Pimonidazole binding in C6 rat brain glioma: relation with lipid droplet detection

In C6 rat brain glioma, we have investigated the relation between hypoxia and the presence of lipid droplets in the cytoplasm of viable cells adjacent to necrosis. For this purpose, rats were stereotaxically implanted with C6 cells. Experiments were carried out by the end of the tumour development. A multifluorescence staining protocol combined with digital image analysis was used to quantitatively study the spatial distribution of hypoxic cells (pimonidazole), blood perfusion (Hoechst 33342), total vascular bed (collagen type IV) and lipid droplets (Red Oil) in single frozen sections. All tumours (n=6) showed necrosis, pimonidazole binding and lipid droplets. Pimonidazole binding occurred at a mean distance of 114 microm from perfused vessels mainly around necrosis. Lipid droplets were principally located in the necrotic tissue. Some smaller droplets were also observed in part of the pimonidazole-binding cells surrounding necrosis. Hence, lipid droplets appeared only in hypoxic cells adjacent to necrosis, at an approximate distance of 181 microm from perfused vessels. In conclusion, our results show that severe hypoxic cells accumulated small lipid droplets. However, a 100% colocalisation of hypoxia and lipid droplets does not exist. Thus, lipid droplets cannot be considered as a surrogate marker of hypoxia, but rather of severe, prenecrotic hypoxia.     

Zonal image analysis of tumour vascular perfusion,hypoxia, and necrosis

A number of laboratories are utilising both hypoxia and perfusion markers to spatially quantify tumour oxygenation and vascular distributions, and scientists are increasingly turning to automated image analysis methods to quantify such interrelationships. In these studies, the presence of regions of necrosis in the immunohistochemical sections remains a potentially significant source of error. In the present work, frozen MCa-4 mammary tumour sections were used to obtain a series of corresponding image montages. Total vessels were identified using CD31 staining, perfused vessels by DiOC(7) staining, hypoxia by EF5/Cy3 uptake, and necrosis by haematoxylin and eosin staining. Our goal was to utilise image analysis techniques to spatially quantitate hypoxic marker binding as a function of distance from the nearest blood vessel. Several refinements to previous imaging methods are described: (1) hypoxia marker images are quantified in terms of their intensity levels, thus providing an analysis of the gradients in hypoxia with increasing distances from blood vessels, (2) zonal imaging masks are derived, which permit spatial sampling of images at precisely defined distances from blood vessels, as well as the omission of necrotic artifacts, (3) thresholding techniques are applied to omit holes in the tissue sections, and (4) distance mapping is utilised to define vascular spacing.     

Changes in tumour morphology with alterations in oxygen availability: further evidence for oxygen as a limiting substrate.

The ability of cancer cells to survive at a distance from blood vessels should be dependent on the local supply of nutrients to each vessel. The corded growth of tumour cells around blood vessels within regions of necrosis in the RH carcinoma in the mouse allows the limit to which cells can be supported by individual vessels to be observed. The thickness of individual tumour cords was measured in conventionally stained tumour sections using a scanning technique to determine the distance between the blood vessel wall and the most distant viable cell adjacent to necrosis. Cord radius was found to vary with the oxygen supply conditions. Control animals had a mean radius of 105 +/- 2 microns while animals that had breathed 10% oxygen had significantly narrower cords (93 +/- 3 microns after 48 h) and animals breathing 100% oxygen had significantly wider cords (117 +/- 3 microns after 24 h). Mice made anaemic (mean hct. 28%) by phlebotomy and plasma transfusion had cord radii that were not significantly different from controls at any time up to 48 h. We conclude that this relatively slow growing mouse tumour is capable of rapid morphological adaptation (less than 3 h) to changes in nutrient availability and that oxygen is probably the limiting substrate.     

