial neoplasia was characterized by thickening of the urothelium and presence of few atypical urothelial cells, without loss of cell polarity (Fig. 4d). Animals treated with PRP associated to BCG immunotherapy clearly showed better histopathological recovery from the cancer state than those observed in the MNU + PRP and MNU + BCG groups, showing decrease of bladder neoplastic lesions progression in 70% of the animals (Table 1). Normal bladder tissue morphology was found in 20% of the animals (Table 1). Furthermore, flat hyperplasia (Fig. 4e) and lowgrade intraurothelial neoplasia were found in 40% and 10% of the animals, respectively (Table 1). The most frequent neoplastic lesions found in this group were pTis, pTa and pT1 (Fig. 4f) in 10%, 10% and 10% of the animals, respectively (Table 1). Benign lesions, such as flat hyperplasia were characterized by thickening of the urothelium without cellular atypia (Fig. 4e).

Fig. 2. a–b: (a) Graph demonstrating the number of platelets (Platelets/ mm3) in both whole blood and concentrated PRP samples. (b) Graph demonstrating bladder carcinoma cells (HTB-9) viability after PRP 5%, BCG 5% and PRP + BCG 5% treatments. Different lowercase letters (a, b) indicate significant differences (p < 0.05) between the groups after the Tukey test.

2.6. Statistical analyses Quantitative results were expressed as the mean ± standard deviation whenever possible. In vitro cytotoxicity analysis was compared among groups by one-way analysis of variance (ANOVA) followed by the Tukey test, with the level of significance set at 5% (p < 0.05). Histopathological and Immunohistochemistry results were compared with a proportion test. The difference between the two proportions was tested using test of proportion with a type-I error of 1%.

3. Results 3.1. Methods of preparation of PRP and platelet recovery were effective and decreased cell viability in bladder grade II carcinoma cells (HBT-9) The PRP methodology performed were able to significantly concentrate a large number of platelets, being 2–3 times higher in PRP samples than whole blood (Fig. 2a). The mean values of platelet concentrate were between 328 × 103/mm3 and 549 × 103/mm3 (Fig. 2a). Also, mean values of platelet concentrate did not differ significantly among healthy human volunteer (Fig. 2a). The obtained PRP were viable and cytotoxic to bladder carcinoma cells (HBT-9) (Fig. 2b). PRP treatment alone or associated with BCG, at 5% concentration, demonstrated important cytotoxicity in bladder carcinoma cells (HBT-9), with approximately 35% and 32%, respectively, inhibition of cell HTB-9 proliferation when compared to BCG treatment alone (100% cell HTB-9 proliferation) (Fig. 2b). Also, BCG treatment was not cytotoxic to HTB-9 cells (Fig. 2b).

3.3. PRP treatment associated with BCG immunotherapy modulated TLR2 and 4, and increased interferon signaling pathway immunoreactivities TLR2 and 4 immunoreactivities were significantly intense in the Control group (Fig. 5a, d) when compared to Control + PRP (Fig. 5b, e), MNU + BCG (Fig. 6a, d) and MNU + PRP (Fig. 6b, e) group, which showed moderate immunoreactivities (Table 2). In response to PRP plus BCG treatment, these immunoreactivities were significantly intense than either treatment alone (Fig. 6c, f; Table 2). In addition, TLR2 and 4 immunoreactivities were weak in the MNU group (Fig. 5c, f; Table 2). Intensified MyD88 immunoreactivities were found in the Control group (Fig. 5g; Table 2) in relation to MNU group, which showed weak immunoreactivities (Fig. 5i; Table 2). The animals treated with PRP alone (Control + PRP and MNU + PRP groups) exhibited moderate 20

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Fig. 3. a–h: Representative photomicrographs of urinary bladder from Control (a, b), Control + PRP (c, d), and MNU (e, f, g, h) groups. (a)–(d) Three different cell types composed the normal bladder urothelium: basal cell layer (arrowhead), intermediate cell layer (arrow) and surface cell layer (or umbrella cells, open arrowhead). (c), (d) Inflammatory infiltrate (asterisks) in the urothelium and lamina propria. (e), (f) pT1 tumor: cancer cells (arrows) invading the lamina propria. (g) pTa tumor: cancer cells show slender papillae with frequent branching, minimal fusion, and variations in nuclear polarity, size, shape, and chromatin pattern and with the presence of nucleoli. (h) pTis tumor: flat lesion in the urothelium surface characterized by large and pleomorphic cells, severe nuclear atypia (arrows) and loss of cellular polarity. a–h: Lp – lamina propria, M – muscle layer, Ur – urothelium.

that received combined treatment with PRP and BCG when compared to other experimental groups (Fig. 6r; Table 2). The animals from Control, Control + PRP, MNU + BCG and MNU + PRP groups showed moderate IFN-γ immunoreactivities (Figs. 5 and 6p, q,) in relation to MNU group (Fig. 5r), which exhibited weak immunoreactivities (Table 2).

