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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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LYVE-1 immunostaining. A shows mean lymphatic vessel density, and B shows mean vessel area, both of which are indicators of lymphovascular capacity. Both measures were found to have unequal variance between groups, and therefore although the groups were not equivalent, ANOVA could not be used to verify their differences. While 3 week old MFP tumours had the highest mean lymphatic vessel density, 5 week old SC tumours had greater mean vessel size, both of which contribute to overall lymphatic drainage capacity. All data are shown as the mean of n = 4 animals ± SD. Representative images of fluid-filled spaces lined with collagen (violet) but not with endothelial cells (negative for CD31, brown)are shown in: C 3 week MFP and D 5 week SC tumours. Several of these spaces, which indicate lymphedema, are highlighted with black arrows; blue staining represents cell nuclei. Scale bars represent 200 μm.
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Figure 5: LYVE-1 immunostaining. A shows mean lymphatic vessel density, and B shows mean vessel area, both of which are indicators of lymphovascular capacity. Both measures were found to have unequal variance between groups, and therefore although the groups were not equivalent, ANOVA could not be used to verify their differences. While 3 week old MFP tumours had the highest mean lymphatic vessel density, 5 week old SC tumours had greater mean vessel size, both of which contribute to overall lymphatic drainage capacity. All data are shown as the mean of n = 4 animals ± SD. Representative images of fluid-filled spaces lined with collagen (violet) but not with endothelial cells (negative for CD31, brown)are shown in: C 3 week MFP and D 5 week SC tumours. Several of these spaces, which indicate lymphedema, are highlighted with black arrows; blue staining represents cell nuclei. Scale bars represent 200 μm.

Mentions: LYVE-1 staining was used to detect lymphatic vessels in tumour tissue. Lymphatic vessels provide a network to drain protein rich interstitial fluid back into circulation. By the nature of their function, these vessels are porous to allow macromolecules to be transported [35], and therefore nanocarrier accumulation in tumour tissue may increase when their expression is impaired. Mouse models of lymphatic impairment can be generated by surgically ablating lymphatic vessels in the tail, resulting in lymphedema. In these models, the surrounding tissue attempts to restore homeostasis by generating new lymphatic vessels and dilating the remaining lymphatic vessels, suggesting that both density and diameter impact drainage capacity [36]. LYVE-1 stained sections were used to quantify lymphatic vessel size and density (Figure 5A-B). Both of these measures gave different variances between groups (P < 0.05 by Bartlett’s test of equality of variances) meaning that the groups tested were not equivalent. While the mean lymphatic vessel density was highest in the 3 week old MFP tumours, the 5 week old SC tumours demonstrated the highest mean lymphatic vessel area. These factors counterbalance one another, as density and capacity each contribute to overall drainage.


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)

LYVE-1 immunostaining. A shows mean lymphatic vessel density, and B shows mean vessel area, both of which are indicators of lymphovascular capacity. Both measures were found to have unequal variance between groups, and therefore although the groups were not equivalent, ANOVA could not be used to verify their differences. While 3 week old MFP tumours had the highest mean lymphatic vessel density, 5 week old SC tumours had greater mean vessel size, both of which contribute to overall lymphatic drainage capacity. All data are shown as the mean of n = 4 animals ± SD. Representative images of fluid-filled spaces lined with collagen (violet) but not with endothelial cells (negative for CD31, brown)are shown in: C 3 week MFP and D 5 week SC tumours. Several of these spaces, which indicate lymphedema, are highlighted with black arrows; blue staining represents cell nuclei. Scale bars represent 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: LYVE-1 immunostaining. A shows mean lymphatic vessel density, and B shows mean vessel area, both of which are indicators of lymphovascular capacity. Both measures were found to have unequal variance between groups, and therefore although the groups were not equivalent, ANOVA could not be used to verify their differences. While 3 week old MFP tumours had the highest mean lymphatic vessel density, 5 week old SC tumours had greater mean vessel size, both of which contribute to overall lymphatic drainage capacity. All data are shown as the mean of n = 4 animals ± SD. Representative images of fluid-filled spaces lined with collagen (violet) but not with endothelial cells (negative for CD31, brown)are shown in: C 3 week MFP and D 5 week SC tumours. Several of these spaces, which indicate lymphedema, are highlighted with black arrows; blue staining represents cell nuclei. Scale bars represent 200 μm.
Mentions: LYVE-1 staining was used to detect lymphatic vessels in tumour tissue. Lymphatic vessels provide a network to drain protein rich interstitial fluid back into circulation. By the nature of their function, these vessels are porous to allow macromolecules to be transported [35], and therefore nanocarrier accumulation in tumour tissue may increase when their expression is impaired. Mouse models of lymphatic impairment can be generated by surgically ablating lymphatic vessels in the tail, resulting in lymphedema. In these models, the surrounding tissue attempts to restore homeostasis by generating new lymphatic vessels and dilating the remaining lymphatic vessels, suggesting that both density and diameter impact drainage capacity [36]. LYVE-1 stained sections were used to quantify lymphatic vessel size and density (Figure 5A-B). Both of these measures gave different variances between groups (P < 0.05 by Bartlett’s test of equality of variances) meaning that the groups tested were not equivalent. While the mean lymphatic vessel density was highest in the 3 week old MFP tumours, the 5 week old SC tumours demonstrated the highest mean lymphatic vessel area. These factors counterbalance one another, as density and capacity each contribute to overall drainage.

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