Induction of endothelial cell apoptosis by the antivascular agent 5,6-Dimethylxanthenone-4-acetic acid

5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg x kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed. Apoptosis of central vessels in splenic white pulp was also detected in tumour-bearing mice but not in mice without tumours. Apoptosis was not observed in liver tissue. No apoptosis was observed with the inactive analogue 8-methylxanthenone-4-acetic acid. Positive TUNEL staining of tumour vascular endothelium was evident in one patient in a Phase I clinical trial, from a breast tumour biopsy taken 3 and 24 h after infusion of 5,6-Dimethylxanthenone-4-acetic acid (3.1 mg x m(-2)). Tumour necrosis and the production of tumour tumour necrosis factor were not observed. No apoptotic staining was seen in tumour biopsies taken from two other patients (doses of 3.7 and 4.9 mg x m(-2)). We conclude that 5,6-Dimethylxanthenone-4-acetic acid can induce vascular endothelial cell apoptosis in some murine and human tumours. The action is rapid and appears to be independent of tumour necrosis factor induction.     

Antivascular therapy of cancer: DMXAA

The vascular endothelium of tumour tissue, which differs in several ways from that of normal tissues, is a potential target for selective anticancer therapy. By contrast with antiangiogenic agents, antivascular agents target the endothelial cells of existing tumour blood vessels, causing distortion or damage and consequently decreasing tumour blood flow. DMXAA (5,6-dimethylxanthenone-4-acetic acid), a low-molecular-weight drug, has a striking antivascular and in some cases curative effect in experimental tumours. Its action on vascular endothelial cells seems to involve a cascade of events leading to induction of tumour haemorrhagic necrosis. These events include both direct and indirect effects, the latter involving the release of further vasoactive agents, such as serotonin, tumour necrosis factor, other cytokines, and nitric oxide from host cells. Phase I clinical trials of DMXAA have been completed and the next challenge to face is how the antivascular effect of this drug should be exploited for the treatment of human cancer.     

Expression of tumour necrosis factor alpha and its receptors in carcinoma of the breast.

The expression of tumour necrosis factor alpha (TNF-alpha) and its two distinct receptors, TNF-R p55 and TNF-R p75, was assessed by immunocytochemistry in 28 primary breast cancer and three reduction mammoplasty specimens (''normal'' breast tissue). Expression of TNF-alpha or TNF-R p75 was not detectable in normal breast tissue or in non-malignant breast tissue adjacent to the tumours. By contrast, TNF-R p55 was expressed by occasional stromal cells in normal tissue. TNF-alpha was expressed focally in 50% of the tumours studied, being largely localised to macrophage-like cells in the stroma. TNF-R p55 was expressed by a population of stromal cells in all the tumours examined, and a varying proportion of neoplastic cells in 75% of these tissues. TNF-R p75 was detected in about 70% of the tumours, immunoreactivity being confined mainly to cells in the stroma. In this preliminary study there was no association between the above cytokine parameters and such measures of tumour biology as lymph node status, tumour grade, proliferative activity or degree of angiogenesis. However, there was a correlation between the expression of TNF-R p55 by blood vessels and the number of leucocytes present.     

The tumour stroma of oral squamous cell carcinomas show increased vascularity compared with adjacent host tissue.

For tumours to grow they must acquire an adequate blood supply, and the use of drugs to inhibit tumour vascularization is one promising approach to anti-cancer therapy. Clear information is therefore required on the vascular architecture of human tumours and animal tumour models used for testing anti-angiogenic therapies. Many previous studies on animal tumour models have shown that carcinomas are least vascular in their centres and that host tissues become more vascular with proximity to the tumour. However, we have previously found that many human colorectal carcinomas do not show this pattern. The present study on human oral squamous cell carcinomas (SCCs) again reveals significant differences. Paraffin sections from 24 SCCs were immunostained using the QBEnd-10 monoclonal antibody to demonstrate blood vessels, and these were quantified by interactive morphometry using a Kontron Videoplan system. In most carcinomas, viable tumour tissue was no less vascular in the tumour centre than in the tumour periphery. Although tumours are known to release angiogenic factors, viable tumour tissue was less vascular than adjacent host tissues. However, the tumour stroma, by itself, was more vascular than adjacent host tissues. Host tissue adjacent to tumour showed no obvious increase in vascular density with increasing proximity to the tumour edge, which suggests that tumour-released angiogenic factors are only effective over a short distance.     

Tumour endothelial marker 8 (TEM-8) in human colon cancer and its association with tumour progression.

