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In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis.

Ganai S, Arenas RB, Sauer JP, Bentley B, Forbes NS - Cancer Gene Ther. (2011)

Bottom Line: From 12 and 48 h after injection, the average distance between bacterial colonies and functional vasculature significantly increased from 130 to 310 μm.All observed metastases contained Salmonella and the extent of bacterial colocalization with metastatic tissue was 44% compared with 0.5% with normal liver parenchyma.These results demonstrate that Salmonella can penetrate tumor tissue and can selectively target metastases, two critical characteristics of a targeted cancer therapeutic.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Baystate Medical Center, Tufts University School of Medicine, Springfield, MA, USA.

ABSTRACT
Motile bacteria can overcome diffusion resistances that substantially reduce the efficacy of standard cancer therapies. Many reports have also recently described the ability of Salmonella to deliver therapeutic molecules to tumors. Despite this potential, little is known about the spatiotemporal dynamics of bacterial accumulation in solid tumors. Ultimately this timing will affect how these microbes are used therapeutically. To determine how bacteria localize, we intravenously injected Salmonella typhimurium into BALB/c mice with 4T1 mammary carcinoma and measured the average bacterial content as a function of time. Immunohistochemistry was used to measure the extent of apoptosis, the average distance of bacteria from tumor vasculature and the location of bacteria in four different regions: the core, transition, body and edge. Bacteria accumulation was also measured in pulmonary and hepatic metastases. The doubling time of bacterial colonies in tumors was measured to be 16.8 h, and colonization was determined to delay tumor growth by 48 h. From 12 and 48 h after injection, the average distance between bacterial colonies and functional vasculature significantly increased from 130 to 310 μm. After 48 h, bacteria migrated away from the tumor edge toward the central core and induced apoptosis. After 96 h, bacteria began to marginate to the tumor transition zone. All observed metastases contained Salmonella and the extent of bacterial colocalization with metastatic tissue was 44% compared with 0.5% with normal liver parenchyma. These results demonstrate that Salmonella can penetrate tumor tissue and can selectively target metastases, two critical characteristics of a targeted cancer therapeutic.

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Salmonella accumulated in distinct tumor regions over timeA, Composite images of 4T1 Tumors, 12 hours after systemic bacterial injection. Serial 4μm sections were dual label stained using a combination of Salmonella immunohistochemistry and biotinylated tomato lectin (left) to visualize bacteria (red) and perfused vasculature (brown) or stained using cleaved caspase-3 immunohistochemistry (middle) to visualize regions of apoptosis (brown). Scale bars are 1mm. Based on histological staining, four regions with distinct microenvironments were observed in each tumor: edge (within 1mm of tumor periphery), body, transition, or core. B, Salmonella accumulated as colonies in the edge (left) and body (middle left) tumor regions. Bacteria were seen dispersed throughout the extracellular space, shown here in the body region (middle right). Arrows indicate colony edges (left, middle left) and individual bacteria in the extracellular space (middle right). Salmonella were observed both extracellularly and intracellularly (white arrows, right). The black arrow highlights a single extracellular bacterium (right). Scale bars are 50μm (left, middle left), 25μm (middle right), and 10μm (right). C, Dynamics of bacterial accumulation were determined by measuring pixel density per image and normalized to the edge microenvironment at 12 hours. At 12 and 48 hours, there was significantly greater accumulation within the edge than all other regions (*, p<0.05; †, p<0.01; except the core at 48 hrs). At 96 hours, there was greater accumulation within the tumor transition zone compared to all other regions (‡, p<0.01). D, At late time points Salmonella accumulated as stratified bands in the tumor transition zone (between arrows, left and highlighted, right). Arrows indicate boundary of the transition zone. Sparse accumulation in the necrotic core is present in D-left. Scale bars are 1mm (left) and 100μm (right).
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Figure 2: Salmonella accumulated in distinct tumor regions over timeA, Composite images of 4T1 Tumors, 12 hours after systemic bacterial injection. Serial 4μm sections were dual label stained using a combination of Salmonella immunohistochemistry and biotinylated tomato lectin (left) to visualize bacteria (red) and perfused vasculature (brown) or stained using cleaved caspase-3 immunohistochemistry (middle) to visualize regions of apoptosis (brown). Scale bars are 1mm. Based on histological staining, four regions with distinct microenvironments were observed in each tumor: edge (within 1mm of tumor periphery), body, transition, or core. B, Salmonella accumulated as colonies in the edge (left) and body (middle left) tumor regions. Bacteria were seen dispersed throughout the extracellular space, shown here in the body region (middle right). Arrows indicate colony edges (left, middle left) and individual bacteria in the extracellular space (middle right). Salmonella were observed both extracellularly and intracellularly (white arrows, right). The black arrow highlights a single extracellular bacterium (right). Scale bars are 50μm (left, middle left), 25μm (middle right), and 10μm (right). C, Dynamics of bacterial accumulation were determined by measuring pixel density per image and normalized to the edge microenvironment at 12 hours. At 12 and 48 hours, there was significantly greater accumulation within the edge than all other regions (*, p<0.05; †, p<0.01; except the core at 48 hrs). At 96 hours, there was greater accumulation within the tumor transition zone compared to all other regions (‡, p<0.01). D, At late time points Salmonella accumulated as stratified bands in the tumor transition zone (between arrows, left and highlighted, right). Arrows indicate boundary of the transition zone. Sparse accumulation in the necrotic core is present in D-left. Scale bars are 1mm (left) and 100μm (right).

