Metastatic prostate cancer cell-specific phage-like particles as a targeted gene-delivery system.

BACKGROUND: One of the cardinal requirements for effective therapeutic management of tumors is the selective delivery of cancer drugs to the right site by ligand-decorated nanomedicines. Screening of 2 × 109 clone landscape phage library provides a reliable avenue for generating protein ligands specific for tumor cells. It was shown that selective phage proteins derived from landscape phage libraries against breast and prostate cancer cells are able to navigate drug or siRNA loaded liposomes to corresponding cancer cells with minimal toxicity to non-neoplastic cells. In an alternative platform, glioma cell-specific phage proteins were used for assembling in vivo cancer-specific phage-like particles, named 'phagemid infective particles' as targeted gene-delivery vehicles. METHODS: To extend the panel of anticancer cell phages, we have screened a 2 × 109 clone landscape phage library f8/8 to select phage clones specific for metastatic prostate cancer cell PC-3M. The phage clones were characterized for their selective interaction with PC-3M cells using phage capture assay, immunofluorescence microscopy and electron microscopy. A prostate cancer selective phage was converted to phage-like particles harboring emerald green fluorescent protein. RESULTS: Phage clone EPTHSWAT (designated by the sequence of inserted peptide) was found to be most selective for PC-3M cells and was observed to internalize PC-3M cells as revealed by immunofluorescence microscopy and electron microscopy. Conversion of this phage to phage-like particles harboring emerald green fluorescent protein and the expression of emerald green fluorescent protein in the phage-like particles treated PC-3M cells showed potential of adoption of this phage-like particle in prostate cancer therapeutic gene delivery. CONCLUSION: Successful employment of phage-like particles expressing emerald green fluorescent protein genes targeted to prostate cancer cells PC-3M confirms a prospect of their use for targeted delivery of therapeutic genes to cancer cells.

Evaluations of CRC2631 toxicity, tumor colonization, and genetic stability in the TRAMP prostate cancer model.

Conventional cancer chemotherapies are not fully efficacious and do not target tumors, leading to significant treatment-related morbidities. A number of genetically attenuated cancer-targeting bacteria are being developed to safely target tumors in vivo. Here we report the toxicological, tumor-targeting, and efficacy profiles of Salmonella enterica serovar Typhimurium CRC2631 in a syngeneic and autochthonous TRAMP model of aggressive prostate cancer. CRC2631 preferentially colonize primary and metastatic tumors in the TRAMP animals. In addition, longitudinal whole genome sequencing studies of CRC2631 recovered from prostate tumor tissues demonstrate that CRC2631 is genetically stable. Moreover, tumor-targeted CRC2631 generates an anti-tumor immune response. Combination of CRC2631 with checkpoint blockade reduces metastasis burden. Collectively, these findings demonstrate a potential for CRC2631 in cancer immunotherapy strategies.

Development of Salmonella strains as cancer therapy agents and testing in tumor cell lines.

Despite significant progress in the development of new drugs and radiation, deaths due to cancer remain high. Many novel therapies are in clinical trials and offer better solutions, but more innovative approaches are needed to eradicate the various subpopulations that exist in solid tumors. Since 1997, the use of bacteria for cancer therapy has gained increased attention. Salmonella Typhimurium strains have been shown to have a remarkably high affinity for tumor cells. The use of bacterial strains to target tumors is a relatively new research method that has not yet reached the point of clinical success. The first step in assessing the effectiveness of bacterial tumor therapy will require strain development and preclinical comparisons of candidate strains, which is the focus of this chapter. Several investigators have developed strains of Salmonella with reduced toxicity and capacity to deliver anti-tumor agents. Although methods for obtaining safe therapeutic strains have been relatively successful, there is still need for further genetic engineering before successful clinical use in human patients. As described by Forbes et al. in 2003, the main stumbling block is that, while bacteria preferentially embed within tumor cells, they fail to spread within the tumor and finish the eradication process. Further engineering might focus on creating Salmonella that remove motility limitations, including increased affinity toward tumor-generated chemotactic attractants and induction of matrix-degrading enzymes.

Whole-Genome Shotgun Sequences of Salmonella enterica Serovar Typhimurium Lilleengen Type Strains LT1, LT18, LT19, LT20, LT21, and LT22.

