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Tumor invasion of Salmonella enterica serovar Typhimurium is accompanied by strong hemorrhage promoted by TNF-alpha.

Leschner S, Westphal K, Dietrich N, Viegas N, Jablonska J, Lyszkiewicz M, Lienenklaus S, Falk W, Gekara N, Loessner H, Weiss S - PLoS ONE (2009)

Bottom Line: A rapid increase of TNF-alpha in blood was observed at that time, in addition to other pro-inflammatory cytokines.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany. sara.leschner@helmholtz-hzi.de

ABSTRACT

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.

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Related in: MedlinePlus

Inhibition of TNF-α in S. Typhimurium-infected BALB/c mice retards blood influx into tumors and bacterial colonization.(a) TNF-α concentration in the blood of S. Typhimurium-infected, CT26 tumor-bearing BALB/c mice (black bars) and of S. Typhimurium-infected, anti-TNF-α treated, CT26 tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (b) Photographs of CT26 tumors of an S. Typhimurium-infected tumor (left set of pictures) and an S. Typhimurium-infected, anti-TNF-α treated tumor (right set of pictures). The left column of each set of pictures show the fur side of the tumors, the right pictures show the ventral side of the tumors. (c) Hemoglobin content in CT26 tumors of S. Typhimurium-infected BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 6 h p.i. the difference of hemoglobin content in the tumors is significant with p<0.05. (d) Bacterial number per g tumor tissue in S. Typhimurium-infected tumor-bearing BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 12 h p.i. the difference of bacterial numbers between differently treated tumors is significant with p<0.05. Results are representative for at least two independent experiments with 3–5 mice per group. The second experiment also included an isotype control where normal rat IgG was injected into the mice of the control group (data not shown).
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pone-0006692-g005: Inhibition of TNF-α in S. Typhimurium-infected BALB/c mice retards blood influx into tumors and bacterial colonization.(a) TNF-α concentration in the blood of S. Typhimurium-infected, CT26 tumor-bearing BALB/c mice (black bars) and of S. Typhimurium-infected, anti-TNF-α treated, CT26 tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (b) Photographs of CT26 tumors of an S. Typhimurium-infected tumor (left set of pictures) and an S. Typhimurium-infected, anti-TNF-α treated tumor (right set of pictures). The left column of each set of pictures show the fur side of the tumors, the right pictures show the ventral side of the tumors. (c) Hemoglobin content in CT26 tumors of S. Typhimurium-infected BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 6 h p.i. the difference of hemoglobin content in the tumors is significant with p<0.05. (d) Bacterial number per g tumor tissue in S. Typhimurium-infected tumor-bearing BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 12 h p.i. the difference of bacterial numbers between differently treated tumors is significant with p<0.05. Results are representative for at least two independent experiments with 3–5 mice per group. The second experiment also included an isotype control where normal rat IgG was injected into the mice of the control group (data not shown).

Mentions: We then wanted to test whether TNF-α is the sole cytokine responsible for the blood influx and bacterial colonization of solid tumors. Therefore, we neutralized TNF-α by injecting rat anti-mouseTNF-α i.v. 20 min before infection. As intended, the majority of TNF-α in blood was neutralized this way (Fig. 5a). The consequence was a retardation of blood influx into the tumor (Fig. 5b). While tumors from Salmonella-infected mice became dark red within 2 h p.i., tumors of anti-TNF-α treated mice began to show this phenomenon between 4 and 6 h p.i.. No resolution of blood influx became apparent within 30 h p.i..


Tumor invasion of Salmonella enterica serovar Typhimurium is accompanied by strong hemorrhage promoted by TNF-alpha.

Leschner S, Westphal K, Dietrich N, Viegas N, Jablonska J, Lyszkiewicz M, Lienenklaus S, Falk W, Gekara N, Loessner H, Weiss S - PLoS ONE (2009)

Inhibition of TNF-α in S. Typhimurium-infected BALB/c mice retards blood influx into tumors and bacterial colonization.(a) TNF-α concentration in the blood of S. Typhimurium-infected, CT26 tumor-bearing BALB/c mice (black bars) and of S. Typhimurium-infected, anti-TNF-α treated, CT26 tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (b) Photographs of CT26 tumors of an S. Typhimurium-infected tumor (left set of pictures) and an S. Typhimurium-infected, anti-TNF-α treated tumor (right set of pictures). The left column of each set of pictures show the fur side of the tumors, the right pictures show the ventral side of the tumors. (c) Hemoglobin content in CT26 tumors of S. Typhimurium-infected BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 6 h p.i. the difference of hemoglobin content in the tumors is significant with p<0.05. (d) Bacterial number per g tumor tissue in S. Typhimurium-infected tumor-bearing BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 12 h p.i. the difference of bacterial numbers between differently treated tumors is significant with p<0.05. Results are representative for at least two independent experiments with 3–5 mice per group. The second experiment also included an isotype control where normal rat IgG was injected into the mice of the control group (data not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2724709&req=5

pone-0006692-g005: Inhibition of TNF-α in S. Typhimurium-infected BALB/c mice retards blood influx into tumors and bacterial colonization.(a) TNF-α concentration in the blood of S. Typhimurium-infected, CT26 tumor-bearing BALB/c mice (black bars) and of S. Typhimurium-infected, anti-TNF-α treated, CT26 tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (b) Photographs of CT26 tumors of an S. Typhimurium-infected tumor (left set of pictures) and an S. Typhimurium-infected, anti-TNF-α treated tumor (right set of pictures). The left column of each set of pictures show the fur side of the tumors, the right pictures show the ventral side of the tumors. (c) Hemoglobin content in CT26 tumors of S. Typhimurium-infected BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 6 h p.i. the difference of hemoglobin content in the tumors is significant with p<0.05. (d) Bacterial number per g tumor tissue in S. Typhimurium-infected tumor-bearing BALB/c mice (black bars) and S. Typhimurium-infected, anti-TNF-α treated tumor-bearing BALB/c mice (grey bars). Error bars show standard deviations of means. (*) At 12 h p.i. the difference of bacterial numbers between differently treated tumors is significant with p<0.05. Results are representative for at least two independent experiments with 3–5 mice per group. The second experiment also included an isotype control where normal rat IgG was injected into the mice of the control group (data not shown).
Mentions: We then wanted to test whether TNF-α is the sole cytokine responsible for the blood influx and bacterial colonization of solid tumors. Therefore, we neutralized TNF-α by injecting rat anti-mouseTNF-α i.v. 20 min before infection. As intended, the majority of TNF-α in blood was neutralized this way (Fig. 5a). The consequence was a retardation of blood influx into the tumor (Fig. 5b). While tumors from Salmonella-infected mice became dark red within 2 h p.i., tumors of anti-TNF-α treated mice began to show this phenomenon between 4 and 6 h p.i.. No resolution of blood influx became apparent within 30 h p.i..

Bottom Line: A rapid increase of TNF-alpha in blood was observed at that time, in addition to other pro-inflammatory cytokines.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany. sara.leschner@helmholtz-hzi.de

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus