Mycobacteria are the unique group of bacteria that are currently used in antitumoral immunotherapy. Specifically, intravesical instillation of viable cells of Mycobacterium bovis Bacillus Calmette-Guérin (BCG), after transurethral resection of non-muscle invasive bladder cancer, is the most efficacious treatment for avoiding recurrence and progression of the disease. BCG has been used for the last 35 years for bladder cancer treatment, but other mycobacteria or mycobacteria components are currently under preclinical and clinical studies for the immunotherapeutic treatment of non-invasive bladder cancer and also of other types of tumors located at the urinary system. Those are, for instance, cell wall extracts or heat-killed forms from BCG or other mycobacteria such as Mycobacterium phlei or Mycobacterium indicus pranii (MIP) or even viable cells from non-pathogenic mycobacteria such us Mycobacterium brumae. A review of the literature in which mycobacteria components, non-viable mycobacteria, and viable mycobacteria have been used for these different cancers will be performed. In this chapter, the function of mycobacteria as antitumor agents will be then analyzed, awarding the audience a broad knowledge of one of the beneficial applications of mycobacteria, which are usually introduced as dangerous microorganisms.
- bladder cancer
- Mycobacterium brumae
- Mycobacterium phlei
Since Science journal chose cancer immunotherapy as the breakthrough of the year in 2013, with only a few evidence of its efficacy and consequences, this field is now in fashion . Immunotherapies follow extremely diverse strategies, and the only point they have in common is that all of them activate somehow the cancer patient’s immune system to attack tumor cells. In fact, tumor cells are supposed to be recognized by the immune system as foreign, however, in the cases in which cancer progresses because the tumor and the immune system reach equilibrium that drives to immunotolerance. Immunotherapies include antibodies against tumor epitopes, cytokines, checkpoint inhibitors that break the equilibrium, oncolytic virus, T cell therapy using T cells removed from the patient and modified with chimeric antigen receptors (CARs), and finally, therapeutic vaccines and adjuvants which are the most ancient immunotherapies that exist.
1.1. Prechemotherapeutic era: the first association between mycobacteria and cancer
The first thoughts about a possible intervention of the immune system in the clearance of tumors were made at the beginning of the nineteenth century (Figure 1). It was observed that in cancer patients who underwent a gas gangrene, caused by
The phenomenon described in the case of erysipelas disease was also observed for tuberculosis (TB) patients. In 1929, Pearl published a large series of studies describing the inverse relationship between patients suffering from cancer and TB based on the evaluation of hundreds of autopsies . Almost in parallel, 8 years before the publication of Pearl’s studies, the first girl was being administered with three doses of an attenuated strain of
Therefore, studies on the use of BCG for cancer treatment started right after, and in 1935, Holmgren was the first scientist to report success in cancer patients . As a continuation of the experiments he had begun in 1913 in which he evaluated the sensitivity to tuberculin in more than 600 gastric cancer patients, he intravenously injected repeated BCG doses in 28 cancer patients, most of them were gastric cancer patients . In the 1950s, Rosental observed specifically lower incidence of leukemia in people who had received BCG at birth . However, once the patient developed the tumor, there was no chance to previously immunize the patient, so the key point was to know whether the BCG had a curative role . In the following years, many authors proved the efficacy of BCG in several cancer animal models including the bladder . However, the spreading of the modern chemotherapy and radiotherapy for the treatment of cancer weakened the investigations on BCG as tumor treatment.
1.2. Post-chemotherapeutic era: consolidation studies
During the 1970–1980s, hundreds of articles were published in the field of immunotherapy using of BGC or BCG components for the treatment of cancer following different strategies. First, as was done until that moment, BCG was directly used intratumorally, and many studies in different cancers during these decades continued in this line of investigation. Following another strategy, some other scientists assayed the antitumoral effect of BCG as adjuvant in patients undergoing chemotherapy or radiotherapy for the treatment of lung , melanoma , cervical , head and neck , or ovarian cancers , for instance. Another completely different strategy was the use of BCG as an adjuvant administering it together with tumor cell lysates . In that period there were some authors who evaluated the antitumor efficacy of some mycobacterial fractions or other mycobacterium species in different cancer models, for instance, of hepatoma or sarcoma .
In 1974, Zbar and Rapp established the favorable conditions for obtaining a positive outcome using a guinea pig model . They determined the amount of bacilli that should be administered, that it was a mandatory a close contact between BCG and the tumor, and that BCG worked better against small tumors and immunocompetency of the host which permit to mount an immune response against the BCG . Thanks to this postulates, in 1976, the urologist Morales and collaborators published the results of a successful small clinical trial in which they evaluated the intravesical administration of BCG in bladder cancer (BC) patients. It was not until 1990 that BCG was finally approved by the Food and Drug Administration (FDA) for the treatment of superficial BCG (Figure 1).
