Bacterial delivery of therapeutic proteins to the nuclei of cancer cells.

Targeting nucleic targets with therapeutic proteins would enhance the treatment of hard-to-treat cancers. However, exogenous proteins are excluded from the nucleus by both the cellular and nuclear membranes. We have recently developed Salmonella that deliver active proteins into the cytoplasm of cancer cells. Here, we hypothesized that bacterially delivered proteins accumulate within nuclei, nuclear localization sequences (NLSs) increase delivery, and bacterially delivered proteins kill cancer cells. To test this hypothesis, we developed intranuclear delivering (IND) Salmonella and quantified the delivery of three model proteins. IND Salmonella delivered both ovalbumin and green fluorescent protein to nuclei of MCF7 cancer cells. The amount of protein in nuclei was linearly dependent on the amount delivered to the cytoplasm. The addition of a NLSs increased both the amount of protein in each nucleus and the number of nuclei that received protein. Delivery of Omomyc, a protein inhibitor of the nuclear transcript factor, Myc, altered cell physiology, and significantly induced cell death. These results show that IND Salmonella deliver functional proteins to the nucleus of cancerous cells. Extending this method to other transcription factors will increase the number of accessible targets for cancer therapy.

Targeting of pancreatic cancer cells and stromal cells using engineered oncolytic Salmonella typhimurium.

Pancreatic cancer is resistant to conventional therapeutic interventions, mainly due to abundant cancer stromal cells and poor immune cell infiltration. Here, we used a targeted cancer therapy approach based on attenuated Salmonella typhimurium engineered to express cytolysin A (ClyA) to target cancer stromal cells and cancer cells and treat pancreatic cancer in mice. Nude mice bearing subcutaneous or orthotopic human pancreatic cancers were treated with engineered S. typhimurium expressing ClyA. The tumor microenvironment was monitored to analyze stromal cell numbers, stromal cell marker expression, and immune cell infiltration. The attenuated bacteria accumulated and proliferated specifically in tumor tissues after intravenous injection. The bacteria secreted ClyA into the tumor microenvironment. A single dose of ClyA-expressing Salmonella markedly inhibited growth of pancreatic cancer both in subcutaneous xenograft- and orthotopic tumor-bearing nude mice. Histological analysis revealed a marked decrease in expression of stromal cell markers and increased immune cell (neutrophils and macrophages) infiltration into tumors after colonization by ClyA-expressing bacteria. ClyA-expressing S. typhimurium destroyed cancer stromal cells and cancer cells in mouse models of human pancreatic cancer. This approach provides a novel strategy for combining anticancer and anti-stromal therapy to treat pancreatic cancer.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

Engineered bacteria detect spatial profiles in glucose concentration within solid tumor cell masses.

Tumor heterogeneity makes cancer difficult to treat. Many small molecule cancer drugs target rapidly dividing cells on the periphery of tumors but have difficulty in penetrating deep into tumors and are ineffective at treating entire tumors. Targeting both rapidly dividing and slower growing regions of tumors is essential to effectively treat cancer. A cancer drug carrier that penetrates deep into tumors and identifies metabolically activity could supply treatment to those areas based on the local microenvironment. We hypothesized that glucose sensing bacteria could identify sugar gradients in solid tumors. To test this hypothesis, a genetic circuit was designed to trigger expression of a green fluorescent protein (GFP) reporter through the chemotaxis-osmoporin fusion protein, Trz1, a receptor for sensing glucose and ribose sugars. E. coli equipped with the Trz1-GFP expression system, were administered to an in vitro model of a continuously perfused tumor tissue that mimics systemic delivery and clearance of bacteria through a blood vessel adjacent to a solid tumor. The level of GFP expressed, per bacterium, was time independent and indicated the glucose concentration as a function of penetration depth within the microfluidic tumors. The measured glucose concentration, correlated (P-value = 2.6 × 10(-5) ) with tumor cell viability as a function of depth. Mathematical analysis predicted drug delivery by glucose-sensing bacteria would eliminate a higher percentage of the viable tumor cell population than a systemically administered drug. Glucose-sensing bacteria could deliver cancer therapies with increased drug penetration and nutrient-dependent dosing to continuously treat viable regions of cancer tissue that have a higher prevalence for metastatic dissemination. Biotechnol. Bioeng. 2016;113: 2474-2484. © 2016 Wiley Periodicals, Inc.

