Diagnosis of cancer multidrug resistance by bacterium-mediated imaging
Introduction
Multidrug resistance (MDR) is a phenomenon expressed by many tumors, representing the main cause of chemotherapy failure. MDR is defined as the intrinsic or acquired resistance of cancer cells to structurally and functionally unrelated drugs [1]. Many mechanisms are involved in MDR including Adenosine triphosphate Binding Cassette (ABC) transporters up-regulation [2], [3], [4] and limited drug penetration in solid tumors [5].
ABC transporters, especially P-glycoprotein (Pgp) and Multi Drug Resistance associated protein (MRP), are major contributors to MDR. They work by an efflux mechanism maintaining a very low concentration of their substrates (such as chemotherapeutic agents), insufficient to carry out their effect [3], [6]. These proteins are usually over-expressed in patients refractory to chemotherapy; this overexpression is generally associated with poor prognosis [2], [4], [7]. Thus, MDR phenotype can affect the treatment decision of cancer. For instance, if the tumor is MDR, then one of three strategies should be considered during therapy: avoiding the MDR pumps, using drugs that are not substrate of the pumps; reversing the resistance, using MDR pump inhibitors [4], [8]; or even exploiting it, using protector drugs substrate to the MDR pumps [6], [8].
Cancer MDR implication goes beyond cancer to affect antibacterial chemotherapy. Since MDR reduces the intracellular accumulation of some antibiotics (substrate of the MDR pumps), it reduces their activity against intracellular bacteria: increasing their minimum inhibitory concentration (MIC) [6], [9]. These antibiotics are diverse including and belong to various classes: macrolides, azalides, ciprofloxacin, ofloxacin, clindamycin, rifampicin, chloramphenicol, doxycycline, and trimethoprim [9], [10]. Again, MRP and Pgp play an important role in this reduced intracellular activity, where their selective inhibitors reverse this reduced activity [10].
So far, the methods developed for MDR diagnosis are mostly invasive. They involve testing the tolerance of cancer cells (isolated from primary tissues) to chemotherapeutic drugs. This can be indicated by the change in the half inhibitory concentration, cell growth curve, cell proliferation index, resistance index, or apoptosis index. They can also assess cytotoxic drugs (such as doxorubicin) pump out rate or detect multidrug resistance genes [1].
Radio-labeled substrates of MDR proteins have been investigated as non-invasive alternatives for functional imaging of MDR. Technetium (99mTc) labeled sestamibi ([99mTc] MIBI) is the first and most studied of these radio-labeled substrates. It is a 99mTc-labeled lipophilic cation originally introduced for imaging myocardial perfusion [11], [12]. [99mTc] MIBI passively diffuses into the cell, accumulating in the mitochondria; however, in MDR cells, it is expelled by the MDR proteins i.e. its retention in tumors correlates inversely with the degree of MDR proteins expression [11]. As it diffuses passively into the cells, we should question its ability to differentiate between resistances due to up-regulation of MDR proteins, and limited drug penetration (due to poor distribution caused by distended distance between cancer cells and vasculatures in tumors), which in turn can affect the therapy decision. However, this approach is not clinically employed yet, and in vivo approaches are still needed to clinically evaluate MDR in early stages of the disease [1], [11].
Microbial-mediated imaging is a noninvasive novel approach that is being investigated for imaging of cancer [13], [14], [15], [16]. It depends on the natural preference of some bacteria and viruses for the tumor microenvironment. This preference is attributed to the nutrient-rich, hypoxic and immune-hiding niches provided by tumors’ microenvironment [17]. Thus, attenuated bacteria and viruses bearing reporter genes are successfully employed to reveal locations of solid tumors and metastases in animal studies. These reporter genes are expressed to produce signals such as: luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which can be imaged by sensitive photon detectors [13], [16], [18]; magnetic resonance imaging (MRI) contrast enhancers like ferritin, which can be imaged by MRI [19]; and radionuclides, which can be imaged by positron emission tomography (PET) [15]. The viruses and bacteria studied as tumor-colonizing live vector are diverse including vaccinia viruses, Bifidobacterium breve, Escherichia coli, Vibrio cholera, and Salmonella species [14], [16].
Salmonella typhimurium is one of the most studied bacteria employed in targeting cancer. The wild-type has the ability to infect a wide host range—including human and mice—and can be easily engineered to carry foreign genes [20]. Moreover, Salmonella preferentially colonizes tumor xenografts in mice rather than normal tissues achieving tumor implant/normal tissue ratios of 10,000:1 [21]. Being a facultative anaerobe, Salmonella can survive in both oxygenated and hypoxic conditions; thus, it could accumulate in small metastasis and solid tumors [13], [17], [20].
To assure safety and increase tumor-targeting capabilities, the attenuated S. typhimurium, VNP20009, was developed by msbB (Lipid- A- modified) and purI (purine auxotrophs) mutations, respectively [22], [23]. The msbB mutation largely decreased host TNF-α induction, reducing the induction of proinflammatory cytokines, and purI mutation made Salmonella auxotrophic for purine, which is abundant in tumors [22]. S. typhimurium is also susceptible to a variety of antibiotics including azithromycin and ciprofloxacin [24], which are substrates to the MDR proteins: Pgp and MRP, respectively [10].
Section snippets
Hypothesis
We propose that bacterium-mediated imaging of cancer can diagnose MDR by using antibiotics substrate to the MDR proteins: monitoring the change in the concentration required to eliminate the intracellular bacteria. The reporter bacteria (such as bioluminescent bacteria) proliferating within MDR cancer cells would require a larger dose of antibiotics to be eliminated, than those present in non-MDR cancer cells; this elimination would be reflected in the cease of the signals produced by the
Significance and potentials
Considering the great implication of MDR on cancer prognosis and treatment options, an early, convenient and cost effective diagnostic method of MDR is needed. Bacterium-mediated imaging of cancer is a noninvasive, safe, efficient, versatile and relatively simple method [14], [25] that can satisfy this need. It involves the administration of reporter bacteria intravenously, and its selective proliferation in cancer is detected by a suitable imaging system after a predetermined time [16]. To
Evaluation of the hypothesis
The preclinical evaluation of this proposed potential of bacterium-mediate imaging of cancer should involve in vitro assessment of the employed bacteria ability to invade the cancer cells, followed by in vivo overall assessment of the approach using BLI as a preclinical imaging modality. In vitro, the ability of VNP20009—as an ideal model of this proposed approach—to invade cancer cells and the extent of this invasion can be assessed by gentamicin protection assay. Gentamicin selectively kills
Conclusion
Bacterium-mediated imaging of cancer has shown promising results in animal studies. Antibiotics substrates to MDR proteins could expand its utility in the area of functional imaging of MDR. As there is a great need for such approach in the study and diagnosis of MDR, this proposed feature of bacterium-mediated imaging of cancer could be of a great value for preclinical studies and clinical practice. The early diagnosis of MDR would allow prompt choice of the most appropriate therapy: avoiding
Authors’ contributions
Elkadi, O.A: Conceived the hypothesis and its evaluation, literature search, wrote the manuscript. Abdelbasset, M: Biolumenscent imaging and VNP20009 literature search.
Conflict of interest
None declared.
Acknowledgments
We wish to express our sincere gratitude to Dr. Ayman Yassin from the microbiology and immunology department, faculty of pharmacy, Cairo University for proofreading this work and for his constructive and valuable comments.
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