The Lilleengen type (LT) collection of Salmonella enterica serovar Typhimurium strains has served the scientific community as a group of model organisms for basic genetic and biochemical pathway research. Here, we report the whole-genome shotgun sequences of Salmonella enterica serovar Typhimurium strains LT1, LT18, LT19, LT20, LT21, and LT22.

Regulation of site-specific recombination by the C-terminus of lambda integrase.

Site-specific recombination catalyzed by bacteriophage lambda integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein-protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.

Strains, Mechanism, and Perspective: Salmonella-Based Cancer Therapy.

Recently, investigation of bacterial-based tumor therapy has regained focus due to progress in molecular, cellular, and microbial biology. Many bacteria such as Salmonella, Listeria, Escherichia, and Clostridium have proved to have tumor targeting and in some cases even tumor-destroying phenotypes. Furthermore, bacterial clinical treatments for cancer have been improved by combination with other therapeutic methods such as chemotherapeutic drugs and radioactive agents. Synthetic biology techniques have also driven the development of new bacterial-based cancer therapies. However, basic questions about the mechanisms of bacterial-mediated tumor targeting and destruction are still being elucidated. In this review, we focus on three tumor-therapeutic Salmonella models, the most intensively studied bacterial genus in this field. One of these Salmonella models is our Salmonella enterica serovar Typhimurium LT2 derived strain CRC2631, engineered to minimize toxicity but maximize tumor-targeting and destruction effects. The other two are VNP20009 and A1-R. We compare the means by which these therapeutic candidate strain models were selected for study, their tumor targeting and tumor destruction phenotypes in vitro and in vivo, and what is currently known about the mechanisms by which they target and destroy tumors.

Salmonella Bacterial Monotherapy Reduces Autochthonous Prostate Tumor Burden in the TRAMP Mouse Model.

Attenuated Salmonella typhimurium injected in the circulatory system of mammals selectively targets tumors. Using weekly intraperitoneal injections of attenuated Salmonella strain CRC2631, we tested for regression and/or inhibition of tumor development in the TRAMP prostate tumor mouse model, which utilizes SV40 early region expression for autochthonous formation of prostate tumors that progress into metastatic, poorly differentiated prostatic carcinomas in an immunocompetent murine model. Thirteen weekly intraperitoneal administrations of 105-107 CFU CRC2631 into 10 week old mice were well tolerated by the TRAMP model. Sacrifice and histological analysis of TRAMP prostates at 22 weeks indicated that Salmonella monotherapy at administrated levels decrease visible tumor size (>29%) but did not significantly inhibit previously described SV40 expression-driven TRAMP tumor progression to undifferentiated carcinomas when histologically examined. In conclusion, this work demonstrates baseline results for CRC2631 Salmonella monotherapy using the immunocompetent TRAMP prostate tumor model in preparation for study of combination therapies that resolve autochthonously generated TRAMP prostate tumors, further reduce tumor size, or inhibit prostate tumor progression.

Direct attachment of nanoparticle cargo to Salmonella typhimurium membranes designed for combination bacteriotherapy against tumors.

Nanoparticle technology is an emerging approach to resolve difficult-to-manage internal diseases. It is highly regarded, in particular, for medical use in treatment of cancer due to the innate ability of certain nanoparticles to accumulate in the porous environment of tumors and to be toxic to cancer cells. However, the therapeutic success of nanoparticles is limited by the technical difficulty of fully penetrating and thus attacking the tumor. Additionally, while nanoparticles possess seeming-specificity due to the unique physiological properties of tumors themselves, it is difficult to tailor the delivery of nanoparticles or drugs in other models, such as use in cardiac disease, to the specific target. Thus, a need for delivery systems that will accurately and precisely bring nanoparticles carrying drug payloads to their intended sites currently exists. Our solution to this engineering challenge is to load such nanoparticles onto a biological "mailman" (a novel, nontoxic, therapeutic strain of Salmonella typhimurium engineered to preferentially and precisely seek out, penetrate, and hinder prostate cancer cells as the biological delivery system) that will deliver the therapeutics to a target site. In this chapter, we describe two methods that establish proof-of-concept for our cargo loading and delivery system by attaching nanoparticles to the Salmonella membrane. The first method (Subheading 1.1) describes association of sucrose-conjugated gold nanoparticles to the surface of Salmonella bacteria. The second method (Subheading 1.2) biotinylates the native Salmonella membrane to attach streptavidin-conjugated fluorophores as example nanoparticle cargo, with an alternative method (expression of membrane bound biotin target sites using autodisplay plasmid vectors) that increases the concentration of biotin on the membrane surface for streptavidin-conjugated nanoparticle attachment. By directly attaching the fluorophores to our bacterial vector through biocompatible, covalent, and stable bonds, the coupling of bacterial and nanoparticle therapeutic approaches should synergistically lead to improved tumor destruction.