Persistent enhancement of bacterial motility increases tumor penetration.

Motile bacteria can overcome the transport limitations that hinder many cancer therapies. Active bacteria can penetrate through tissue to deliver treatment to resistant tumor regions. Bacterial therapy has had limited success, however, because this motility is heterogeneous, and within a population many individuals are non-motile. In human trials, heterogeneity led to poor dispersion and incomplete tumor colonization. To address these problems, a swarm-plate selection method was developed to increase swimming velocity. Video microscopy was used to measure the velocity distribution of selected bacteria and a microfluidic tumor-on-a-chip device was used to measure penetration through tumor cell masses. Selection on swarm plates increased average velocity fourfold, from 4.9 to 18.7 µm/s (P < 0.05) and decreased the number of non-motile individuals from 51% to 3% (P < 0.05). The selected phenotype was both robust and stable. Repeating the selection process consistently increased velocity and eliminated non-motile individuals. When selected strains were cryopreserved and subcultured for 30.1 doublings, the high-motility phenotype was preserved. In the microfluidic device, selected Salmonella penetrated deeper into cell masses than unselected controls. By 10 h after inoculation, control bacteria accumulated in the front 30% of cell masses, closest to the flow channel. In contrast, selected Salmonella accumulated in the back 30% of cell masses, farthest from the channel. Selection increased the average penetration distance from 150 to 400 µm (P < 0.05). This technique provides a simple and rapid method to generate high-motility Salmonella that has increased penetration and potential for greater tumor dispersion and clinical efficacy.

Effect of Antibiotic Treatment on Attenuated Salmonella typhimurium VNP20009 Mediated Schwannoma Growth Control.

BACKGROUND/AIM: This study evaluated the effect of enrofloxacin antibiotic treatment on the ability of an attenuated Salmonella typhimurium (S. typhimurium) strain VNP20009 to control schwannoma growth in a preclinical mouse schwannoma tumor model. MATERIALS AND METHODS: The antitumor efficacy of VNP20009 intratumoral (i.t.) injection was assessed in a syngeneic mouse-NF2 schwannoma model, with and without subcutaneous (s.c.) injection of enrofloxacin beginning on day-1 or day-8 post-VNP20009 injection. S. typhimurium colonization was assessed in excised tumor samples following antibiotic treatment. RESULTS: I.t. injection of the VNP20009 significantly decreased the growth of schwannoma tumors in mice compared to PBS-treated controls. Treatment of mice with enrofloxacin on day-1 post-VNP20009 injection resulted in abrogation of VNP20009-mediated tumor growth control. In contrast, tumor growth in i.t. VNP20009-injected mice infused with enrofloxacin beginning on day 8 was significantly decreased compared to i.t. PBS-injected controls. Enrofloxacin significantly reduced the number of viable VNP20009 bacteria in excised tumor samples within one day of antibiotic infusion. Viable bacteria were either few or essentially eliminated at the end of the experiment in antibiotic-treated animals compared to VNP20009-only. CONCLUSION: Viable VNP20009 can persist for as long as 2.5 weeks following intratumoral injection of schwannoma, during which time tumor growth is retarded. Antibiotic treatment starting 1-day following i.t. VNP20009 abrogated bacterial tumor growth control, whereas initiation of antibiotics 8-days following i.t. VNP20009 was associated with control of tumor growth, albeit less than seen in animals unexposed to antibiotics.

Culture of attenuated Salmonella Typhimurium VNP20009 in animal-product-free media does not alter schwannoma growth control.

Schwannomas are slow-growing benign peripheral nerve sheath tumors derived from Schwann-lineage cells that develop in association with NF2-related schwannomatosis (NF2) and schwannomatosis (NF3), as well as spontaneously. Individuals affected with NF2 and NF3 have multiple schwannomas with tumors arising throughout life. Surgical resection, the standard management, is limited in scope and efficacy and is itself associated with significant morbidity. We have previously shown that direct intratumoral injection of attenuated Salmonella Typhimurium (S. Typhimurium), strain VNP20009, showed a potent anti-tumor effect in preclinical NF-2 schwannoma models. The United States Federal Drug Administration (FDA) requires that bacterial products utilized in clinical trials be produced without exposure to animal-derived-products. In this context, we developed, characterized, and tested the antitumor efficacy of an attenuated S. Typhimurium serially passaged in animal-product-free media, naming it VNP20009-AF for "VNP20009-animal-product-free." Our in vitro data did not indicate any significant changes in the viability, motility, or morphology of VNP20009-AF, compared to its parental strain. In vivo efficacy data demonstrated that VNP20009-AF and VNP20009 controlled tumor growth to the same degree in both human NF2-schwannoma xenograft and murine-NF2 schwannoma allograft models. Together, these data support the use of VNP20009-AF for the translation of bacterial schwannoma therapy into clinical trials.

