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Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours.

Ho KS, Poon PC, Owen SC, Shoichet MS - BMC Cancer (2012)

Bottom Line: For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model.Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo.A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering & Applied Chemistry, 200 College Street, Toronto, ON M5S 3E5, Canada.

ABSTRACT

Background: Human tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically (mammary fat pad, MFP) or ectopically (subcutaneous, SC), and the organ environment experienced by the tumour cells has been shown to influence their behaviour.

Methods: To evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels.

Results: SC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for P < 0.10). Immunostaining revealed greater vascular density and thinner basement membranes in the MFP tumour model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid.

Conclusions: Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

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CD31 and collagen IV immunostaining. Mean blood vessel wall thickness visualized through A CD31 (endothelial cells) and B collagen IV (basement membrane). Both are abnormally thick as compared to healthy liver control tissue, which is denoted by the dashed line. C shows that mean blood vessel density assayed using CD31 staining is greatest in 3 week old MFP tumours. D indicates mean vascular area as a measure of blood vessel size and capacity. Their small size categorizes them as microvasculature. All data are shown as the mean of n = 4 animals ± SD. Starred lines connecting bars denote statistical significance, P < 0.05.
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Figure 3: CD31 and collagen IV immunostaining. Mean blood vessel wall thickness visualized through A CD31 (endothelial cells) and B collagen IV (basement membrane). Both are abnormally thick as compared to healthy liver control tissue, which is denoted by the dashed line. C shows that mean blood vessel density assayed using CD31 staining is greatest in 3 week old MFP tumours. D indicates mean vascular area as a measure of blood vessel size and capacity. Their small size categorizes them as microvasculature. All data are shown as the mean of n = 4 animals ± SD. Starred lines connecting bars denote statistical significance, P < 0.05.

Mentions: To better understand the underlying vascular pathophysiology present in both tumour models, tumour slices were immunostained to provide information on the blood and lymphatic vessels present. Tissue was stained for CD31, an endothelial cell marker, to locate and characterize blood vessels. In normal blood vessels, an intact monolayer of endothelials cells is expected, whereas hyperpermeable tumour blood vessels are characterized by multiple layers of discontinuous endothelial cells that may sprout outwards or project into the vessel lumen [10,13]. The CD31 staining revealed greater vessel wall thickness across all groups when compared to liver tissue (represented by a dashed line) which was used as a healthy tissue control (Figure 3A). This observation suggests that blood vessels present in all models, whether they are existing vessels that have been remodeled or newly formed vessels, have the abnormal multi-layered endothelial cell structure associated with solid tumours. The vessel thickness was highest in the 3 week old MFP tumours, indicating a greater level of endothelial cell disorganization in this group. It is possible that this led to the increased permeability observed in the 3 week MFP tumours using a relatively large model nanocarrier (~80 nm), an effect that is more pronounced in other studies utilizing models such as albumin (~7 nm) [30,31].


Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours.

Ho KS, Poon PC, Owen SC, Shoichet MS - BMC Cancer (2012)

CD31 and collagen IV immunostaining. Mean blood vessel wall thickness visualized through A CD31 (endothelial cells) and B collagen IV (basement membrane). Both are abnormally thick as compared to healthy liver control tissue, which is denoted by the dashed line. C shows that mean blood vessel density assayed using CD31 staining is greatest in 3 week old MFP tumours. D indicates mean vascular area as a measure of blood vessel size and capacity. Their small size categorizes them as microvasculature. All data are shown as the mean of n = 4 animals ± SD. Starred lines connecting bars denote statistical significance, P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539979&req=5

Figure 3: CD31 and collagen IV immunostaining. Mean blood vessel wall thickness visualized through A CD31 (endothelial cells) and B collagen IV (basement membrane). Both are abnormally thick as compared to healthy liver control tissue, which is denoted by the dashed line. C shows that mean blood vessel density assayed using CD31 staining is greatest in 3 week old MFP tumours. D indicates mean vascular area as a measure of blood vessel size and capacity. Their small size categorizes them as microvasculature. All data are shown as the mean of n = 4 animals ± SD. Starred lines connecting bars denote statistical significance, P < 0.05.
Mentions: To better understand the underlying vascular pathophysiology present in both tumour models, tumour slices were immunostained to provide information on the blood and lymphatic vessels present. Tissue was stained for CD31, an endothelial cell marker, to locate and characterize blood vessels. In normal blood vessels, an intact monolayer of endothelials cells is expected, whereas hyperpermeable tumour blood vessels are characterized by multiple layers of discontinuous endothelial cells that may sprout outwards or project into the vessel lumen [10,13]. The CD31 staining revealed greater vessel wall thickness across all groups when compared to liver tissue (represented by a dashed line) which was used as a healthy tissue control (Figure 3A). This observation suggests that blood vessels present in all models, whether they are existing vessels that have been remodeled or newly formed vessels, have the abnormal multi-layered endothelial cell structure associated with solid tumours. The vessel thickness was highest in the 3 week old MFP tumours, indicating a greater level of endothelial cell disorganization in this group. It is possible that this led to the increased permeability observed in the 3 week MFP tumours using a relatively large model nanocarrier (~80 nm), an effect that is more pronounced in other studies utilizing models such as albumin (~7 nm) [30,31].

Bottom Line: For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model.Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo.A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering & Applied Chemistry, 200 College Street, Toronto, ON M5S 3E5, Canada.

ABSTRACT

Background: Human tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically (mammary fat pad, MFP) or ectopically (subcutaneous, SC), and the organ environment experienced by the tumour cells has been shown to influence their behaviour.

Methods: To evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels.

Results: SC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for P < 0.10). Immunostaining revealed greater vascular density and thinner basement membranes in the MFP tumour model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid.

Conclusions: Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

Show MeSH
Related in: MedlinePlus