Tumor Vascular Bed Research Articles

Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature.

Antibody-based therapy of solid tumors has met with limited success, chiefly because solid tumors are relatively impermeable to macromolecules. This problem could be circumvented by attacking the readily accessible endothelial cells of the tumor vascular bed. We have developed a model to test this "vascular targeting" approach in which cytokine gene transfection of the tumor cells causes them to induce an experimental marker selectively on tumor vascular endothelium. An anti-tumor endothelial cell immunotoxin caused complete occlusion of the tumor vasculature and dramatic regressions of large solid tumors. By contrast, a conventional anti-tumor cell immunotoxin of equivalent in vitro potency produced only minor, transient antitumor effects but, when combined, the two immunotoxins induced permanent complete remissions in over half of the animals. These experiments indicate that immunotoxins directed against recently described markers on vascular endothelial cells in human tumors could provide a general treatment for solid tumors in humans.

The effects of tumour necrosis factor alpha on the vascular bed and blood flow in an experimental rat hepatoma.

The influence of TNF alpha on tumour growth rate has been attributed to its effects on the vascular bed and blood flow. The aim of our study was to investigate the effects of pharmacological doses of TNF alpha on the tumour vascular bed and to quantify blood flow in an experimental hepatoma during a more extended period after TNF-alpha exposure than hitherto reported. In Lister rats, a syngeneic rat hepatoma was implanted on the dorsum of the right hind foot. TNF alpha was given i.v. The injection was repeated after 24 hr. Tumour blood flow was estimated before and 1, 24, and 96 hr after TNF-alpha administration with the 133Xe-washout technique. The passage of microspheres through the tumour vascular bed (non-entrapment), as a measure of vascular occlusion, was estimated 4 and 96 hr after TNF-alpha administration. Tumour growth rate was measured. The tumours were subjected to histological examination and the sensitivity to TNF alpha in vitro was tested. A reduction of tumour blood flow was observed in TNF-alpha-treated groups. Tumour growth rate was equally increased after 96 hr in both the TNF-alpha groups as compared with controls. There was no significant change in non-entrapment for the TNF-alpha-treated rats as compared with controls. Histology revealed extensive necrosis and thrombosis in tumours. TNF alpha had no effect on the viability of the cloned hepatoma cell line in vitro.

Tumour necrosis factor and cancer

TNF is a cytokine whose diverse actions are dependent on the local microenvironment. As a member of the cytokine network, TNF plays an important role in infection and inflammation, but excessive and deregulated production can contribute to disease processes. Likewise in malignant disease, TNF may have a role in cancer therapy and contribute to host response against tumours, but it may also be involved in the progression and spread of the cancer. In experimental models, recombinant TNF can induce significant haemorrhagic necrosis, localised to the tumour vasculature and specific tumour immunity. Although the historical background and preclinical data are promising, systemic therapy with TNF in human cancer has proved highly toxic and is inactive against all tumour types so far tested. Local therapy, particularly isolated limb perfusion, has resulted in complete and long lasting tumour regressions with necrotic activity confined solely to the tumour vascular bed. However, in several animal models, TNF contributes to malignant progression and there is evidence that TNF may have autocrine or paracrine actions in human ovarian cancer.

Fluctuations in Tumor Blood Flow under Normotension and the Effect of Angiotensin II‐induced Hypertension

To elucidate the significance of angiotensin II (AID‐induced hypertension chemotherapy, changes of tissue blood flow both in normal subcutis and in tumors (AH109A, LY80) were measured with the hydrogen gas clearance method. A newly‐developed anesthetic machine was used to keep the animals' condition constant. Tissue blood flow in normal subcutis and tumors always fluctuated with time under normotension. The nature and the rate of fluctuation in tumor Wood flow were almost identical in two different types of tumors. However, the fluctuation of blood flow in tumor and that in normal subcutis were almost always inversely related when blood flows in these different tissues were measured simultaneously, i.e., when tissue blood flow in normal subcutis decreased, tumor blood flow increased, and vice versa. The findings supported the idea that the connection mode between the tumor vascular bed and normal vascular bed is a parallel circuit. Vascular resistance in the normal vascular bed under All‐induced hypertension seemed to be greater than that under normotension, because the All‐increased tumor blood flow always exceeded the maximum tumor blood flow under normotension. Due to the fluctuations of tumor blood flow, no‐flow or low‐flow areas, resistant to delivery of anti‐cancer drugs, moved sporadically within the tumor under the normotensive condition. However, good conditions for drug delivery to tumor tissue were induced by All‐induced hypertension.