ECM-targeting bacteria enhance chemotherapeutic drug efficacy by lowering IFP in tumor mouse models.

Bacterial cancer therapies aim to manipulate bacteria to effectively deploy therapeutic payloads to tumors. Attenuated bacteria alone often cannot eradicate solid tumors. Attenuated Salmonella can be engineered to deliver cytotoxic drugs to either trigger an immune response or increase antitumor efficacy when combined with chemotherapeutic drugs. However, the extracellular matrix (ECM) surrounding cancer cells forms a barrier that often limits the ability of chemotherapeutic and cytotoxic drugs to penetrate and eliminate tumors. To overcome this limitation, we developed a strategy to combine chemotherapy with an attenuated Salmonella typhimurium strain engineered to secrete HysA protein (from Staphylococcus aureus; Hyaluronidase, HAase) in tumors. The engineered Salmonella effectively degraded hyaluronan (HA), which is a major ECM constituent in tumors, and suppressed tumor growth in mouse models of pancreatic adenocarcinoma (ASPC-1) and breast cancer (4T1). Furthermore, it prolonged survival when combined with chemotherapeutic drugs (doxorubicin or gemcitabine). Upon bacterial colonization, the HAase-mediated ECM degradation decreased interstitial fluid pressure (IFP) in the tumor microenvironment. Additionally, HA degradation using HAase-expressing bacteria in vivo led to decreased binding to the receptor, CD44, expressed in tumors. This may modulate proliferation- and apoptosis-related signal pathways. Therefore, ECM-targeting bacteria can be used as a synergistic anticancer therapeutic agent to maximize chemotherapeutic drug delivery into highly invasive tumors.

Blautia coccoides JCM1395T Achieved Intratumoral Growth with Minimal Inflammation: Evidence for Live Bacterial Therapeutic Potential by an Optimized Sample Preparation and Colony PCR Method.

We demonstrate that Blautia coccoides JCM1395T has the potential to be used for tumor-targeted live bacterial therapeutics. Prior to studying its in vivo biodistribution, a sample preparation method for reliable quantitative analysis of bacteria in biological tissues was required. Gram-positive bacteria have a thick outer layer of peptidoglycans, which hindered the extraction of 16S rRNA genes for colony PCR. We developed the following method to solve the issue; the method we developed is as follows. The homogenates of the isolated tissue were seeded on agar medium, and bacteria were isolated as colonies. Each colony was heat-treated, crushed with glass beads, and further treated with restriction enzymes to cleave DNAs for colony PCR. With this method, Blautia coccoides JCM1395T and Bacteroides vulgatus JCM5826T were individually detected from tumors in mice intravenously receiving their mixture. Since this method is very simple and reproducible, and does not involve any genetic modification, it can be applied to exploring a wide range of bacterial species. We especially demonstrate that Blautia coccoides JCM1395T efficiently proliferate in tumors when intravenously injected into tumor-bearing mice. Furthermore, these bacteria showed minimal innate immunological responses, i.e., elevated serum tumor necrosis factor α and interleukin-6, similar to Bifidobacterium sp., which was previously studied as a therapeutic agent with a small immunostimulating effect.

Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria.

Bacterial cancer therapy is a promising next-generation modality to treat cancer that often uses tumor-colonizing bacteria to deliver cytotoxic anticancer proteins. However, the expression of cytotoxic anticancer proteins in bacteria that accumulate in the nontumoral reticuloendothelial system (RES), mainly the liver and spleen, is considered detrimental. This study examined the fate of the Escherichia coli strain MG1655 and an attenuated strain of Salmonella enterica serovar Gallinarum (S. Gallinarum) with defective ppGpp synthesis after intravenous injection into tumor-bearing mice (~108 colony forming units/animal). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 109 colony forming units/g tissue, whereas those in the RES died off. RNA analysis revealed that tumor-associated E. coli activated rrnB operon genes encoding the rRNA building block of ribosome needed most during the exponential stage of growth, whereas those in the RES expressed substantially decreased levels of this gene and were cleared soon presumably by innate immune systems. Based on this finding, we engineered ΔppGpp S. Gallinarum to express constitutively a recombinant immunotoxin comprising TGFα and the Pseudomonas exotoxin A (PE38) using a constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1. The construct exerted anticancer effects on mice grafted with mouse colon (CT26) or breast (4T1) tumor cells without any notable adverse effects, suggesting that constitutive expression of cytotoxic anticancer protein from rrnB P1 occurred only in tumor tissue.