2. Mycobacteria as antitumor agents in the last 25 years
Although more than a century of research led finally only to the standard use of live BCG for BC treatment, the research trying to use mycobacteria components for cancer treatment has not been abandoned. In fact, in the last 25 years, several attempts for using different mycobacteria as immunotherapeutic agents for bladder cancer treatment have been carried out (Figure 1).
Despite the molecule or molecules of BCG responsible for its antitumor effect are still unknown, several genus-specific antigens have been described in mycobacteria cells, and most of them are known stimulators of the immune system. Specifically, the mycobacteria cell wall is rich in a variety of exclusive lipids, glycolipids, lipoproteins, glycans, and proteins that are recognized by immune receptors. Zlotta et al.  demonstrated that not only mycobacterial cell wall components are responsible for the antitumor effect but other fractions also triggered the production of Th1 cytokines and stimulated the cytotoxic activity against T24 BC cells by peripheral blood cells. Antigens from different cell fractions are recognized by surface-located receptors present in antigen-presenting cells.
Molecules such as lipoarabinomannan (LAM), phosphatidylinositol mannosides, or heat-shock proteins (HSP) are recognized by Toll-like receptors (TLR) 2 and 4 or mannose receptors; or antigens like trehalose mono- and dimycolate are agonist of C-type lectin receptors. Other mycobacteria antigens interact to specific intracellular immune receptors after being internalized to the cell, such as unmethylated cytosine-guanosine nucleotide (CpG)-rich DNA motifs, muramyl dipeptide (MDP), or cytosolic DNA which bind to TLR-9, NOD2, or cyclic GMP-AMP synthase (cGAS), respectively. After being processed inside the cells, some antigens are presented to T cells via CD1 receptors such as mycolic acids (MA) or trehalose and glucose mycolates. Signaling through these receptors can induce the production of cytokines and/or chemokines favoring a desirable pro-inflammatory profile in tumor microenvironment .
Nevertheless, not all mycobacteria possess all the mentioned antigens, and even the structure of each antigen can vary between different species. Although some of these molecules such as LAM, trehalose dimycolate (TDM), or MA are present in all mycobacteria, the structure, for example, the presence or not of mannose residues in LAM structure, the length or the presence of unsaturations or oxygenated groups in the lipidic chains of TDM or MA, etc., determine the interaction to the corresponding immune receptor, and in consequence, the immune response is generated. The complexity of mycobacteria antigenicity is enormous leading even to the fact that different strains of the same species can have different antigenic pattern. The case of the antigenic profile of BCG is a good example. As it is known, from the seed strain originated in France by Albert Calmette and Camille Guérin, different strains were originated after subculturing the original BCG in different countries. Before being preserved by freezing, decades of subculturing originate deletions of some genome regions or even the duplication of some other genome regions. Today, we count about a decennium of different BCG strains  that are used broadly for BC treatment as well as for TB vaccination. Each different BCG strain possess or not some immunogenic antigens like phenolglycolipids, phthiocerol dimycocerosates, and MBT64 protein antigen, or even they have two different MA profiles: some strains possess alpha, methoxy- and keto-MA, while some other possesses only alpha- and methoxy-MA. The relevance of these differences has tried to be related to BCG efficacy as antitumor agent or TB vaccine, but until nowadays the critical antigens are still not known.
In the following sections, the use of different mycobacteria or their components in the last 25 years for urinary tract cancers will be reviewed. We will mainly focus on the use of these components as unique antitumor agents, although the inclusion of studies in which mycobacterial antigens are used as adjuvants for tumor antigens or other therapies will also be mentioned.