immunoreactivities for this antigen (Figs. 5 and 6h, Table 2). Furthermore, BCG treatment alone (Fig. 6g) or combined with PRP (Fig. 6i) increased MyD88 immunoreactivities (Table 2). The animals from Control and Control + PRP groups showed intense and moderate TRIF and IRF3 immunoreactivities, respectively (Fig. 5j, k, m, n) in relation to MNU group (Fig. 5l, o), which exhibited weak immunoreactivities (Table 2). The animals from MNU + BCG (Fig. 6j, m) and MNU + PRP (Fig. 6k, n) exhibited moderate immunoreactivities for theses antigens (Table 2). The combined treatment with PRP and BCG was able to increase TRIF and IRF3 immunoreactivities (Fig. 6l, o; Table 2). Intensified IFN-γ immunoreactivities were verified in the animals

4. Discussion and conclusions The growth factors (GFs) present in PRP are released from the alpha granules of activated platelets and their therapeutic effects have been extensively studied in nerves and nerve cell regeneration (Küçük et al., 21

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Table 1 Histopathological changes in the urinary bladder of rats from the Control, Control + PRP, MNU, MNU + BCG, MNU + PRP and MNU + PRP + BCG groups. Histopathology

Normal Flat hyperplasia Low-grade intraurothelial neoplasia Flat carcinoma in situ (pTis) Papillary carcinoma in situ (pTa) high-grade urothelial cancer invading the lamina propria (pT1)

Groups Control (n = 10)

Control + PRP (n = 10)

MNU (n = 10)

MNU + BCG (n = 10)

MNU + PRP (n = 10)

MNU + PRP + BCG (n = 10)

10(100%)* – – – – –

10(100%)* – – – – –

– – – 2(20%) 4(40%) 4(40%)*

– 1(10%) 1(10%)* 5(50%)* 3(30%) –

1(10%) 1(10%) 1(10%)* 2(20%) 5(50%)* –

2(20%) 4(40%)* 1(10%)* 1(10%) 1(10%) 1(10%)

The histopathological alterations are expressed as a percentage of the number of rats (n) examined in each group. * P < 0.0001 (proportions test). Benign lesions: flat hyperplasia; Preneoplastic lesions: low-grade intraurothelial neoplasia; Neoplastic lesions: pTis, pTa and pT1. Fig. 4. a–f: Representative photomicrographs of urinary bladder from MNU + BCG (a, b), MNU + PRP (c, d) and MNU + PRP + BCG (e, f) groups. (a) pTis tumor: flat lesion in the urothelium surface characterized by large and pleomorphic cells, severe nuclear atypia (arrows) and loss of cellular polarity. (b), (c) pTa tumor: cancer cells show slender papillae with frequent branching, minimal fusion, and variations in nuclear polarity, size, shape, and chromatin pattern and with the presence of nucleoli. (d) Low-grade intraurothelial neoplasia (circle) characterized by thickening of the urothelium and presence of few atypical urothelial cells, without loss of cell polarity. (e) Flat hyperplasia (circle) characterized by thickening of the urothelium without cellular atypia. (f) pT1 tumor: cancer cells (arrows) invading the lamina propria. a–f: Lp – lamina propria, M – muscle layer, Ur – urothelium.

higher when compared to blood from rats. Kim et al. (2017) reported a series of studies with human PRP applied in animal models and demonstrated that there is no immunogenic response in animals receiving heterologous PRP. The method proposed by Perez et al. (2013) and performed in this study was reproducible, being possible to obtain platelet-rich and leukocyte-poor plasma, with mean values of platelet concentrate between 328 × 103/mm3 and 549 × 103/mm3. These values were similar to the values found by Semeraro et al. (2007).

2014), tendons (Foster et al., 2009; Dallaudière et al., 2013; Kim et al., 2017), ligaments (Foster et al., 2009), endometriosis (Marini et al., 2016), erosive-ulcerative lesions (Anandan et al., 2016), orthopedic surgeries and articular cartilages repair (Foster et al., 2009). Here, our results demonstrated a new application of PRP in the oncology field, specifically in the NMIBC treatment. In the present study, the choice of obtaining heterologous PRP from human blood is related to volume of blood drawn, being 4–5 times

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Fig. 5. a–r: Immunolabelled antigen intensities of the urinary bladder from the Control (a, d, g, j, m, p), Control + PRP (b, e, h, k, n, q) and MNU (c, f, i, l, o, r) groups. (a), (b), (c) TLR2 immunoreactivities (asterisks) in the urothelium. (d), (e), (f) TLR4 immunoreactivities (asterisks) in the urothelium. (g), (h), (i) MyD88 immunoreactivities (asterisks) in the urothelium. (j), (k), (l) TRIF immunoreactivities (asterisks) in the urothelium. (m), (n), (o) IRF3 immunoreactivities (asterisks) in the urothelium. (p), (q), (r) IFN-γ immunoreactivities (asterisks) in the urothelium. a–r: Lp – lamina propria, Ur – urothelium.

