Exploiting bacteria for cancer immunotherapy.

Immunotherapy has revolutionized the treatment of cancer but continues to be constrained by limited response rates, acquired resistance, toxicities and high costs, which necessitates the development of new, innovative strategies. The discovery of a connection between the human microbiota and cancer dates back 4,000 years, when local infection was observed to result in tumour eradication in some individuals. However, the true oncological relevance of the intratumoural microbiota was not recognized until the turn of the twentieth century. The intratumoural microbiota can have pivotal roles in both the pathogenesis and treatment of cancer. In particular, intratumoural bacteria can either promote or inhibit cancer growth via remodelling of the tumour microenvironment. Over the past two decades, remarkable progress has been made preclinically in engineering bacteria as agents for cancer immunotherapy; some of these bacterial products have successfully reached the clinical stages of development. In this Review, we discuss the characteristics of intratumoural bacteria and their intricate interactions with the tumour microenvironment. We also describe the many strategies used to engineer bacteria for use in the treatment of cancer, summarizing contemporary data from completed and ongoing clinical trials. The work described herein highlights the potential of bacteria to transform the landscape of cancer therapy, bridging ancient wisdom with modern scientific innovation.

Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

PURPOSE: A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. PROCEDURES: We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. RESULTS: Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. CONCLUSIONS: We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

Engineering of an EPHA2-Targeted Monobody for the Detection of Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common cancer worldwide, and is second only to lung cancer with respect to cancer-related deaths. Noninvasive molecular imaging using established markers is a new emerging method to diagnose CRC. The human ephrin receptor family type-A 2 (hEPHA2) oncoprotein is overexpressed at the early, but not late, stages of CRC. Previously, we reported development of an E1 monobody that is specific for hEPHA2-expressing cancer cells both in vitro and in vivo. Herein, we investigated the ability of the E1 monobody to detect hEPHA2 expressing colorectal tumors in a mouse model, as well as in CRC tissue. MATERIALS AND METHODS: The expression of hEPHA2 on the surface of CRC cells was analyzed by western blotting and flow cytometry. The targeting efficacy of the E1 monobody for CRC cells was examined by flow cytometry, and immunofluorescence staining. E1 conjugated to the Renilla luciferase variant 8 (Rluc8) reporter protein was used for in vivo imaging in mice. Additionally, an enhanced green fluorescence protein (EGFP) conjugated E1 monobody was used to check the ability of the E1 monobody to target CRC tissue. RESULTS: The E1 monobody bound efficiently to hEPHA2-expressing CRC cell lines, and E1 conjugated to the Rluc8 reporter protein targeted tumor tissues in mice transplanted with HCT116 CRC tumor cells. Finally, E1-EGFP stained tumor tissues from human CRC patients, showing a pattern similar to that of an anti-hEPHA2 antibody. CONCLUSION: The E1 monobody has utility as an EPHA2 targeting agent for the detection of CRC.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Reprogramming a Doxycycline-Inducible Gene Switch System for Bacteria-Mediated Cancer Therapy.

PURPOSE: Attenuated Salmonella typhimurium is a potential biotherapeutic antitumor agent because it can colonize tumors and inhibit their growth. The present study aimed to develop a doxycycline (Doxy)-inducible gene switch system in attenuated S. typhimurium and assess its therapeutic efficacy in various tumor-bearing mice models. PROCEDURES: A Doxy-inducible gene switch system comprising two plasmids was engineered to trigger the expression of cargo genes (Rluc8 and clyA). Attenuated S. typhimurium carrying Rluc8 were injected intravenously into BALB/c mice bearing CT26 tumors, and bioluminescence images were captured at specified intervals post-administration of doxycycline. The tumor-suppressive effects of bacteria carrying clyA were evaluated in BALB/c mice bearing CT26 tumors and in C57BL/6 mice bearing MC38 tumors. RESULTS: Expression of the fimE gene, induced only in the presence of Doxy, triggered a unidirectional switch of the POXB20 promoter to induce expression of the cargo genes. The switch event was maintained over a long period of bacterial culture. After intravenous injection of transformed Salmonella into mice bearing CT26 tumors, the bacteria transformed with the Doxy-inducible gene switch system for Rluc8 targeted only tumor tissues and expressed the payloads 2 days after Doxy treatment. Notably, bacteria carrying the Doxy-inducible gene switch system for clyA effectively suppressed tumor growth and prolonged survival, even after just one Doxy induction. CONCLUSIONS: These results suggest that attenuated S. typhimurium carrying this novel gene switch system elicited significant therapeutic effects through a single induction triggering and were a potential biotherapeutic agent for tumor therapy.

Yb Substitution and Ultralow Thermal Conductivity of the Ca3-xYbxAlSb3 (0 ≤ x ≤ 0.81(1)) System.