3. Urinary tract cancers and mycobacteria
3.1. Bladder cancer and live BCG: a fruitful relationship
The immunotherapeutic ability of mycobacteria against cancer has the most successful example in the case of the use of BCG for the treatment of high-risk non-muscle invasive bladder cancer (NMIBC) patients. All the conditions described by Zbar and Rapp are accomplished in intravesical BC treatment: a close cavity where mycobacteria can be loaded and being in close contact to tumor cells triggering also an immune response . BC is one of the most common malignances in urology. The number of new cases and deaths of BC was 20.1 and 4.4 per 100,000 men and women per year, being in 2013 estimated 587,426 people living with BC in the USA . In 2016, 76,960 new cases of BC were estimated, and around 70% of them present as NMIBC. Whether NMIBC patients are not treated after transurethral resection (TUR) of tumor, as much as 80% will experience disease recurrence and/or progression. Therefore, after TUR, the standard treatment for high-risk NMIBC patients consists in the intravesical instillation of live BCG. The bladder cavity allows a closed contact between the possible remaining tumor cells and the bacilli. For a short period of time (approximately 2 h), a high concentration of microorganisms (between 107 and 109 bacilli depending on the commercially available preparations of BCG strains), besides being in contact to possible remaining tumor cells, initiates an immune cascade of events. Although the detailed chronogram and magnitude of these events are not totally understood, numerous studies have provided information about the immune cells and signals implicated in the action of BCG. Several excellent reviews have covered this field, explaining what is known until today, see [22, 23]. In summary, BCG firstly interacts with remaining bladder tumor cells inducing apoptosis and/or cell cycle arrest as demonstrated in
Intravesical BCG therapy is then successful. In fact, the same protocol of instillations has been used in high-risk NMIBC patients for the last 30 years. But, although efficacious, BCG is not the perfect treatment. On the one hand, a percentage of patients do not respond to BCG for unknown reasons. On the other hand, a high percentage of patients suffer adverse events during the treatment.
3.2. Increasing efficacy
Several strategies are tried to solve the problem of unresponsive BCG patients. The main strategy consists of improving the immune response triggered by BCG, by combining BCG and immunomodulators or modifying genetically the bacterium for expressing these immunomodulators. For instance, BCG plus an optimized interleukin (IL)-15 mutant significantly increased immune activation and reduced tumor burden and angiogenesis compared to the single agents in the carcinogen-induced rat NMIBC model . The list of modified BCGs is long and comprises the expression of cytokines and chemokines—IL-2, interferon (IFN)-γ, GM-CSF, etc.—or immunodominant mycobacteria antigens like alpha-crystallin antigen (fibronectin-binding protein) complex (Ag85) [9, 22, 34]. These strategies seem to be promising for improving the efficacy of BCG alone as
The study of the immune response triggered by BCG inside the bladder has revealed that an excess of IL-10 production in tumor microenvironment is detrimental for BCG efficacy. Another successful strategy is then the use of anti-IL-10 antibodies in combination to BCG for an improved effect. To block IL-10 receptor together with intravesical BCG reach high tumor regression rates in the murine orthotopic model of BC, enhancing also a systemic specific antitumor immune response compared to BCG alone .
Another strategy is the combination of BCG with TLR agonists. BCG together with TLR4 agonist such as polyporus polysaccharide triggers the expression of activation molecules like CD80 and CD40. In rat BC models, this combination therapy showed a synergic effect reducing invasiveness of cancer together with reduced adverse events originated by BCG alone . Similarly BCG plus a TLR3 agonist
Finally, it has been also demonstrated that the combination of live BCG together with chemotherapeutic treatments improves disease survival compared to BCG alone. Although some works showed no impact on the progression or survival rates of the patients [16, 17], recent study has demonstrated the beneficial effect of sequential intravesical treatment with BCG and mitomycin C. While in mice experiments an augmentation of beneficial M1 tumor-associated macrophages on tumor-bearing treated mice was observed, in treated patients increasing IL-2, IL-8, IL-10, and TNF-α urine levels during treatment and increased efficacy over BCG treatment alone were observed .
3.3. Reducing adverse events
The most critical adverse event regarding intravesical instillations of BCG is the possibility of the patient to be infected. Numerous cases of BCGosis have been described in the literature in the last 5 years [41, 42]. The main reason is a traumatic instillation that can lead to the dissemination of BCG throughout the systemic circulation. As soon as BCG showed to be an excellent option for BC treatment, researchers tried to compare its effect to non-viable mycobacteria, cell wall extracts, or even purified antigens from mycobacteria.
