Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic

Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasi

Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic

Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasia

Tumor Invasion of Salmonella enterica Serovar Typhimurium Is Accompanied by Strong Hemorrhage Promoted by TNF-α

BACKGROUND:Several facultative anaerobic bacteria with potential therapeutic abilities are known to preferentially colonize solid tumors after systemic administration. How they efficiently find and invade the tumors is still unclear. However, this is an important issue to be clarified when bacteria should be tailored for application in cancer therapy. METHODOLOGY/PRINCIPAL FINDINGS:We describe the initial events of colonization of an ectopic transplantable tumor by Salmonella enterica serovar Typhimurium. Initially, after intravenous administration, bacteria were found in blood, spleen, and liver. Low numbers were also detected in tumors associated with blood vessels as could be observed by immunohistochemistry. A rapid increase of TNF-alpha in blood was observed at that time, in addition to other pro-inflammatory cytokines. This induced a tremendous influx of blood into the tumors by vascular disruption that could be visualized in H&E stainings and quantified by hemoglobin measurements of tumor homogenate. Most likely, together with the blood, bacteria were flushed into the tumor. In addition, blood influx was followed by necrosis formation, bacterial growth, and infiltration of neutrophilic granulocytes. Depletion of TNF-alpha retarded blood influx and delayed bacterial tumor-colonization. CONCLUSION:Our findings emphasize similarities between Gram-negative tumor-colonizing bacteria and tumor vascular disrupting agents and show the involvement of TNF-alpha in the initial phase of tumor-colonization by bacteria

Composing a Tumor Specific Bacterial Promoter.

Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy

Therapeutic Potential of Bacteria against Solid Tumors.

Intentional bacterial infections can produce efficacious antitumor responses in mice, rats, dogs, and humans. However, low overall success rates and intense side effects prevent such approaches from being employed clinically. In this work, we titered bacteria and/or the proinflammatory cytokine TNFα in a set of established murine models of cancer. To interpret the experiments conducted, we considered and calibrated a tumor-effector cell recruitment model under the influence of functional tumor-associated vasculature. In this model, bacterial infections and TNFα enhanced immune activity and altered vascularization in the tumor bed. Information to predict bacterial therapy outcomes was provided by pretreatment tumor size and the underlying immune recruitment dynamics. Notably, increasing bacterial loads did not necessarily produce better long-term tumor control, suggesting that tumor sizes affected optimal bacterial loads. Short-term treatment responses were favored by high concentrations of effector cells postinjection, such as induced by higher bacterial loads, but in the longer term did not correlate with an effective restoration of immune surveillance. Overall, our findings suggested that a combination of intermediate bacterial loads with low levels TNFα administration could enable more favorable outcomes elicited by bacterial infections in tumor-bearing subjects. Cancer Res; 77(7); 1553-63. ©2017 AACR

Schematic representation of promoter structure.

<p>Left: binding motifs for factors TGIF, FNR and NagC are shown in green, yellow and brown, respectively. Basal promoter is shown as a directed arrow. Knockout of essential nucleotides within motifs were according to the literature and are represented by crossing lines. Mutation of non-essential nucleotides within motifs was random and is represented by dashed crossing lines. Nucleotides outside motifs were mutated randomly. Right: representative flow-cytometric analyses of GFP-expression in tumor and spleen. Each green point on the blots represents GFP expression levels of an individual bacterial cell. Displayed values of expression are relative to the expression values of the original promoter P0.212_1. All nucleotide substitutions are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s002" target="_blank">S2 Fig</a>.</p

Schematic representation of artificial promoters and their expression in tumor environment.

<p>Promoters were compiled by introduction of the respective elements taken from promoters P0.212, P1.6, P0.134 as well as consensus sequences into the template sequence. Additionally, regions around TSS were enriched for A/T to facilitate DNA melting. Expression values are relative to the expression of P0.212_1. Given are mean±SD. Only promoter P2.3 showed expression in tumor. No expression in tumor or spleen could be observed for the other promoters. Nucleotide sequences are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s003" target="_blank">S3 Fig</a>. Foot note: <b>I, II, III</b>—corresponding sequences were taken from promoters P0.212, P1.6, P0.134 respectively. <b>cons</b>—consensus sequences for the corresponding elements. <b>a, b</b>—modification of loci around putative start of transcription to facilitate DNA melting by random substitution of "C/G"s by "A/T"s.</p

Therapeutic benefit of Salmonella attributed to LPS and TNF-α is exhaustible and dictated by tumor susceptibility.

The potential of bacteria-mediated tumor therapy (BMTT) is highlighted by more than a century of investigation. Attenuated Salmonella has prevailed as promising therapeutic agents. For BMTT - categorized as an immune therapy - the exact contribution of particular immune reactions to the therapeutic effect remains ambiguous. In addition, one could argue for or against the requirement of bacterial viability and tumor targeting. Herein we evaluate the isolated therapeutic efficacy of purified LPS and TNF-α, which together account for a dominant immunogenic pathway of gram negative bacteria like Salmonella. We show that therapeutic efficacy against CT26 tumors does not require bacterial viability. Analogous to viable Salmonella SL7207, tumor regression by a specific CD8+ T cell response can be induced by purified LPS or recombinant TNF-α (rTNF-α). Conversely, therapeutic effects against RenCa tumors were abrogated upon bacterial avitalization and limited using isolated adjuvants. This argues for an alternative mechanistic explanation for SL7207 against RenCa that depends on viability and persistence. Unable to boost bacterial therapies by co-injection of rTNF-α suggested therapeutic effects along this axis are exhausted by the intrinsic adjuvanticity of bacteria alone. However, the importance of TNF-α for BMTT was highlighted by its support of tumor invasion and colonization in concert with lower infective doses of Salmonella. In consideration, bacterial therapeutic effectiveness along the axis of LPS and TNF-α appears limited, and does not offer the necessary plasticity for different tumors. This emphasizes a need for recombinant strengthening and vehicular exploitation to accommodate potency, plasticity and distinctiveness in BMTT

Identification and analysis steps of tumor specific promoters.

<p>A schematic overview. Two programs for basal promoter recognition, one based on sequence alignment kernel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.ref011" target="_blank">11</a>] and another on Hidden Markov Model (HMM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.ref012" target="_blank">12</a>] were applied to the TSP set (DNA sequences are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s006" target="_blank">S1 Text</a>). As a negative control, a set of DNA fragments that do not initiate expression either in tumor or in spleen was selected (negative promoters, NP). Both programs recognize potential promoters in either dataset and the number of predictions greatly depends on user-defined threshold parameters. To evaluate the specificity of predictions, it was assumed that a basal promoter should be recognized in at least 75% of TSP and at most 50% of NPs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#sec014" target="_blank">Methods</a>). This will ensure the generality and specificity of the recognized feature.</p

Expression of promoters P1.6 and P0.212_1 in tumor, spleen and liver.

<p>Homogenates were analyzed via two color flow cytometry and plating to allow normalization. Given are mean and SD. Expression of P1.6 in spleen and liver can be considered negligible compared to expression in tumor. Therefore expression of promoter P1.6 was accepted as tumor specific.</p