A series of Yb-substituted Zintl phases in the Ca3-xYbxAlSb3 (0 ≤ x ≤ 0.81(1)) system has been synthesized by initial arc melting and post-heat treatment, and their isotypic crystal structures were characterized by both powder and single crystal X-ray diffraction analysis. All four title compounds adopted the Ca3AlAs3-type structure (space group Pnma, Pearson code oP28, Z = 4). The overall structure can be described as a combination of the 1-dimensional (1D) infinite chain of ∞1[Al(Sb2Sb2/2)] formed by two vertices sharing [AlSb4] tetrahedral moieties and three Ca2+/Yb2+ mixed sites located in between these 1D chains. The charge balance and the resultant independency of the 1D chains in the title system were explained by the Zintl-Klemm formalism [Ca2+/Yb2+]3[(4b-Al1-)(1b-Sb2-)2(2b-Sb1-)2/2]. A series of DFT calculations proved that (1) the band overlap between the d-orbital states from two types of cations and the p-orbital states from Sb at the high symmetry Γ point implied a heavily doped degenerate semiconducting behavior of the quaternary Ca2YbAlSb3 model and (2) the site preference of Yb for the M1 site was due to the electronic-factor criterion based on the Q values of each atomic site. The electron localization function calculations also proved that the two different shapes of lone pairs of the Sb atoms─the "umbrella-shape" and the "C-shape"─are determined by local geometry and the coordination environment on the anionic frameworks. Thermoelectric measurements of the quaternary title compound Ca2.19(1)Yb0.81AlSb3 showed an approximately two times larger ZT value than that of ternary Ca3AlSb3 at 623 K due to increased electrical conductivity and ultralow thermal conductivity originated from Yb substitution for Ca.

Synergistic cancer immunotherapy utilizing programmed Salmonella typhimurium secreting heterologous flagellin B conjugated to interleukin-15 proteins.

The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver therapeutic payloads. The present study describes the engineering of an attenuated strain of Salmonella typhimurium deficient in ppGpp biosynthesis (SAM) that could secrete Vibrio vulnificus flagellin B (FlaB) conjugated to human (hIL15/FlaB) and mouse (mIL15/FlaB) interleukin-15 proteins in the presence of L-arabinose (L-ara). These strains, named SAMphIF and SAMpmIF, respectively, secreted fusion proteins that retained bioactivity of both FlaB and IL15. SAMphIF and SAMpmIF inhibited the growth of MC38 and CT26 subcutaneous (sc) tumors in mice and increased mouse survival rate more efficiently than SAM expressing FlaB alone (SAMpFlaB) or IL15 alone (SAMpmIL15 and SAMphIL15), although SAMpmIF had slightly greater antitumor activity than SAMphIF. The mice treated with these bacteria showed enhanced macrophage phenotype shift, from M2-like to M1-like, as well as greater proliferation and activation of CD4+ T, CD8+ T, NK, and NKT cells in tumor tissues. After tumor eradication by these bacteria, ≥50% of the mice show no evidence of tumor recurrence upon rechallenge with the same tumor cells, indicating that they had acquired long-term immune memory. Treatment of mice of 4T1 and B16F10 highly malignant sc tumors with a combination of these bacteria and an immune checkpoint inhibitor, anti-PD-L1 antibody, significantly suppressed tumor metastasis and increased mouse survival rate. Taken together, these findings suggest that SAM secreting IL15/FlaB is a novel therapeutic candidate for bacterial-mediated cancer immunotherapy and that its antitumor activity is enhanced by combination with anti-PD-L1 antibody.

The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy.

L-Asparaginase (L-ASNase), a bacterial enzyme that degrades asparagine, has been commonly used in combination with several chemical drugs to treat malignant hematopoietic cancers such as acute lymphoblastic leukemia (ALL). In contrast, the enzyme was known to inhibit the growth of solid tumor cells in vitro, but not to be effective in vivo. We previously reported that two novel monobodies (CRT3 and CRT4) bound specifically with calreticulin (CRT) exposed on tumor cells and tissues during immunogenic cell death (ICD). Here, we engineered L-ASNases conjugated with monobodies at the N-termini and PAS200 tags at the C-termini (CRT3LP and CRT4LP). These proteins were expected to possess four monobody and PAS200 tag moieties, which did not disrupt the L-ASNase conformation. These proteins were expressed 3.8-fold more highly in E. coli than those without PASylation. The purified proteins were highly soluble, with much greater apparent molecular weights than expected ones. Their affinity (Kd) against CRT was about 2 nM, 4-fold higher than that of monobodies. Their enzyme activity (∼6.5 IU/nmol) was similar to that of L-ASNase (∼7.2 IU/nmol), and their thermal stability was significantly increased at 55 °C. Their half-life times were > 9 h in mouse sera, about 5-fold longer than that of L-ASNase (∼1.8 h). Moreover, CRT3LP and CRT4LP bound specifically with CRT exposed on tumor cells in vitro, and additively suppressed the tumor growth in CT-26 and MC-38 tumor-bearing mice treated with ICD-inducing drugs (doxorubicin and mitoxantrone) but not with a non-ICD-inducing drug (gemcitabine). All data indicated that PASylated CRT-targeted L-ASNases enhanced the anticancer efficacy of ICD-inducing chemotherapy. Taken together, L-ASNase would be a potential anticancer drug for treating solid tumors.