Mentions: Composite images were generated for all tumors to observer macroscopic bacterial behavior (Figure 2A). Immunohistochemical staining identified regions of Salmonella accumulation (red in Figure 2, left), functional vasculature (brown in Figure 2A, left), and apoptosis (brown in Figure 2A, middle). Negative controls without primary antibody and without tomato lectin perfusion confirmed stain specificity for functional vasculature. Positive controls performed on tumors with intratumoral injections of Salmonella confirmed the specificity of the antibody stain.


In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis.

Ganai S, Arenas RB, Sauer JP, Bentley B, Forbes NS - Cancer Gene Ther. (2011)

Salmonella accumulated in distinct tumor regions over timeA, Composite images of 4T1 Tumors, 12 hours after systemic bacterial injection. Serial 4μm sections were dual label stained using a combination of Salmonella immunohistochemistry and biotinylated tomato lectin (left) to visualize bacteria (red) and perfused vasculature (brown) or stained using cleaved caspase-3 immunohistochemistry (middle) to visualize regions of apoptosis (brown). Scale bars are 1mm. Based on histological staining, four regions with distinct microenvironments were observed in each tumor: edge (within 1mm of tumor periphery), body, transition, or core. B, Salmonella accumulated as colonies in the edge (left) and body (middle left) tumor regions. Bacteria were seen dispersed throughout the extracellular space, shown here in the body region (middle right). Arrows indicate colony edges (left, middle left) and individual bacteria in the extracellular space (middle right). Salmonella were observed both extracellularly and intracellularly (white arrows, right). The black arrow highlights a single extracellular bacterium (right). Scale bars are 50μm (left, middle left), 25μm (middle right), and 10μm (right). C, Dynamics of bacterial accumulation were determined by measuring pixel density per image and normalized to the edge microenvironment at 12 hours. At 12 and 48 hours, there was significantly greater accumulation within the edge than all other regions (*, p<0.05; †, p<0.01; except the core at 48 hrs). At 96 hours, there was greater accumulation within the tumor transition zone compared to all other regions (‡, p<0.01). D, At late time points Salmonella accumulated as stratified bands in the tumor transition zone (between arrows, left and highlighted, right). Arrows indicate boundary of the transition zone. Sparse accumulation in the necrotic core is present in D-left. Scale bars are 1mm (left) and 100μm (right).
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Figure 2: Salmonella accumulated in distinct tumor regions over timeA, Composite images of 4T1 Tumors, 12 hours after systemic bacterial injection. Serial 4μm sections were dual label stained using a combination of Salmonella immunohistochemistry and biotinylated tomato lectin (left) to visualize bacteria (red) and perfused vasculature (brown) or stained using cleaved caspase-3 immunohistochemistry (middle) to visualize regions of apoptosis (brown). Scale bars are 1mm. Based on histological staining, four regions with distinct microenvironments were observed in each tumor: edge (within 1mm of tumor periphery), body, transition, or core. B, Salmonella accumulated as colonies in the edge (left) and body (middle left) tumor regions. Bacteria were seen dispersed throughout the extracellular space, shown here in the body region (middle right). Arrows indicate colony edges (left, middle left) and individual bacteria in the extracellular space (middle right). Salmonella were observed both extracellularly and intracellularly (white arrows, right). The black arrow highlights a single extracellular