BACKGROUND AND AIMS: Tumour endothelial marker-8 (TEM-8) is endothelial cell surface marker that may be specific to tumour endothelial cells. This study examined the role of TEM-8 in human colon cancer and its correlation with tumour prognosis. METHODOLOGY: Specimens of colorectal tissue (normal and cancer) were stained immunohistochemically with an anti-TEM-8 antibody, newly developed in our laboratory, and with anti-vonWillebrand Factor antibody. RNA was extracted from frozen sections for gene amplification. The anti-TEM-8 antibody specificity tested by using slot blotting with irrelevant antibody, and western blotting with different cell lines. The expression of TEM-8 was assessed using RT-PCR, and the level of TEM-8 was quantified using real-time-quantitative PCR (Q-RT-PCR). RESULTS: TEM-8 staining was primarily seen in endothelial cells. TEM-8 identified more micro-vessels in colon tumour tissue, than in normal colon tissues, (p=0.002). Whereas, fewer vessels were stained positive for TEM-8 in normal tissues stained positive for vonWillebrand Factor (factor-8), (p=0.008). Malignant cells in tumour tissues were found to be stained strongly positive for TEM-8 compared with the epithelial cells in normal colon tissues. The level of TEM-8 expression was significantly higher in the tumour tissues compared to the normal colon mucosa (p=0.001). TEM-8 mRNA expression was also found to be more elevated in patients with advanced tumour, Dukes C (Dukes A vs. Dukes C, p=0.01). CONCLUSION: TEM-8 is a marker that identifies tumour associated micro-vessels in colon cancer. The levels of expression of TEM-8 in invasive colon cancer are linked to disease progression. This suggests that TEM-8 has significant prognostic and therapeutic values in colon cancer.     

Effects of tumour cells on angiogenesis and vasoconstrictor responses in sponge implants in mice.

The effects of tumour cells (Colon 26) on the development and response of new blood vessels to different vasoconstrictors (platelet activating factor; PAF, endothelin-1, angiotensin II, adrenalin and 5-hydroxytryptamine) have been investigated. Sponge implants in mice were used to host tumour cells while washout of 133Xe was employed to assess local blood flow in the implanted sponges. By 14 days after implantation the response of vessels in tumour-bearing implants to the various vasoconstrictors generally was decreased compared to that obtained in control sponge implants or adjacent normal skin. Thus at this time point the t1/2 for 133Xe washout from control sponges treated with adrenalin (0.5 micrograms) was 30 +/- 4 min whereas in tumour-bearing sponges it was 5 +/- 1 min. This decreased sensitivity in tumour vessels was probably not due to a complete lack of contractile elements since actin was demonstrated by immunohistochemistry around blood vessels in both types of implant. The results of the present study have shown that the pharmacological responses of blood vessels in a growing tumour, Colon 26, differed from the responses of vessels of a similar age in non-neoplastic tissue. These results appear to suggest that the different angiogenic stimuli released from tumour tissue may markedly influence pharmacological reactivity of newly formed blood vessels.     

Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia, and myeloid cells

The NGR peptide motif is an aminopeptidase N (CD13) ligand that targets angiogenic blood vessels. NGR-containing peptides have proven useful for delivering cytotoxic drugs, proapoptotic peptides, and tumor necrosis factor-alpha(TNF) to tumor vasculature. Given that CD13 is not only expressed in the angiogenic endothelium but also in other cell types, the mechanism(s) for the tumor-homing properties of NGR-drug conjugates remains elusive. We have examined the expression of CD13 in normal and neoplastic human tissues and cells by using two anti-CD13 monoclonal antibodies. The immunoreactivity patterns obtained with cultured cells and tissue sections from kidney, breast, and prostate carcinomas suggest that different CD13 forms are expressed in myeloid cells, epithelia, and tumor-associated blood vessels. Both, direct binding assays with a CNGRCG-TNF conjugate (NGR-TNF) and competitive inhibition experiments with anti-CD13 antibodies showed that a CD13 isoform expressed in tumor blood vessels could function as a vascular receptor for the NGR motif. In contrast, CD13 expressed in normal kidney and in myeloid cells failed to bind to NGR-TNF. Consistently with these results, neither murine(125)I-NGR-TNF nor (125)I-TNF accumulated in normal organs containing CD13-expressing cells after administration to mice. These findings may explain the selectivity and the tumor-homing properties of NGR-drug conjugates and may have important implications in the development of vascular-targeted therapies based on the NGR/CD13 system.     

