Most of the bladder tumors (>90%) are urothelial carcinomas for they originate in the urothelium [1] . An electronic microscope analysis revealed that the basal cell layer is always in contact with the basal membrane; the intermediate cell layer, occasionally, but never involving the superficial cells or umbrella cells [2] . This bladder epithelium acts as a constant permeable barrier and protects the blood from toxic urinary substances [3] .

In over 70% of the cases, bladder cancer (BC) is superficial (pTa and pT1) or non-muscle invasive, and the occurrence of a muscle invasive disease is less frequent [4] . Over 50% of the non-muscle invasive tumors recur in 4 years after the initial treatment, of which 11% develop into the invasive form [5] .

The high recurrence frequency of the urinary bladder urothelial cells carcinoma, as well as its heterogeneous presentation support the hypothesis that adult stem cells (or cells that acquire stem characteristics by mutation) may be involved in the development and recurrence of this type of tumor [6] .

In different types of cancers, a very small population of cells has been recognized as healthy stem cells (HSC) and cancer stem cells (CSC), for they have self-renewal and differentiation capacities [7] . Both the origin and the frequency of the stem cells are not yet clear [8] . In the bladder, the hypothesis is that these cells originate in the basal lamina [9] or in the epithelial-mesenchymal transition [10] .

Since the discovery of stem cells, researches have focused on identifying markers for these cells. However, although many of the markers have already been described, none have proven to be exclusive or specific [11] . Several cell surface markers have been used to characterize healthy and cancer stem cells, especially the surface antigens CD44, CD117 and CD133 and the ATP Binding Cassette membrane transporter (ABCG2) [12] .

In turn, the toll-like receptors (TLRs) are a family of transmembrane receptors that recognizes conserved molecular patterns of microbial origin, known for their molecular patterns associated to pathogens [13] . They also have a fundamental role in tissue repair due to inflammation [14] . The TLRs classic function is antimicrobial: they recruit leukocytes for infected tissues and subsequently induce adaptive immune responses. The TLR signal transduction is made via different adaptive proteins that activate signaling cascades involving the κB (NF-κB) nuclear transition factor.

Initially, the TLRs we identified only in immune and epithelial cells. However, recent studies have demonstrated that these receptors may also play a functional, yet controversial, role in cancer, for there are conflicting data that point them out as negative or positive carcinogenesis regulators [15] .

Thus, the main objectives of this study were to characterize and compare putative cancer urothelial stem cells (healthy and cancer cells) and TLRs profiles in the urinary bladder of men with and without non-muscle-inva- sive and muscle-invasive tumors.

In this study, we used 30 samples of urinary bladder tissue of men between 50 and 80 years of age (average 61 years old), with and without diagnosis of urothelial lesions, obtained from The Teaching Hospital of the School of Medicine, University of Campinas (UNICAMP). Ten urinary bladder samples were from necropsy of patients without diagnosis of urothelial lesions and/or urologic disease. The remaining 20 urinary bladder samples came from patients submitted to transurethral resection and radical cystectomy. The patients were distributed into 3 groups (n = 10 samples per group): Normal Group (without lesion); Non-Muscle Invasive Bladder Cancer Group and Muscle Invasive Cancer Group. The samples were further processed and submitted to histopathological and immunohistochemical analyses.

Approximately 70% to 80% of the bladder tumors are non-muscle invasive, the majority of which stage Ta and the remaining T1 or Tis. The Ta tumors are generally low-grade while most of the T1 tumors are high-grade. By definition, thecarcinoma in situ (Tis) is a high-grade tumor and represents 10% of the bladder tumors. In 50% of the cases, the Tis is associated with papillary or invasive tumor (secondary Tis), while the other 50% occur isolatedly (primary Tis) [18] . Most of the cases of bladder cancer have high recurrence probability [19] .

A relevant number of risk factors have been associated to the development of bladder cancer, such as occupational exposure to aromatic amines and to organic chemical substances in various professional activities. However, smoking is one of the most important risk factors, responsible for over 33% of the cases of bladder cancer [20] [21] . Furthermore, the cancers may possibly originate from the CSCs, and, hypothetically, the latter are responsible for the occurrence of metastasis and tumor relapse after an initial treatment, as well as for the invasive characteristics of the tumors determined by the stem cell ability to interact with the stroma [22] . The recent identification of cancer stem cells in the bladder as mediators of resistance to conventional therapies make these cells candidates to biological target therapies [1] .

Epithelial tumors contain a heterogeneous cell population and this may be characterized by histopathological and functional properties differences, such as anchorage-independent growth, proliferative and apoptotic capacities and responses to therapy. Recent evidences point out to the existence of a cell hierarchy in epithelial tumors, and the cancer stem cells are at the top of this hierarchy [22] .