Modification of Tumour Blood Flow—a Review

Tumour vascularization is based on two types of vessels, incorporated normal host tissue vessels and newly formed vessels, and is characterized by a wide heterogeneity. No adrenergic innervation has been related to newly formed tumour vessels but may still be found in incorporated normal vessels. In most studies vasoconstricting drugs were found to decrease tumour blood flow, while vasodilating drugs had no significant influence on tumour blood flow. From this it was concluded that the tumour vascular bed is normally in a state close to maximal dilatation, and this is supported by observations of hypoxia and local acidosis in tumour tissue. Some conflicting results have been reported with an increased tumour blood flow after administration of, for example, calcium channel blockers. Tumour blood modification is of interest in radiotherapy but also in tumour hyperthermia, where circulatory disturbances are explained on a multifactorial basis. Based on conflicting observations, with results varying with tumour-host systems studied and techniques for flow recording, it seems reasonable to concentrate further on methodological studies to develop clinically relevant techniques for tumour blood flow recording.

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration.

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.

Monoclonal Antibody-Defined Human Endothelial Antigens as Vascular Markers

A review is given of human endothelial antigens recognized by monoclonal antibodies and used as vascular markers. These antigens can be classified tentatively into two categories that partly overlap: 1) differentiation markers and 2) antigens involved in specific cellular functions. Monoclonal antibodies recognizing endothelial differentiation markers reacting with all types of human endothelium can be regarded as constitutive endothelial markers. Other differentiation markers have a restricted distribution that is associated with a subtype of endothelium. Although sensitivity of the markers is high in general, specificity for endothelium is not absolute, based on distribution studies in tissues or in cell lines. With the exception of PAL-E and EN-3/EN-4, it is not clear from the literature whether the antibodies also react with lymphatic endothelium. Immunohistochemical examination of other species indicate that only BW 200 is restricted to humans. Immunoelectron microscopy of microvascular cells in tissue specimens has revealed that the monoclonal antibodies recognizing differentiation antigens show different subcellular distribution patterns. PAL-E and BW 200 react with the luminal endothelial surface, in a local and diffuse pattern, respectively. Anti-Von Willebrand factor (i.e., Factor VIII-related ag) antibodies react with Weibel-Palade bodies but also with subendothelial structures. Applications of immunohistochemistry using monoclonal antibodies in diagnostic pathology include assessment of vascular invasion by cancer cells, and identification of endothelial neoplasms and related disorders. Because anti-Factor VIII-related antigen and BW 200 are applicable on formaldehyde-fixed and paraplast-embedded tissue, they are most suitable for histodiagnostic application. Immunohistochemistry using monoclonal antibodies recognizing endothelial antigens involved in specific cellular functions also may contribute to pathobiologic research on the characterization of blood-tissue barriers, e.g., in the tumor vascular bed.

Monoclonal antibody-defined human endothelial antigens as vascular markers.

A review is given of human endothelial antigens recognized by monoclonal antibodies and used as vascular markers. These antigens can be classified tentatively into two categories that partly overlap: 1) differentiation markers and 2) antigens involved in specific cellular functions. Monoclonal antibodies recognizing endothelial differentiation markers reacting with all types of human endothelium can be regarded as constitutive endothelial markers. Other differentiation markers have a restricted distribution that is associated with a subtype of endothelium. Although sensitivity of the markers is high in general, specificity for endothelium is not absolute, based on distribution studies in tissues or in cell lines. With the exception of PAL-E and EN-3/EN-4, it is not clear from the literature whether the antibodies also react with lymphatic endothelium. Immunohistochemical examination of other species indicate that only BW 200 is restricted to humans. Immunoelectron microscopy of microvascular cells in tissue specimens has revealed that the monoclonal antibodies recognizing differentiation antigens show different subcellular distribution patterns. PAL-E and BW 200 react with the luminal endothelial surface, in a local and diffuse pattern, respectively. Anti-Von Willebrand factor (i.e., Factor VIII-related ag) antibodies react with Weibel-Palade bodies but also with subendothelial structures. Applications of immunohistochemistry using monoclonal antibodies in diagnostic pathology include assessment of vascular invasion by cancer cells, and identification of endothelial neoplasms and related disorders. Because anti-Factor VIII-related antigen and BW 200 are applicable on formaldehyde-fixed and paraplast-embedded tissue, they are most suitable for histodiagnostic application. Immunohistochemistry using monoclonal antibodies recognizing endothelial antigens involved in specific cellular functions also may contribute to pathobiologic research on the characterization of blood-tissue barriers, e.g., in the tumor vascular bed.