Phenotypic evolution of therapeutic Salmonella enterica serovar Typhimurium after invasion of TRAMP mouse prostate tumor.

Salmonella has been of interest in cancer research due to its intrinsic ability to selectively target and colonize within tumors, leading to tumor cell death. Current research indicates promising use of Salmonella in regular administrations to remove tumors in mouse models while minimizing toxic side effects. However, selection of mutants during such long-term tumor colonization is a safety concern, and understanding selection of certain phenotypes within a tumor is an important consideration in predicting the long-term success of bacterium-based cancer treatment strategies. Thus, we have made an initial examination of selected phenotypes in a therapeutic Salmonella enterica serovar Typhimurium population developed from an archival wild-type LT2 strain and intraperitoneally injected into a 6-month-old TRAMP (transgenic adenocarcinoma of mouse prostate) mouse. We compared the original injected strain to isolates recovered from prostate tumors and those recovered from the spleen and liver of non-tumor-bearing TRAMP mice through phenotypic assessments of bacteriophage susceptibility, motility, growth rates, morphology, and metabolic activity. Tumor isolate traits, particularly the loss of wild-type motility and flagella, reflect the selective pressure of the tumor, while the maintenance of bacteriophage resistance indicates no active selection to remove this robust trait. We posit that the Salmonella population adopts certain strategies to minimize energy consumption and maximize survival and proliferation once within the tumor. We find these insights to be nonnegligible considerations in the development of cancer therapies involving bacteria and suggest further examinations into the evolution of therapeutic strains during passage through tumors. Importance: Salmonella is of interest in cancer research due to its intrinsic abilities to selectively target, colonize, and replicate within tumors, leading to tumor cell death. However, mutation of strains during long-term colonization within tumors is a safety concern, and understanding their evolution within a tumor is an important consideration in predicting the long-term success of bacterium-based cancer treatment strategies. Thus, we have made an initial examination of phenotypically diverse Salmonella colonies recovered from a therapeutic Salmonella strain that we developed and injected into prostate tumor-bearing mice. We compared the bacteriophage susceptibility, motility, growth rates, morphology, and metabolic activity of the original therapeutic strain to those of strains recovered from prostate tumors of tumor-bearing mice and the liver and spleen of non-tumor-bearing mice. Our results suggest that the Salmonella population adopts certain strategies to minimize energy consumption and maximize survival and proliferation once within the tumor, leading to phenotypic changes in the strain.

Salmonella-host cell interactions, changes in host cell architecture, and destruction of prostate tumor cells with genetically altered Salmonella.

Increasingly, genetically modified Salmonella are being explored as a novel treatment for cancer because Salmonella preferentially replicate within tumors and destroy cancer cells without causing the septic shock that is typically associated with wild-type S. typhimurium infections. However, the mechanisms by which genetically modified Salmonella strains preferentially invade cancer cells have not yet been addressed in cellular detail. Here we present data that show S. typhimurium strains VNP20009, LT2, and CRC1674 invasion of PC-3M prostate cancer cells. S. typhimurium-infected PC-3M human prostate cancer cells were analyzed with immunofluorescence microscopy and transmission electron microscopy (TEM) at various times after inoculation. We analyzed microfilaments, microtubules, and DNA with fluorescence and immunofluorescence microscopy. 3T3 Phi-Yellow-mitochondria mouse 3T3 cells were used to study the effects of Salmonella infestation on mitochondria distribution in live cells. Our TEM results show gradual destruction of mitochondria within the PC-3M prostate cancer cells with complete loss of cristae at 8 h after inoculation. The fluorescence intensity in YFP-mitochondria-transfected mouse 3T3 cells decreased, which indicates loss of mitochondria structure. Interestingly, the nucleus does not appear affected by Salmonella within 8 h. Our data demonstrate that genetically modified S. typhimurium destroy PC-3M prostate cancer cells, perhaps by preferential destruction of mitochondria.