There are two proposed mechanisms for the occurrence of CSC in different tumors: oncogenic mutations, which inactivate the expansion of normal stem cells or in a cell that is more differentiated by oncogenic mutations that may cause a continuous proliferation of cells that will not go into a differentiated postmitotic state, creating a population of self-renewal cells in which these mutations may accumulate [23] . Cancer treatment failures may be associated to the low effects of these agents over the CSCs, which maintain their total ability to reestablish tumoral cell masses [24] .

Cell surface markers, such as CD44, CD133 and ABCG2, are considered the most common markers to identify normal and cancer stem cells [25] . CD44 is a transmembrane glycoprotein that is expressed in practically all cell types and acts in cell-cell and cell-extracellular matrix interactions; it also participates in growth regulation by presenting growth factors to its cell surface receptors [26] . Several studies have demonstrated that the CD44 expression seems to play a fundamental role in the migration of cancer cells and tumor progression, and also has prognostic value in certain types of cancer [27] .

In turn, CD133 (prominin-1) is a plasma membrane glycoprotein that is expressed in cell protrusions of several organs and tumors. Cells that are positive for CD133 have 3 important stem cell features: they are rare, they have high proliferation potential and they are able to reconstitute highly branched ductal-like structures [28] . CD117, also known as c-Kit, is a potential stem cell marker that is mainly expressed in the basal epithelial com- partment of the prostate area near the urethra [29] .

The ATP Binding Cassette (ABCG2) membrane transporter has been used as a possible CSC marker and is as- sociated to the development of multi-resistance to drugs [30] .

In addition, TLRs are expressed both in the normal and neoplastic urothelium. Ayari et al. [31] identified TLRs 2 and 4 in healthy urothelium and non-invasive tumors with much lower intensities in the last detection. This expression was markedly reduced in invasive muscle urothelial tumors [31] . Very similar results were found in this study, averaging around 10% in the normal group and 90% in groups with tumors for both recep- tors intensities. Also, Fávaro et al. [32] demonstrated in appropriate animal model for non-muscle invasive blad- der cancer significant decrease of TLRs 2 and 4 protein levels in animals with non-muscle invasive cancer in re- lation to normal animals.

The TLR role in cancer is a controversial issue, for there are conflicting data that point them out as negative or positive carcinogenesis regulators. They also have a fundamental role in tissue repair due to inflammation [14] . TLR activation may cause tumor regression by increasing vascular permeability and through the recruitment of leukocytes, which determines tumor cell lysis by natural killer (NK) and cytotoxic T cells [13] - [15] . The contradictory evidence that TLR promotes carcinogenesis, whereas in others it exerts antitumor effects, could be explained by the different intensity and nature of the inflammatory response [15] . In fact, chronic inflammatory processes are milder than acute inflammatory responses, which are aimed at inducing pathogen clearance. In most cases, cancer-associated inflammation is similar to chronic inflammation, including the production of factors that stimulate tissue repair and cancer cell survival and proliferation [14] [15] . However, if the inflammatory response develops into acute inflammation, an immune effector mechanism is activated, and cancer regression takes place [14] [15] . Among the different elements that control neoplastic processes, a major role is attributed to members of the chemokine superfamily. Chemokines expressed by tumor cells and by host cells play a critical role in determining the fate of the developing tumor by regulating the migration of different leukocyte subtypes [14] [15] . The relative proportion of each defense cell type (macrophages, T cells, NK cells, dendritic cells, or other leukocyte subtypes) within the tumor largely dictates the immune profile at the tumor site; local production of numerous inflammatory mediators is crucial for the recruitment and activation of leukocytes in addition to macrophages and mast cells [14] [15] . In particular, CD8 T cells and some types of innate immune cells, such as NK cells, can protect against experimental tumor growth [14] [15] .

These studies demonstrate that activation of TLR4 signaling in cancer cells correlates with tumor progression through various mechanisms, by escape immune surveillance thereby preventing the attack of the host defense system.

This study demonstrated that TLR2 and TLR4 immunoreactivities were significantly lower in low-grade non- muscle invasive (pTa stage) and muscle invasive (pT2 stage) tumors, and this may contribute to the high tumor relapse and progression rates.

This study leads us to the conclusion that cancer stem cell occurrence was sensitive to the decreased in TLR2 and TLR4 immunoreactivities. Also, TLR2 and TLR4 demonstrated their involvement in the regulation of the different biomarkers for healthy and cancer urothelial stem cells, probably acting as negative regulators of uro- thelial carcinogenesis. Taken together data obtained suggest that use of TLRs agonists could be a promising alter- native for the treatment of non-muscle and muscle invasive bladder tumors.

Support by São Paulo Research Foundation (FAPESP―Process Number 2012/50014-5 and 2011/14223-6).