Comparison of Anticancer Activities and Biosafety Between Salmonella enterica Serovar Typhimurium ΔppGpp and VNP20009 in a Murine Cancer Model.

Salmonella Typhimurium defective in guanosine 5'-diphosphate-3'-diphosphate (ppGpp) synthesis (ΔppGpp) is an attenuated strain with good biosafety and excellent anticancer efficacy. It has been widely applied in preclinical studies of anticancer therapy for various types of solid cancer. VNP20009 is another genetically modified auxotrophic strain with 108-kb deletion, purI- , msbB- , and many single nucleotide polymorphisms (SNPs); it has shown promising therapeutic efficacy in various preclinical tumor models and entered phase I clinical trials. Here, the invasion activities and virulence of ΔppGpp were obviously lower than those of the VNP20009 strain when tested with cancer cells in vitro. In addition, the MC38 tumor-bearing mice showed comparable cancer suppression when treated with ΔppGpp or VNP20009 intravenously. However, the ΔppGpp-treated mice showed 16.7% of complete cancer eradication and prolonged survival in mice, whereas VNP20009 showed higher toxicity to animals, even with equal tumor size individually. Moreover, we found decreased levels of inflammatory cytokines in circulation but strengthened immune boost in tumor microenvironments of ΔppGpp-treated mice. Therefore, the engineered ΔppGpp has high potential for cancer therapeutics, and it is a promising option for future clinical cancer therapy.

Tumor Colonization and Therapy by Escherichia coli Nissle 1917 Strain in Syngeneic Tumor-Bearing Mice Is Strongly Affected by the Gut Microbiome.

In the past, different bacterial species have been tested for cancer therapy in preclinical and clinical studies. The success of bacterial cancer therapy is mainly dependent on the ability of the utilized bacteria to overcome the host immune defense system to colonize the tumors and to initiate tumor-specific immunity. In recent years, several groups have demonstrated that the gut microbiome plays an important role of modulation of the host immune response and has an impact on therapeutic responses in murine models and in cohorts of human cancer patients. Here we analyzed the impact of the gut microbiome on tumor colonization and tumor therapy by the Escherichia coli Nissle 1917 (EcN) strain. This EcN strain is a promising cancer therapy candidate with probiotic properties. In our study, we observed significantly better tumor colonization by EcN after antibiotic-induced temporal depletion of the gut microbiome and after two intranasal applications of the EcN derivate (EcN/pMUT-gfp Knr) in 4T1 tumor-bearing syngeneic BALB/c mice. In addition, we demonstrated significant reduction in tumor growth and extended survival of the EcN-treated mice in contrast to phosphate-buffered saline (PBS)-treated tumor-bearing control animals. Multispectral imaging of immune cells revealed that depletion of the gut microbiome led to significantly lower infiltration of cytotoxic and helper T cells (CD4 and CD8 cells) in PBS tumors of mice pretreated with antibiotics in comparison with antibiotic untreated PBS-or EcN treated mice. These findings may help in the future advancement of cancer treatment strategies using E. coli Nissle 1917.

Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments.

Bacteria equipped with genetically encoded lactate biosensors are promising tools for biopharmaceutical production, diagnostics, and cellular therapies. However, many applications involve glucose-rich and anoxic environments, in which current whole-cell lactate biosensors show low performance. Here we engineer an optimized, synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite and anoxic repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expressions to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), can operate in glucose-rich, aerobic and anoxic conditions. We show that ALPaGA works reliably in the probiotic chassisEscherichia coliNissle 1917 and can detect endogenous l-lactate produced by 3D tumor spheroids with an improved dynamic range. In the future, the ALPaGA system could be used to monitor bioproduction processes and improve the specificity of engineered bacterial cancer therapies by restricting their activity to the lactate-rich microenvironment of solid tumors.