Use of a carbocyanine dye as a marker of functional vasculature in murine tumours

An intravenously administered fluorescent carbocyanine dye, DiOC7(3), has been evaluated for use in conjunction with Hoechst 33342 as a marker of murine tumour vasculature. DiOC7(3) stains cells immediately adjacent to blood vessels and thus, like Hoechst 33342, outlines perfused tumour vasculature. The different fluorescence excitation and emission properties of DiOC7(3) and Hoechst 33342 permit discrimination of the stains in the same tissue section. Mice tolerate a DiOC7(3) dose of 1 mg kg-1 i.v. with no ill effects. The dye has a distribution half-life in blood of 180s and staining of perivascular tumour cells is sufficiently stable to allow visualisation of vasculature for up to 30 min after DiOC7(3) injection. However, DiOC7(3) causes a 75% reduction in tumour blood flow as measured by laser Doppler techniques. Consequently, the compound appears to be most suitable as a second vascular marker, administered at some time after Hoechst 33342, to detect temporal and spatial fluctuations in tumour perfusion.     

A simple fluorescent technique for the location of tumour cells in frozen sections of the head and neck region.

Tumour cells possess cell surface proteases referred to as guanidinobenzoatase (GB) which are closely similar to plasminogen activator. Previous studies have demonstrated different isoenzymic forms of GB on tumour cells and normal cells which can be recognised by cytoplasmic protein inhibitors extracted from frozen sections of appropriate tissues. We now show that normal human serum possesses inhibitors which selectively recognise the isoenzymic forms of GB associated with normal cell surfaces but do not recognise the GB on tumour cell surfaces in frozen sections of tissue obtained from the head and neck regions.     

Photodynamic therapy: An effective,but non-selective treatment for superficial cancers of the oral cavity

It has often been claimed that photodynamic therapy (PDT) produces selective destruction of small cancers without affecting the adjacent normal tissue. The objective of our work was to treat small cancers of the oral cavity with PDT and subsequently excise the treated areas for histological studies of tumour and adjacent normal tissue exposed to the same light dose. Eleven patients with histologically proven T1NO oral squamous-cell carcinomas were treated with PDT, using Photofrin as a sensitiser. The tumours plus a surrounding cuff of normal tissue were exposed to 50 J/cm² non-thermal laser light at 630 nm delivered by surface illumination and the treated areas subsequently excised. Histological staining and image analysis were used to determine the nature and extent of injury. No macroscopic distinction was evident between tumour and normal tissue exposed to light. Histologically, replacement of superficial epithelium, tumour and connective tissue with a fibrinous necrotic slough was seen. There was also loss of endothelium from small vessels, with haemorrhage and thrombosis. Preservation of subepithelial collagen and elastin was demonstrated with EVG staining. No evidence of selective tumour necrosis was found. Although depth of injury was variable, full thickness mucosal necrosis occurred in all cases.Int. J. Cancer 71: 937-942, 1997. © 1997 Wiley-Liss Inc.     

CD133 identifies perivascular niches in grade II–IV astrocytomas

The aim of the present study was to investigate the localization and distribution of the putative brain tumour stem cell marker CD133 in formalin fixed paraffin embedded astrocytomas. A retrospective analysis of 114 grade II, III and IV astrocytomas was undertaken. The immunohistochemical expression of CD133 in paraffin sections was analysed using morphometry. In all grades, CD133 was expressed on tumour and endothelial cells. Tumour cells were found in perivascular niches, as dispersed single cells and in pseudopalisade formations around necrosis. There was no correlation between the mean volume fraction of CD133⁺ niches and all CD133⁺ tumour cells and tumour grade. However, the volume fraction of CD133⁺ blood vessels increased significantly from 0.4% in diffuse astrocytomas to 2.2% in glioblastomas. Neither of them was related to patient survival. Double immunofluorescence stainings showed that the CD133⁺ niches both contained CD133⁺ cells with and without co-expression of the intermediate filament protein marker nestin, and only few CD133⁺/MIB-1⁺ proliferating cells were found. In conclusion, a CD133⁺ perivascular stem cell-like entity exists in astrocytomas. CD133⁺ tumour vessels may play an important role in a brain tumour stem cell context, while CD133 alone does not appear to be a specific tumour stem cell marker related to patient survival.     

Cell proliferation and vascularization in human breast carcinomas.

Cell proliferation and vascularization were studied in 10 human breast carcinomas by an immunoenzyme technique. The monoclonal antibody (MAb) Ki-67 was used as a marker for proliferating cells and a polyclonal antibody directed against human von-Willebrand factor to identify blood vessels. The proportion of Ki-67-labelled cells varied from 1% to 20%, the number of small blood vessels from 4.4/mm2 to 57.6/mm2. Within single histological sections of individual tumours the percentage of proliferating cells was not related to the number of small blood vessels. However, after evaluation of 5 sections of each tumour, the average values showed that tumours with a high grade of vascularization had a higher percentage of Ki-67-positive cells than poorly vascularized samples. The influence of vascular density on cell proliferation was investigated in a selected area of one of the tumours (in 2-dimensions) and with regard to the over- and underlying sections (in 3-dimensions). After 2-dimensional evaluation, distances from proliferating cells to the closest blood vessel between 10 and 390 microns were observed, and after 3-dimensional evaluation none of the proliferating cells measured was located more than 130 microns away from the closest vessel.     

Histochemical probes for the detection of hypoxic tumour cells.

Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate dehydrogenase activity, endogenous lactate and accumulation of neutral fat. An ascites tumour (Yoshida hepatoma) and a solid tumour (Ehrlich carcinoma) were used as the experimental models. A gel film technique was used for visualizing lactate dehydrogenase and "nothing dehydrogenase" (or endogenous lactate). The fluorescent dyes Nile Red and Acridine Orange were used to demonstrate lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or LDHk isoenzymes bound to membranous structures; 2) an intense granular positivity of "Nothing Dehydrogenase" due to high concentrations of endogenous lactate; 3) neutral lipid droplets emitting an intense yellow fluorescence in Nile Red-stained preparations; 4) a yellow cytoplasmic fluorescence in Acridine Orange-stained sections, attributable to a low cellular RNA content. Electron microscopy revealed moderately osmiophilic lipid globules in close association with damaged mitochondria. Better oxygenated cells showed: (a) a reddish-blue diffuse pattern of LDH, ascribed to moderately active soluble LDH isoenzymes containing H subunits; (b) almost no "Nothing Dehydrogenase" positivity; (c) no cytoplasmic lipid droplets; and (d) an intense orange-red fluorescence in the cytoplasm of Acridine Orange-stained specimens, due to high concentrations of cellular RNA. Nile Red fluorescence showed that the lipids of the solid tumour membranes were more hydrophobic than in the normal surrounding tissue. This suggests that there are abnormal domains of neutral lipids in the tumour cell membranes. In solid tumours, cells with the characteristics attributable to hypoxia were usually observed on the edge of necrosis of cuff-like formations. In very advanced growth stages, however, they were also seen surrounding (and occasionally clogging) blood vessels, or in tentacular formations coming from a necrosis border and polarized towards the vessels. Lipid-loaded cells were also seen in blood vessels distant from the tumour. These observations point towards a chemotactic process of hypoxic cells towards better environments.     

Critical evaluation of histochemical and immunochemical methods for the demonstration of vascular supply in rectal and oesophageal cancer

The vascularisation of rectal and oesophageal carcinomas and of normal mucosa was studied using histochemical and immunohistochemical methods. Endothelial cells were stained for alkaline phosphatase (AP) using an azo-dye procedure. Histochemical results were compared with the immunohistochemical identification of endothelial cells using the monoclonal antibody BW 200 recognising an epitope restricted to human endothelial cells. In the AP experiments the simultaneous reactivity of stromal tissue often precluded the exact evaluation of tumour blood vessels. Immunohistochemistry facilitated the identification of vessels in neoplastic tissues and allowed a quantitative analysis of vascular volume by means of point-counting. Vascular volumes of normal tissues exceeded those of tumours by a factor of 1.6. This immunohistochemical technique has potential application in studying the importance of tumour blood supply in man, especially in relation to radiotherapy.     

Reduced capacity of tumour blood vessels to produce endothelium-derived relaxing factor: significance for blood flow modification.

The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.