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Fig. 6. a–r: Immunolabelled antigen intensities of the urinary bladder from the MNU + BCG (a, d, g, j, m, p), MNU + PRP (b, e, h, k, n, q) and MNU + PRP + BCG (c, f, i, l, o, r) groups. (a), (b), (c) TLR2 immunoreactivities (asterisks) in the urothelium. (d), (e), (f) TLR4 immunoreactivities (asterisks) in the urothelium. (g), (h), (i) MyD88 immunoreactivities (asterisks) in the urothelium. (j), (k), (l) TRIF immunoreactivities (asterisks) in the urothelium. (m), (n), (o) IRF3 immunoreactivities (asterisks) in the urothelium. (p), (q), (r) IFN-γ immunoreactivities (asterisks) in the urothelium. a–r: Lp – lamina propria, Ur – urothelium.

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Table 2 Immunolabelled antigen intensities of the urinary bladder of rats in the different experimental groups. Antigens

TLR2 TLR4 MyD88 TRIF IRF3 IFN-γ

Groups Control (n = 10)

Control + PRP (n = 10)

MNU (n = 10)

MNU + BCG (n = 10)

MNU + PRP (n = 10)

MNU + PRP + BCG (n = 10)

3(95.9%)* 3(96.8%)* 3(94.0%)* 3(88.1%)* 3(82.7%)* 2(51.4%)

2(63.2%) 2(66.5%) 2(48.0%) 2(51.1%) 2(41.0%) 2(43.8%)

1(17.0%) 1(23.5%) 1(15.0%) 1(19.6%) 1(11.2%) 1(18.0%)

2(65.2%) 2(61.4%) 3(77.9%)* 2(65.8%) 2(41.2%) 2(61.1%)

2(56.3%) 2(57.1%) 2(49.6%) 2(44.8%) 2(51.0%) 2(44.7%)

3(85.6%)* 3(89.7%)* 3(89.0%)* 3(85.3%)* 3(85.2%)* 3(77.9%)*

0 (no immunoreactivity), 0% positive urothelial cells; 1(weak immunoreactivity), 1–35% positive urothelial cells; 2 (moderate immunoreactivity), 36-70% positive urothelial cells; 3 (intense immunoreactivity), > 70% positive urothelial cells.

show decreased TLRs expression (Ayari et al., 2011; LaRue et al., 2013; Stopiglia et al., 2015). The BCG antitumor effects in the NMIBC are related to local immunological mechanisms, since after BCG instillation were found in the urine of patients within 24 h several cytokines and activated immunocompetent leukocytes (LaRue et al., 2013). In a series of experiments done by our group using the same animal model to study NMIBC, BCG increased TLR2 and TLR4 protein levels, resulting in activation of MyD88 and NF-κB, with a consequent increase in inflammatory cytokines (TNF-α) (Fávaro et al., 2012; Garcia et al., 2015, 2016). In these studies, the induction of MyD88-dependent pathway by BCG was not effective in reducing urothelial neoplastic lesions progression. Rivadeneyra et al. (2014) demonstrated that platelets expressed TLRs and triggered immune responses by NF-κB activation. TLR4 signaling pathway induces IFN-γ production that has antitumor effects by induction of TNF-related-apoptosis-inducing ligand (TRAIL), a potent inducer of tumor cell death (Luo et al., 2004). Shankaran et al. (2001) showed that tumor suppressor effect of the immune system is depend on the actions of IFN-γ, which module tumorcell immunogenicity. IFN-γ stimulates different antiproliferative and antitumor biochemical pathways in both macrophages and tumor cells, as well as reduces solid tumors growth and metastasis (Li et al., 2007; duPre’ et al., 2008; Martini et al., 2010; Alshaker and Matalka, 2011; Tate et al., 2012). IFN-γ produced by IL-12-activated tumor-infiltrating CD8 + T cells induced apoptosis of mouse hepatocellular carcinoma cells (Komita et al., 2006; Martini et al., 2010). IFN-γ produced by infiltrating T cells in the tumor microenvironment can activate both tumor T cells and also generate inducible nitric oxide synthase (iNOS) (Beatty and Paterson, 2001; Shankaran et al., 2001). Nitric oxide (NO) is considered one of the main factors responsible for cytotoxic activity of macrophages against tumor cells (Koskela et al., 2012; Tate et al., 2012). NO can stimulate cell growth and cell differentiation when present at low concentrations, whereas high concentrations often result in cytotoxic effects (Koskela et al., 2012). Previous studies showed that increased NO concentrations in the urinary bladder from BCG-treated patients suggest that NO is a critical factor in the BCG-mediated antitumor effect (Hosseini et al., 2006; Andrade et al., 2010; Koskela et al., 2012). We have demonstrated here that chemically induced bladder tumors showed weak TLRs 2 and 4 signaling pathways immunoreactivities. In addition, the PRP or BCG treatments alone were not able to restore TLRs 2 and 4 immunoreactivities. However, PRP treatment associated with BCG immunotherapy led to distinct activation of immune system TLRs 2 and 4-mediated, resulting in increased MyD88, TRIF, IRF3, IFNγ immunoreactivities. Thus, the activation of interferon signaling pathway by this therapeutic association was more effective in reducing urothelial neoplastic lesions progression. Martin et al. (2013) demonstrated that a single dose of PRP was ineffective in modulating inflammation and promoting structural changes of tissues and organs. Thus, to ensure efficient modulation of the TLRs in the urothelium, we used 4 vesical instillations of PRP, which were effective in regulating TLRs signaling pathways induced by