3.3.1. Purified mycobacteria antigens and non-viable mycobacteria
The majority of mycobacteria antigens, which are able to stimulate the immune system, have been widely studied in order to find epitopes for vaccines to prevent TB infection and also to develop immunodiagnostic tests for TB infection or disease. Researchers have taken advantage of the knowledge of these molecules for trying to use them as antitumor agents for BC. In this line of research, MPT-64 antigen, 38 kDa protein, Ag85 antigen, or mycobacterial DNA have been evaluated for their ability to treat BC. For MPT-64 a dose-dependent response in survival rates was observed when instilled intravesically. The higher MPT-64 dose administrated provided higher survival rates in tumor-bearing treated mice than non-treated mice, triggering also a favorable IFN-γ systemic response . When the 38 kDa antigen was studied, a cytotoxic activity against T24 BC cells was observed in 38 kDa antigen-activated peripheral blood mononuclear cells (PBMC), also in a dose-dependent manner. Again, 38 kDa antigen-intravesically treated tumor-bearing mice survived longer than non-treated tumor-bearing mice , triggering a systemic response observed when splenocytes from treated mice respond specifically to the instilled antigen. Different works have evaluated the antitumor capacity of Ag85. Initially, the generation of cytolytic CD8 and an antitumor response was observed when cDNA from
Apart from purified antigens, complex extracts of mycobacteria have also been evaluated for BC treatment. The two principal assayed compositions have been a cell wall extract from BCG (composed by the cell wall skeleton or also called SPM-105)  and a mixture of cell wall plus DNA of
On the one hand, the BCG-cell wall skeleton (CWS) has been formulated into liposomes in which their surface was modified by an octaarginine (R8) anchor, an efficient cell-penetrating peptide [54, 55, 56]. Researchers demonstrated that R8-liposome-BCG-CWS binds to MBT-2 murine BC cells inhibiting its growth in a syngeneic subcutaneous tumor model , being also efficacious in an intravesical BC rat model of the disease . Furthermore, the formulated extract is able to inhibit human BC growth
On the other hand,
Finally, whole non-viable mycobacteria have also been evaluated for BC treatment. Contrary to purified antigens or cell extracts, the use of the whole bacteria warrants the presence of the whole antigenic profile, but depending on the inactivation method, some of the possible crucial antigens can be altered or lost. On the one hand, heat-killing form has been the most studied although mycobacteria cells are damaged [63, 64], and on the other hand, γ-irradiation is the treatment which preserves better the integrity of the mycobacteria cell and maintains some metabolic activity .
Using the orthotopic murine intravesical model of BC, HK BCG-treated mice survived similarly to non-treated tumor-beating mice , together with lower production levels of Th1 cytokines  that has also been related to inability to trigger T-cell infiltration into bladder cavity . Interestingly, when live BCG or
In BC patients, HK BCG instilled to previously live BCG nonresponders showed reduced toxicity and no increase in the risk of tumor recurrence . Whole HK MIP has been instilled intravesically in five BC patients undergoing radiation therapy , maintaining 100% survival rates and recurrence-free rates. It has been also instilled in the treatment of BCG-refractory patients .
3.3.2. Live non-BCG mycobacteria
Concluding for the whole work compiled in the literature, it seems that live BCG provides the best option compared to the non-viable mycobacteria or mycobacteria fractions. Therefore, another feasible option as alternative to BCG for BC treatment is to consider the use of live non-pathogenic mycobacteria. However, few studies have considered them for BC treatment. As explained above, the majority of mycobacterium species is saprophytic and potentially share immunomodulatory antigens with BCG.
In a recent work of our research group , several non-pathogenic-considered mycobacteria (
Although antitumor effect of mycobacteria is not considered for muscle-invasive BC, MIP plus radiation therapy in a small number of patients (5) showed disease-free survival more than 2 years .
3.4. Other renal and urinary tract cancers
Immunotherapy using mycobacteria components has been also applied in urinary tract cancers other than NMIBC.
For prostatic cancer, studies using BCG,
In renal cancer, SRL172 has been administrated in patients suffering from metastasis demonstrating low toxicity and similar survival rates compared to patient treatment with cytokines (IL-2 or IFN-α)  and increased rates when synergized both treatments: SRL172 and antibodies . In stage IV renal cell cancer, patients treated with BCG plus irradiated autologous tumor cells, and later infused with autologous activated T cells, showed durable tumor responses .
4. Future perspectives: a long history with room for improvement
The potential beneficial effect of mycobacteria as antitumor agents has been clearly demonstrated after almost a century of observations and experimentation. But even in the case of BCG treatment for NMIBC patients, many issues remain under question: the appropriate schedule to reduce recurrence and progression without increasing adverse events [94, 95, 96]; the reason why a proportion of patients do not respond to BCG treatment; the detail description of immune mechanism which could permit to predict the response to the treatment; the possibility of using other mycobacteria species that have shown similar or increased effective than BCG but with potential increased safety; etc. In fact, the experience using mycobacteria in BC is permitting novel approaches for improving its efficacy. In this sense, a critical point for mycobacteria efficacy is the delivery of mycobacteria or mycobacteria antigens into the tumor site. The optimization of mycobacteria formulation could be critical for reducing adverse-associated events  and/or improving mycobacteria antitumor efficacy . Moreover, the possibility to manipulate genetically mycobacteria for being vehicle for delivery antigens could lead also a chance to get more potent antitumor tools.
This work was funded by the Spanish Ministry of Economics and Competitiveness (SAF2015-63867-R), the European Regional Development Fund (FEDER), and the Generalitat of Catalunya (2014SGR-132).