bacterium (right). Scale bars are 50μm (left, middle left), 25μm (middle right), and 10μm (right). C, Dynamics of bacterial accumulation were determined by measuring pixel density per image and normalized to the edge microenvironment at 12 hours. At 12 and 48 hours, there was significantly greater accumulation within the edge than all other regions (*, p<0.05; †, p<0.01; except the core at 48 hrs). At 96 hours, there was greater accumulation within the tumor transition zone compared to all other regions (‡, p<0.01). D, At late time points Salmonella accumulated as stratified bands in the tumor transition zone (between arrows, left and highlighted, right). Arrows indicate boundary of the transition zone. Sparse accumulation in the necrotic core is present in D-left. Scale bars are 1mm (left) and 100μm (right).
Mentions: Composite images were generated for all tumors to observer macroscopic bacterial behavior (Figure 2A). Immunohistochemical staining identified regions of Salmonella accumulation (red in Figure 2, left), functional vasculature (brown in Figure 2A, left), and apoptosis (brown in Figure 2A, middle). Negative controls without primary antibody and without tomato lectin perfusion confirmed stain specificity for functional vasculature. Positive controls performed on tumors with intratumoral injections of Salmonella confirmed the specificity of the antibody stain.

Bottom Line: From 12 and 48 h after injection, the average distance between bacterial colonies and functional vasculature significantly increased from 130 to 310 μm.All observed metastases contained Salmonella and the extent of bacterial colocalization with metastatic tissue was 44% compared with 0.5% with normal liver parenchyma.These results demonstrate that Salmonella can penetrate tumor tissue and can selectively target metastases, two critical characteristics of a targeted cancer therapeutic.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Baystate Medical Center, Tufts University School of Medicine, Springfield, MA, USA.

ABSTRACT
Motile bacteria can overcome diffusion resistances that substantially reduce the efficacy of standard cancer therapies. Many reports have also recently described the ability of Salmonella to deliver therapeutic molecules to tumors. Despite this potential, little is known about the spatiotemporal dynamics of bacterial accumulation in solid tumors. Ultimately this timing will affect how these microbes are used therapeutically. To determine how bacteria localize, we intravenously injected Salmonella typhimurium into BALB/c mice with 4T1 mammary carcinoma and measured the average bacterial content as a function of time. Immunohistochemistry was used to measure the extent of apoptosis, the average distance of bacteria from tumor vasculature and the location of bacteria in four different regions: the core, transition, body and edge. Bacteria accumulation was also measured in pulmonary and hepatic metastases. The doubling time of bacterial colonies in tumors was measured to be 16.8 h, and colonization was determined to delay tumor growth by 48 h. From 12 and 48 h after injection, the average distance between bacterial colonies and functional vasculature significantly increased from 130 to 310 μm. After 48 h, bacteria migrated away from the tumor edge toward the central core and induced apoptosis. After 96 h, bacteria began to marginate to the tumor transition zone. All observed metastases contained Salmonella and the extent of bacterial colocalization with metastatic tissue was 44% compared with 0.5% with normal liver parenchyma. These results demonstrate that Salmonella can penetrate tumor tissue and can selectively target metastases, two critical characteristics of a targeted cancer therapeutic.

Show MeSH
Related in: MedlinePlus