461 Background: Effective intravesical therapies remain lacking for non-muscle invasive bladder cancer (NMIBC) when Bacillus Calmette-Guerin fails. OncoTherad is a nanostructured inorganic phosphate complex associated with glycosidic protein, developed by the University of Campinas/Brazil, which triggers immunomodulatory and antitumor activities. Previous studies have shown that Platelet Rich Plasma (PRP) acts on immune activation and exerts antitumor effects. This study characterized the effects of the OncoTherad associated with PRP in the treatment of NMIBC chemically induced in mice and the modulation promoted in the Toll-like receptors (TLRs) signaling pathway. Methods: Forty-two C57BL/6J mice were divided into groups: Control; Cancer (N-ethyl-N-nitrosourea carcinogen, 50 mg/ml); PRP (0.1 ml); OncoTherad (20 mg/ml); OncoTherad+PRP 10 mg/ml and OncoTherad+PRP 20 mg/ml. The intravesical doses (0.1 ml) were instilled once a week for 6 weeks after induction. Results: The NMIBC induction decreases (p<0.05) body weight, although after treatments the body weight was recovered similarly to the healthy mice. The treatments did not significantly alter the biochemical patterns of the urine and food and water consumption. There was no acute toxicity or kidney damage, and the presence of hydroureter was variable. The urinary bladders of mice treated with Oncotherad associated or not with PRP showed hyperemia associated with the inflammatory condition. The thickening of the urinary bladder wall in the Cancer group was more evident than in the treated groups, in which there were bladders without thickening or macroscopic lesions. Flat carcinoma in situ (pTis) was present in 100% of the mice in the Cancer group and the intensity of immunoreactivities for TLR2, IL-6, TLR4, and IRF-3 was significantly weaker in comparison with the Control, indicating suppression of the immune system in the tumor microenvironment. When treated intravesically with PRP only, mice showed 28.6% of tumor progression inhibition rate; with OncoTherad 85.7% and with OncoTherad+PRP 10 mg/ml or 20 mg/ml 71.4%. Intravesical treatments led to distinct activation of TLRs 2 and 4-mediated innate immune system in the interleukins (MyD88-dependent) and interferons (TRIF-dependent) signaling pathways. The combined treatment of OncoTherad+PRP increased (p<0.05) the percentage of positive TLR4 urothelial cells and the intensity of immunoreaction for TLR4 compared to the isolated treatments and the immunoreactivities of NF-kB, IL-6, TLR4, IRF-3, and IFN-γ in comparison to the Cancer. Conclusions: Intravesical treatment with OncoTherad plus PRP promoted significant inhibition of tumor progression, possibly due to immunomodulatory activity involving the TLR pathway. This association can constitute a therapeutic strategy for refractory NMIBC patients.

Background: This study characterized and compared the molecular profiles of CX3C chemokine receptor 1 (CX3CR1, a marker of T-cell differentiation), Toll-like receptor 4 (TLR4)-mediated interferon signaling pathway, and immune checkpoints in the different histological stages of non-muscle invasive bladder cancer (NMIBC), aiming the investigation of these biomarkers as a criterion of clinical response to immunotherapy. Methods: Seventy-five formalin-fixed paraffin-embedded samples of bladder were obtained from 34 to 96-year-old patients (mean 65 years) with NMIBC diagnosis in University of Campinas (UNICAMP) and Paulinia Municipal Hospital/ Brazil. Subsequently, the samples were divided into 3 groups (n= 25 samples per group): pTis group, high-grade pTa group, and pT1 group; and submitted to immunohistochemistry analysis: TLR4-mediated IFN-γ production signaling pathway (TRIF, TBK1, IRF-3, IFN-γ), CX3CR1+CD8+ T-cells, immune checkpoints (PD-1/PD-L1 and CTLA-4), and regulatory T (Treg) cells (FOXP3). The retrospective anonymous study was approved by the local ethics committee (Clinical Trial: RBR-6swqd2). Results: pTis group showed the lowest activation of TLR4-mediated IFN-γ signaling pathway when compared (p<0.01) to high-grade pTa and pT1 groups. Both the immunoreaction intensity and positive cells percentage were lower (p<0.01) for TLR4, TRIF, IRF-3, and IFN-γ in the pTis group with respect to other groups. No statistical difference was found between high-grade pTa and pT1 groups for these biomarkers. Likewise, CX3CR1 immunoreactivities were remarkably lower (p<0.01) in the pTis group in comparison with high-grade pTa and pT1 groups, which did not show statistical differences between them. Furthermore, immune checkpoints (PD-1/PD-L1 and CTLA4) and FOXP3+ Treg cells immunoreactivities were significantly higher (p<0.01) in the high-grade pTa and pT1 compared to the pTis group. Conclusions: Our data demonstrated that pTis stage was characterized by an immunosuppressive microenvironment in comparison with pTa and pT1 stages, showing decreased TLR4-mediated interferon signaling pathway and low activation of CX3CR1+CD8+ T-cells; which implies in low sensitivity to immunotherapy. The larger number of FOXP3+ Treg cells in pTa and pT1 was correlated with intensified immune checkpoints immunoreactivities, indicating higher sensitivity to immunotherapy. Finally, these biomarkers may be useful in the clinical management of patients with NMIBC.

This study characterized the effects of the immunotherapy using the nanostructured complex OncoTherad, either associated or not to Platelet-Rich Plasm (PRP) on the non-muscle invasive bladder cancer (NMIBC) chemically induced in mice. The treatment with OncoTherad inhibited the cancer's progression and promoted tumoral regression in 85.7% and the OncoTherad associated with PRP in 71.4% of the animals. Thus, the immunotherapy using OncoTherad associated to PRP raises as a new therapeutic strategy for NMIBC, which allows the treatment's personalization and the reduction of the immunogenicity to patients who either show recurrence, are resistant to the therapy using Bacillus Calmette-Guérin (BCG) or are ineligible to the radical cystectomy.The immune system modulation and the relation of OncoTherad with the activated platelets demand continuity of studies to detail the mechanisms of this association.

e17602 Background: Ovarian cancer (OC) has the most devastating death rate of gynecological cancers with only 44% of women surviving 5 years after diagnosis. Toll-like receptors (TLRs) signaling can play an important role in the OC treatment. TLR activation in immune cells can help activate an effective antitumor response. In this scenario, a new perspective is represented by combined OncoTherad (MRB-CFI-1) immunotherapy and Platelet Rich Plasma (PRP). OncoTherad (MRB-CFI-1) leads to the distinct stimulation of the innate immune system mediated by TLR2 and TLR4, resulting in an increased activation of the IFN signaling pathway. Also, PRP acts on immune activation and exerts antitumor effects. This study characterized the histopathological and molecular effects of the OncoTherad associated with PRP in the treatment of OC chemically induced in rats and described the possible mechanisms of action of this association involving the TLRs 2 and 4 signaling pathways. Methods: Fischer 344 female rats were divided into 5 groups (n = 7 animals per group): Control (Sham surgery) group; Cancer (7,12 dimethylbenzoanthracene – DMBA injection in the ovarian bursa, 1.25 mg/kg) group; OncoTherad (20mg/kg) group; PRP (328.103 – 549.103 platelets/mm3) group and; OncoTherad+PRP (same doses of the isolated treatments) group. Intraperitoneal doses were administered twice a week for 4 weeks. Immunohistochemistry was analyzed as total immunoreactivity and intensity of immunoreaction. Results: Our results showed serous type ovarian carcinoma, follicular atresia, absence of corpora lutea and hyperplasia of the germinal epithelium and tunica albuginea in the Cancer group. The histopathological changes in the PRP group were similar as Cancer group. In contrast, the animals treated with OncoTherad singly or in combination with PRP showed decrease of ovarian neoplastic lesions and histopathological recovery. TLR4 and IL-6 immunoreactivities were significantly lower (p < 0.05) in the Cancer group. PRP treatment reduced (p < 0.05) TLR2. In the Oncotherad+PRP group, the immunoreactivities of TLR4, MyD88, IRF-3 and TNF-α were lower. OncoTherad treatment alone led to stimulation of the innate immune system mediated by TLR2 and TLR4, resulting in an increased activation of the MyD88 signaling pathway (intensified IL-6 and TNF-α immunoreactivities). Conclusions: OncoTherad immunotherapy was able to reduce ovarian lesions and stimulated the TLR2 and 4 signaling pathways in the OC microenvironment, leading to antitumor effects by activating the production of inflammatory cytokines. Thus, OncoTherad immunotherapy could be considered an important therapeutic strategy for OC.

e16611 Background: Bladder cancer (BC) is the most common malignancy of the urinary tract and one of the most prevalent cancers worldwide. As the overall prognosis of BC has not changed over the past 30 years, there is a pressing medical need to develop new diagnostic and therapeutic approaches. At present, efforts to deeply understand the pathogenic mechanisms that support urothelial carcinogenesis could help in identifying new therapeutic targets. Monoamine oxidases (MAOs) A and B are oxidative isoenzymes that catalyze the conversion of biogenic amines into their corresponding aldehydes and ketones through oxidative deamination. Recently, MAOs expression have been associated with metastasis and decreased overall survival in various types of cancer. However, the role of MAOs in BC is still poorly explored. This study characterized and compared the molecular profiles of MAO-A and MAO-B isoenzymes in the different histological stages of non-muscle (NMIBC) and muscle (MIBC) invasive bladder cancer, aiming the adaptation of these biomarkers as a criterion of clinical response, risk stratification and outcome prognosis. Methods: Sixty formalin-fixed paraffin-embedded samples of bladder were obtained from 34 to 96-year-old patients (mean 65 years) with BC diagnosis in University of Campinas and Paulinia Municipal Hospital/ Brazil. Subsequently, the samples were divided into 6 groups: pTis group, low-grade pTa group, high-grade pTa group, pT1 group and pT2 group; and submitted to immunohistochemistry analysis for MAO-A and MAO-B. The retrospective anonymous study was approved by the local ethics committee (Clinical Trial: RBR-6swqd2). Results: All stages and grades of NMIBC and pT2 (MIBC) showed positive immunoreactivity for both MAO-A and MAO-B. MAO-A immunoreactivities were significantly higher (p < 0.01) in the high-grade pTa, pTis and pT2 groups in relation to low-grade pTa and pT1. Likewise, MAO-B immunoreactivities were remarkably higher (p < 0.01) in the high-grade pTa and pT2 groups. No statistical differences were found between pTis and low-grade pTa for MAO-B. Interestingly, pT1 group showed the lowest (p < 0.01) MAO-A and MAO-B immunoreactivities in relation to other stages and grades of BC, probably due to disruption of the extracellular matrix, with consequent reduction in O2 delivery (hypoxia). Conclusions: These findings collectively characterize the contribution of MAOs in BC pathogenesis and suggest that MAOs have potential as a therapeutic target in BC.