Salmonella enterica Typhimurium engineered for nontoxic systemic colonization of autochthonous tumors.

Much of the bacterial anticancer therapy being developed relies on the ability of bacteria to specifically colonise tumours. Initial attempts to translate promising Salmonella enterica Typhimurium (S. Typhimurium) preclinical results to the clinical setting failed, primarily due to lack of tumour colonisation and the significant toxicities from systemically administered Gram-negative bacteria. To address the difference in results between preclinical experiments performed in mice with transplant tumours and clinical trials in human volunteers with autochthonous tumours, a genetically engineered mouse model of breast cancer (BALB-neuT) was utilised to develop a strain of virulence-attenuated S. Typhimurium capable of robust colonisation of autochthonous tumours. Several genes that code for bacterial surface molecules, responsible for signalling a toxic immune response against the bacteria, were mutated. The resulting S. Typhimurium strain, BCT2, allowed non-toxic intravenous administration of 3 × 106 colony forming units of bacteria in tumour-burdened mice when combined with a vascular disruption agent to induce intratumoral necrotic space and facilitate bacterial colonisation.

Virulence-attenuated Salmonella engineered to secrete immunomodulators reduce tumour growth and increase survival in an autochthonous mouse model of breast cancer.

The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.

Recent Progress in the Molecular Imaging of Tumor-Treating Bacteria.

Bacterial cancer therapy (BCT) approaches have been extensively investigated because bacteria can show unique features of strong tropism for cancer, proliferation inside tumors, and antitumor immunity, while bacteria are also possible agents for drug delivery. Despite the rapidly increasing number of preclinical studies using BCT to overcome the limitations of conventional cancer treatments, very few BCT studies have advanced to clinical trials. In patients undergoing BCT, the precise localization and quantification of bacterial density in different body locations is important; however, most clinical trials have used subjective clinical signs and invasive sampling to confirm bacterial colonization. There is therefore a need to improve the visualization of bacterial densities using noninvasive and repetitive in vivo imaging techniques that can facilitate the clinical translation of BCT. In vivo optical imaging techniques using bioluminescence and fluorescence, which are extensively employed to image the therapeutic process of BCT in small animal research, are hard to apply to the human body because of their low penetrative power. Thus, new imaging techniques need to be developed for clinical trials. In this review, we provide an overview of the various in vivo bacteria-specific imaging techniques available for visualizing tumor-treating bacteria in BCT studies.

Bacterial proteolytic activity improves drug delivery in tumors in a size, pharmacokinetic, and binding affinity dependent manner - A mechanistic understanding.

Motile bacteria are able to penetrate in the distal areas of blood vessel, which makes bacteria attractive to researchers as a drug delivery vehicle carrying anti-cancer drugs to tumors. Not only therapeutic bacteria show wide anti-tumor effect but also the combination of therapeutic bacteria and conventional chemotherapy leads to dramatically large synergetic effect. We provide a mechanistic understanding of enhanced drug delivery in tumors by co-administration of chemotherapeutic agents and therapeutic bacteria. In this work, simultaneous delivery of C. novyi-NT and chemotherapeutic agents in tumors is mathematically modeled. Simulated doxorubicin concentration in tumors after Doxil administration with or without bacteria agreed reasonably well with experimental literature. Simulated doxorubicin concentration in tumors by the combination of Doxil and C. novyi-NT is over twice higher than that of Doxil alone. This enhanced doxorubicin concentration in tumors is due to the degradation of extracellular matrix of collagen by bacterial proteolytic activity, which increases hydraulic conductivity of interstitium, reduces interstitial fluid pressure, and thus increases convection through vessel walls. Additionally, it alleviates solid stress, which decompresses blood vessels, and thus increases vessel density. On the other hand, simulated doxorubicin concentration in tumors for non-liposomal free-doxorubicin is not enhanced by C. novyi-NT because vascular permeability of free-doxorubicin is larger than Doxil, and thus increased but relatively small convection across vessel walls is offset by the efflux due to increased interstitial flow. A strategy to further enhance this combination therapy is discussed along with sensitivity analysis.

Inhibition of acute leukemia with attenuated Salmonella typhimurium strain VNP20009.