Genetic Programming by Nitric Oxide-Sensing Gene Switch System in Tumor-Targeting Bacteria.

Recent progress in synthetic biology has enabled bacteria to respond to specific disease signals to perform diagnostic and/or therapeutic tasks. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) colonization of tumors results in increases in nitric oxide (NO) levels, suggesting that NO may act as a candidate inducer of tumor-specific gene expression. The present study describes a NO-sensing gene switch system for triggering tumor-specific gene expression in an attenuated strain of S. Typhimurium. The genetic circuit was designed to sense NO via NorR, thus initiating the expression of FimE DNA recombinase. This was found to lead sequentially to the unidirectional inversion of a promoter region (fimS), which induced the expression of target genes. Target gene expression in bacteria transformed with the NO-sensing switch system was triggered in the presence of a chemical source of NO, diethylenetriamine/nitric oxide (DETA/NO) in vitro. In vivo results revealed that the gene expression is tumor-targeted, and specific to NO generated by inducible nitric oxide synthase (iNOS) after S. Typhimurium colonization. These results showed that NO was a promising inducer to finely tune the expression of target genes carried by tumor-targeting bacteria.

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.

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria.

PURPOSE: In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. PROCEDURES: In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR. RESULTS: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. CONCLUSIONS: Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Description of Anaerostipes faecalis sp. nov., a new segmented filamentous bacterium isolated from swine faeces.

A novel, strictly anaerobic, gram-negative, segmented filamentous bacterium strain AGMB03513T, was isolated from the faeces of a 5-month-old pig. Phylogenetic analysis based on the 16S rRNA gene indicated that the isolate was a member of the family Lachnospiraceae, and the closest strain was Anaerostipes butyraticus. Strain AGMB03513T formed a lineage within the genus Anaerostipes and was closely related to A. butyraticus DSM 22094 T (= KCTC 15125 T, 95.8%), Anaerostipes hadrus DSM 3319 T (= KCTC 15606 T, 95.5%), Anaerostipes caccae DSM 14662 T (= KCTC 15019 T, 94.0%), and Anaerostipes rhamnosivorans DSM 26241 T (= KCTC 15316 T, 93.4%). Strain AGMB03513T grew at temperatures between 30 and 45 °C, within a pH range of 7.0-9.0, and in medium containing up to 1.5% NaCl. Cells were found to utilise D-glucose, D-mannitol, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-mannose, and D-sorbitol, and acetate was identified as the major end product of metabolism. The major components of the cellular fatty acids were C12:0, C16:0, and C18:0. In addition, the bacterium contained meso-diaminopimelic acid in the cell wall. According to the comparative analysis of the whole genome sequence, the DNA G + C content of strain AGMB03513 was 37.0 mol%. In addition, Average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridisation (dDDH) values were obtained in comparisons of strain AGMB03513T with reference strains of species in the genus Anaerostipes. ANI values were found to be between 71.0 and 75.7%, AAI values between 66.6 and 73.2%, and dDDH values between 19.5 and 21.4%. All the data were below the threshold range for species determination. Based on phenotypic, phylogenetic, biochemical, chemotaxonomic, and genomic characteristics, we considered it reasonable to assign a novel species status to strain AGMB03513T, for which we propose the name Anaerostipes faecalis sp. nov. The type strain is AGMB03513T (= KCTC 25020 T = NBRC 114896 T).

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy.

Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein-engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

Imaging Calreticulin for Early Detection of Immunogenic Cell Death During Anticancer Treatment.

Surface-exposed calreticulin (ecto-CRT) is a well-known "eat-me" signal exhibited by dying cells that contributes to their recognition and destruction by the immune system. We assessed the use of a CRT-specific binding peptide for imaging ecto-CRT during immunogenic cell death and its utility for early prediction of treatment response. Methods: A synthetic CRT-specific peptide, KLGFFKR (CRTpep), was labeled with fluorescein isothiocyanate or 18F, and the characteristics of ecto-CRT were evaluated in a colon cancer cell line in vitro and in vivo. Results: In vitro flow cytometry, immunofluorescence staining, and in vivo small-animal PET imaging results showed that CRTpep detected preapoptotic cells treated with immunogenic drugs or radiation but not those treated with the nonimmunogenic drug or a nontherapeutic dose of immunogenic drug. Conclusion: The present results indicate that the CRT-specific peptide would enable the prediction of therapeutic response, thereby facilitating early decisions on continuation or discontinuation of immunogenic treatment.