Platelets play an active role in innate and adaptive immunity through adhesive interactions with leukocytes and endothelial cells via P-selectin, which may lead to proinflammatory events, including leukocytes activation, cytokines cascade production and recruitment of leukocytes to sites of tissue injury (Semple and Freedman, 2010). In addition, studies have shown that both murine and human platelets can express TRL2, TLR4 and TLR9 (Semple and Freedman, 2010). Platelet expression of TLR4 mediates LPS-induced thrombocytopenia and the production of TNF-α by leukocytes, indicating that these events could be responsible for innate immune system activation (Semple and Freedman, 2010). In atherosclerotic disease, the adhesion of activated platelets to the endothelium releases proinflammatory molecules and cytokines, such as IL-1β and CD40L, cytokine CCL5/ RANTES and platelet factor 4, which are deposited in the vascular endothelium by a P-selectin-dependent process, with consequent recruitment of monocytes to the lesion site (Semple and Freedman, 2010). Furthermore, Jiang et al. (2017) demonstrated that association of BCG with an Escherichia coli maltose-binding protein (MBP) stimulated TLRs 2 and 4 through activating MHC class II molecules in dendritic cells, promoting the activation of Th1 cells. The association of MBP and BCG exerted a synergistic effect, increasing the production of lymphocytes in relation to MBP and BCG treatments alone. Similarly, our results demonstrated that PRP treatment associated to BCG led to an additive effect, promoting inhibition of tumor progression in both in vivo and in vitro experiments. PRP treatment alone or associated with BCG triggered significant cytotoxicity in bladder carcinoma cells (HTB9) when compared to BCG treatment alone, which was not cytotoxic to these cells. The BCG intravesical immunotherapy showed decrease of urothelial neoplastic lesions progression and histopathological recovery in 20% of chemically induced NMIBC animals. However, PRP treatment was more effective than BCG and reduced urothelial neoplastic lesions progression in 30% of animals. Animals treated with PRP associated to BCG clearly showed better histopathological recovery from the cancer state and decrease of urothelial neoplastic lesions progression in 70% of animals when compared to groups that received the same therapies administered singly. Compounds or molecules that bind to and activate TLRs are the subjects of deep research and development for the treatment of cancer, including NMIBC (LaRue et al., 2013; Garcia et al., 2015, 2016). Epithelial and immune cells express TLRs, which play important role in activating immune system (Lightfoot et al., 2011; Morales et al., 2015). TLRs signaling consists of two pathways: MyD88-dependent (canonical) and TRIF-dependent (non-canonical) pathways (Akira and Takeda, 2004; Takeda and Akira, 2004; Pradere et al., 2014; Zhao et al., 2014). Except for TLR3, the MyD88-dependent pathway activates NF-kB and MAPK, resulting in inflammatory cytokines release, such as TNF-α and IL-6 (Akira and Takeda, 2004; Takeda and Akira, 2004; Pradere et al., 2014; Zhao et al., 2014). Conversely, the TRIF-dependent pathway activates Interferon Regulatory Factor 3 (IRF3) for the production of interferon (Akira and Takeda, 2004; Takeda and Akira, 2004; Pradere et al., 2014; Zhao et al., 2014). Non-muscle invasive bladder tumors 25

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BCG. Thus, we could be concluded that growth factors present in PRP played an important role in the modulation of TLRs 2 and 4 signaling pathways induced by BCG, resulting in distinct activation of the immune system. The activation of interferon signaling pathway by PRP treatment associated with BCG immunotherapy was effective in reducing urothelial neoplastic lesions progression. Taken together, the data obtained suggest that interferon signaling pathway activation by PRP treatment in combination with BCG immunotherapy may provide novel therapeutic approaches for non-muscle invasive bladder cancer.

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