Acute leukemia is a common hematological malignancy. Despite recent promising progress, the prognosis of acute leukemia patients remains to be improved. New therapies are therefore still needed. Salmonella typhimurium has been shown to be highly effective as an anti-tumor agent in many solid cancer models, but it has not been applied in acute leukemia. Here, we report an attenuated Salmonella typhimurium strain, VNP20009, can induce apoptosis in multiple types of leukemia cells both in vivo and in vitro. Furthermore, VNP20009 significantly inhibited the proliferation of MLL-AF9-induced acute myeloid leukemia cells and prolonged the survival of the AML-carrying mice. VNP20009 restored the counts of white blood cell (WBC) and its five subsets in peripheral blood (PB) to near-physiological values, and elevated the levels of certain cytokines, such as tumor necrosis factor-α (TNF-α), leukemia inhibitory factor (LIF), interferon-γ (IFN-γ), chemokine C-X-C motif ligand-10 (CXCL-10) and C-C motif ligand-2 (CCL-2). Moreover, the ratio of immune cells, including natural killer cells (NKs), CD4+ Th1-type cells and CD8+ IFN-γ-producing effector T cells were highly upregulated in the AML mice treated with VNP20009. The results of the present study potentially provide an alternative therapeutic strategy for hematologic malignancies through boosting the innate and adaptive anti-tumor immunity.

Imaging of tumor colonization by Escherichia coli using 18F-FDS PET.

Tumor-targeting bacteria have been actively investigated as a new therapeutic tool for solid tumors. However, in vivo imaging of tumor-targeting bacteria has not been fully established. 18F-fluorodeoxysorbitol (FDS) positron emission tomography (PET) is known to be capable of imaging Gram-negative Enterobacteriaceae infection. In the present study, we aimed to validate the use of 18F-FDS PET for visualization of the colonization and proliferation of tumor-targeting Escherichia coli (E. coli) MG1655 in mouse tumor models. Methods:E. coli (5 × 107 colony forming unit) were injected intravenously into BALB/c mice bearing mouse colon cancer (CT26). Before and 1, 3, and 5 days after the bacterial injection, PET imaging was performed following i.v. injection of approximately 7.4 MBq of 18F-FDS. Regions of interest were drawn in the engrafted tumor and normal organs including the heart, liver, lung, brain, muscle, and intestine. Semiquantitative analysis was performed using maximum standardized uptake value (SUVmax). Results:18F-FDS uptake was significantly higher in tumors colonized by live E. coli MG1655 than in uncolonized tumors (p < 0.001). The PET signals in the colonized tumors at 3 days after bacterial injection were 3.1-fold higher than those in the uncolonized tumors. Tumoral 18F-FDS uptake correlated very strongly with the number of E. coli in tumors (r = 0.823, p < 0.0001). Cross sectional analysis of autoradiography, bioluminescence, and pathology revealed that the 18F-FDS uptake sites in tumors matched the locations of E. coli MG1655. Conclusion: In conclusion, 18F-FDS PET is expected to be useful for the semiquantitative visualization of tumor-targeting bacteria when bacterial cancer therapy is performed using Gram-negative Enterobacteriaceae such as E. coli.

Targeted Delivery of the Mitochondrial Target Domain of Noxa to Tumor Tissue via Synthetic Secretion System in E. coli.

Targeted delivery of drugs is a key aspect of the successful treatment of serious conditions such as tumors. In the pursuit of accurate delivery with high specificity and low size limit for peptide drugs, a synthetic type 3 secretion system (T3SS) has been repurposed from a native genetic system encoded in Salmonella pathogenicity island-1 (SPI-1) with no virulence effectors. Here, we tested the potential of synthetic T3SS as drug delivery machinery for peptide-based drugs owing to its modular nature. First, the genetic system for synthetic T3SS was introduced into non-native host E. coli, which was chosen for its lack of Salmonella-driven virulence factors. Next, the mitochondrial targeting domain (MTD) of Noxa was tested as a cargo protein with anti-tumor activity. To this end, the gene encoding MTD was engineered for secretion through synthetic T3SS, thereby resulting in the tagged MTD at the N-terminus. When E. coli carrying synthetic T3SS and MTD on plasmids was administered into tumor-bearing mice, MTD with a secretion tag at the N-terminus was clearly detected in the tumor tissue after induction. Also, the tumor growth and mortality of tumor-bearing animals were mitigated by the cytotoxic activity of the delivered. Thus, this work potentiates the use of biotherapeutic bacteria for the treatment of tumors by implanting a dedicated delivery system.