Production of 64Cu-labeled monobody for imaging of human EphA2-expressing tumors.

We previously reported on the monobody E1, which specifically targets the tumor marker hEphA2. In this study, we labeled NOTA-conjugated E1 with 64Cu (64Cu-NOTA-E1) and evaluated biologic characteristics. The uptake of 64Cu-NOTA-E1 in PC3 cells (a human prostate cancer cell line) with high expression of hEphA2 increased in a time-dependent manner. In PC3 xenograft mice, 64Cu-NOTA-E1 injected via the tail vein allowed visualization of tumors on positron emission tomography after 1 h and the highest uptake measured at 24 h post-injection. By contrast, the radioactivity of other tissues either did not increase or decreased over 24 h. This indicates that 64Cu-NOTA-E1 has high tumor uptake and retention, with rapid clearance, and low background values in other tissues. Therefore, 64Cu-NOTA-E1 should be suitable as a novel PET imaging agent for hEphA2-expressing tumors.

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.

Salicylic Acid Reduces OmpF Expression, Rendering Salmonella enterica Serovar Typhimurium More Resistant to Cephalosporin Antibiotics.

Salmonella enterica serovar Typhimurium is one of the most important bacterial pathogens causing diarrhea. The resistance of S. typhimurium to antimicrobial agents, which has recently been isolated from patients, is causing serious problems. We investigated the effects of salicylic acid (Sal) and acetyl salicylate (AcSal) on the susceptibility of S. typhimurium to cephalosporin antibiotics, which are known to increase resistance to cephalosporin and quinolone antibiotics. The MIC of cephalosporin antibiotics was higher than that of the media without Sal. The rate of accumulation of ethidium bromide (EtBr) in the bacteria by the outer membrane protein (Omp) was not different from that of the bacteria cultured in the medium containing Sal. However, Carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an inhibitor of bacterial efflux pumps, significantly reduced the rate of accumulation of EtBr in bacteria cultured on Sal containing medium. In the medium containing CCCP, the MIC of the antimicrobial agent tended to decrease as compared with the control. In addition, the MIC of the bacteria treated with CCCP and Sal was higher than that of the antimicrobial agent against the CCCP treated experimental bacteria. These results suggest that Sal decreases the expression of OmpF in the Omp of S. typhimurium and reduces the permeability of cephalosporin antibiotics to bacteria, which may induce tolerance to cephalosporin antibiotics.

A Mild Form of COG5 Defect Showing Early-Childhood-Onset Friedreich's-Ataxia-Like Phenotypes with Isolated Cerebellar Atrophy.

Progressive cerebellar ataxias are rare diseases during childhood, especially under 6 years of age. In a single family, three affected siblings exhibited Friedreich's-ataxia-like phenotypes before 2 years of age. They had progressive cerebellar atrophy, intellectual disability, and scoliosis. Although their phenotypes were similar to those observed in patients with autosomal recessive cerebellar ataxias, other phenotypes (e.g., seizure, movement disorders, ophthalmologic disturbance, cardiomyopathy, and cutaneous disorders) were not noted in this family. Whole-exome sequencing of the family members revealed one potential heterozygous mutation (c.1209delG, NM_181733.2; p.Met403IlefsX3, NP_859422.2) of the gene encoding conserved oligomeric Golgi complex subunit 5 (COG5). The heterozygous deletion at the fifth base in exon 12 of COG5 caused a frameshift and premature stop. Western blotting of COG5 proteins in the skin tissues from an affected proband showed a significantly decreased level of full length COG5 and smaller, aberrant COG5 proteins. We reported a milder form of COG5 defect showing Friedreich's-ataxia-like phenotypes without hypotonia, microcephaly, and short stature that were observed in most patients with COG5 defect.

A High-Affinity Repebody for Molecular Imaging of EGFR-Expressing Malignant Tumors.

The accurate detection of disease-related biomarkers is crucial for the early diagnosis and management of disease in personalized medicine. Here, we present a molecular imaging of human epidermal growth factor receptor (EGFR)-expressing malignant tumors using an EGFR-specific repebody composed of leucine-rich repeat (LRR) modules. The repebody was labeled with either a fluorescent dye or radioisotope, and used for imaging of EGFR-expressing malignant tumors using an optical method and positron emission tomography. Our approach enabled visualization of the status of EGFR expression, allowing quantitative evaluation in whole tumors, which correlated well with the EGFR expression levels in mouse or patients-derived colon cancers. The present approach can be effectively used for the accurate detection of EGFR-expressing cancers, assisting in the development of a tool for detecting other disease biomarkers.