Endothelial cell pseudopods and angiogenesis of breast cancer tumors
© Cameron et al; licensee BioMed Central Ltd. 2005
Received: 14 July 2004
Accepted: 26 May 2005
Published: 26 May 2005
A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse). Once the tumor grew to 35 mm3, it developed a well-vascularized capsule. Histological sections of tumors greater than 35 mm3 were stained with PAS, with CD-31 antibody (an endothelial cell maker), or with hypoxia inducible factor 1α antibody (HIF). The extent of blood vessel and endothelial cell pseudopod volume density was measured by ocular grid intercept counting in the PAS stained slides.
The tumor area within 100–150 μm of the well-vascularized capsule had few blood vessels and only occasional endothelial cell pseudopods, whereas the area greater than 150 μm from the capsule had more blood vessels, capillaries, and a three-fold increase in volume density of pseudopods sprouting from the capillary endothelial cells. This subcortical region, rich in pseudopods, some of which were observed to have vacuoles/lumens, was strongly positive for presence of HIF. In some larger tumors, pseudopods were observed to insinuate for mm distances through hypoxic regions of the tumor.
The positive correlation between presence of HIF and the increased extent of pseudopods suggests volume density measure of the latter as a quantifiable marker of tumor hypoxia. Apparently, hypoxic regions of the tumor produce HIF leading to production of vascular endothelial growth factors that stimulate sprouting of capillary endothelial cells and formation of endothelial cell pseudopods.
Most tissues in the non-growing adult body have an adequate vascular supply with no need for formation of new blood vessels. Growth in tissue or tumor mass however requires recruitment of endothelial cells to form new blood vessels. In an early report, new blood capillaries were directly observed to form by sprouting of pseudopods from existing endothelial cells . Speidel reported endothelial cell sprouting in the regenerating tail fin of the frog (Rana clamitans) 6 day after amputation. He saw capillary sprouts that progressed for 32 μm through the tissue over a 50-minute period, leaving a solid capillary sprout behind. Up to seven side sprouts or pseudopods also arose along the length of this main capillary sprout during this 50-minute period. He also observed a sprout that acquired a vacuole that fused with the capillary lumen and allowed entry of an erythrocyte from the capillary lumen. This report of morphological events involved in capillary cell sprouting and pseudopod formation remains the most definite description of endothelial cell sprouting and pseudopod formation.
The recruitment of endothelial cells by a tumor is a key early step in tumor angiogenesis . This tumor angiogenic process has become an important target in cancer therapy [2–4]. There is now strong molecular and genetic support for targeting tumor angiogenesis [2–4]. However, there is much more to be learned about tumor angiogenesis. In our own investigation of angiogenesis of the human breast cancer tumors (MDA MB231) growing in the female nude mouse xenograft, we discovered that staining 8 μm thick histological mid-cross sections of the tumor with the periodic acid-Schiff (PAS) technique for glycoproteins  revealed an unexpected abundance of PAS positive endothelial cell pseudopods. The distribution of endothelial cell pseudopods was observed not to be random throughout the tumor. This is the report of a study done to determine the spatial pattern and the possible explanation for this non-random pattern of endothelial cell pseudopods during tumor angiogenesis.
Can the observation of PAS positive endothelial cell pseudopods be generalized to human breast cancer surgical biopsy specimens? To address this question, specimens from 15 breast cancer patients, obtained from the University of Texas Health Science Center at San Antonio Department of Pathology, were examined. Of these 15 patients, nine were diagnosed with ductal confinement intraductal carcinoma, or "DCIC." Although five showed multilayering of cancer cells within the ducts, none showed evidence of endothelial pseudopods within this cancer cell population. Seven of the biopsy specimens were diagnosed as invasive ductal carcinoma, or "IDS." Six of the seven IDS specimens showed evidence of endothelial pseudopods but to a lesser extent than in the MDA MB231 human breast cancer tumor used in the current study. Two IDS tumor specimens had visible regions of necrosis. The region of the tumor adjacent to these necrosis regions had more pseudopods than seen elsewhere in the tumor. Thus, PAS positive endothelial cell pseudopods were commonly present in the human IDS specimens.
The observed localization of HIF in the tumors by immunohistochemistry helped explain the vascular pattern of the encapsulated tumors. The HIF positive reactivity in the tumor was localized in the subcortical areas of the tumor in the same area found to have the most endothelial cell pseudopods (Fig. 2). These findings suggest that this subcortical area of the tumor is hypoxic and is producing angiogenesis growth factors, which in turn acts on the host endothelial cells to sprout pseudopods. Sprouting of cultured endothelial cells in response to vascular endothelial growth factors has been reported . In the same report, Feraud et al. demonstrated that several angiostatic agents could inhibit this endothelial cell sprouting. The presence of pseudopodial like structures in viable areas of a tumor immediately adjacent to necrotic areas of the tumor has been previously reported . The lack of HIF reactivity beneath the well vascularized tumor capsule in the cortical area suggests this cortical area is not hypoxic and not in need of an extensive vasculature. Apparently, the PAS staining of endothelial cell pseudopods in tumors gives quantifiable spatial information on where in the tumor that HIF and vascular endothelial growth factors are located.
Materials and Methods
A detailed account of the materials and methods used in this study has been published elsewhere . Thus, only a summary is given here. Fifteen 6-week-old female athymic nude mice where fed AIN-76 semipurified diet altered to contain 10% corn oil. Two millions human breast cancer cells were inoculated into the inguinal mammary fat pad of each mouse. Once the tumors had grown to greater than 35 mm3 the mice were euthanized by injection of a ketamine/rompun anesthesia supplied by the University of Texas Health Science Center at San Antonio Laboratory Animal veterinarian, then cervically dislocated and exsanguinated by cardiac puncture. The tumors were then removed and fixed in Omni Fix II (Mt. Vernon, NY), dehydrated and embedded in paraffin, sectioned at 4 or 8 μm thickness, mounted on slides, deparaffinized, and stained with H&E or with periodic acid-Schiff (PAS). Additional slides were stained for the immunohistochemical markers for endothelial cells, CD-31 (PECAM-1, PharMingen) or for hypoxia-inducible factor 1-alpha (#OSA-601, Stressgen). Quantification of the volume density of endothelial cell pseudopods was done on the 8 μm thick PAS stained histological sections of the tumors using counts of the number of ocular grid line intercepts.
Archived surgical biopsy specimens that had been fixed in 10% neutralized formalin were dehydrated and embedded in paraffin. Histological sections of the specimens were stained with H&E for routine histopathology examination or with PAS to assess endothelial cell pseudopod distribution.
This work was supported by LSU fund 44096, NIH grant CA7553, and EMF Therapeutics, Inc.
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