Tumor necrosis factor/cachectin-induced intravascular fibrin formation in meth A fibrosarcomas.

Recent studies have indicated that TNF can promote activation of the coagulation mechanism by modulating coagulant properties of endothelial cells. In this report, we demonstrate that infusion of low concentrations of TNF (3 micrograms/animal) into mice bearing meth A fibrosarcomas leads to localized fibrin deposition with formation of occlusive intravascular thrombi in close association with the endothelial cell surface. Studies with 125I-fibrinogen showed tenfold enhanced accumulation of radioactivity in tumor within 2 h after TNF infusion. Western blots of tumor extracts subjected to SDS-PAGE and visualized with a fibrin-specific mAb indicated that fibrin forms in the tumor after the TNF infusion. Electron microscopic studies demonstrated fibrin strands, based on the characteristic 21-nm periodicity, which appeared to be adherent to the endothelial cell surface. Further ultrastructural studies indicated that fibrin formation, first evident within 30 min of the TNF infusion, led to occlusive thrombi limited to the tumor vascular bed (i.e., not in the normal mouse vasculature) within 2 h and was associated with an 80% reduction in tumor perfusion based on studies with Evans blue. In view of previous work concerning TNF induction of endothelial cell procoagulant activity, the hypothesis that tumor cell products prime the response of endothelium to this cytokine was tested. Supernatants of cultured meth A fibrosarcomas obtained serum-free conditions, which had no intrinsic procoagulant activity, considerably enhanced tissue factor induction in endothelium in response to submaximal concentrations of TNF. The factor(s) in the tumor-conditioned medium appeared to be distinct from IL-1, fibroblast growth factor, IFN-gamma, TNF, endotoxin, TGF-alpha, and TGF-beta. These studies delineate a novel model of localized clot formation in which thrombosis is initiated by a pathophysiologic mediator, TNF, and provides an opportunity to examine mechanisms in the microenvironment directing clot formation to the tumor vascular bed.

The antitumor function of tumor necrosis factor (TNF), I. Therapeutic action of TNF against an established murine sarcoma is indirect, immunologically dependent, and limited by severe toxicity.

The ability of murine recombinant tumor necrosis factor (rTNF) and natural TNF in tumor-necrotizing serum (TNS) to cause regression of the SA1 sarcoma was investigated. We found that to cause regression of a 9-d SA1 sarcoma, near lethal quantities of rTNF and TNS had to be given to the host. However, even at these highly toxic doses, rTNF was not reliable at causing complete tumor regression. On the other hand, both types of TNF were reliable at causing a tumor hemorrhagic reaction that resulted in the destruction of greater than 75% of the tumor's center in 24 h. The TNF-induced hemorrhagic reaction involved the development of numerous petechial hemorrhages in the tumor's vascular bed, which apparently resulted from destruction of the tumor's blood vessels. It was possible to follow the development of the hemorrhagic reaction against time after giving rTNF or TNS by measuring the intratumor extravasation of 51Cr-labeled syngeneic red cells. According to this method, TNF-induced intratumor hemorrhaging was in progress within 1 h of giving TNF and continued for about a 6-h period. However, the hemorrhagic reaction was greatly reduced and complete regression of the rim of the living tumor tissue that survived hemorrhagic necrosis failed to occur, if SA1 sarcoma was growing in T cell-deficient (TXB) mice. This indicates that the TNF-induced hemorrhagic reaction is partly dependent, and the tumor regression that follows is completely dependent on host immunocompetence. This suggests in turn, that rTNF does not directly destroy SA1 tumor cells in vivo, even though it was shown that it can destroy SA1 tumor cells in vitro. This interpretation is supported by the additional findings that rTNF was no more therapeutic against a 3-d (3-mm) SA1 than against a 9-d (8-mm) SA1, and was no more therapeutic when injected directly into the tumor than when injected intravenously. Lastly it was possible to completely inhibit the ability of rTNF and TNS to cause tumor hemorrhagic necrosis and regression by infusing the host with a monospecific, polyvalent anti-rTNF antibody that neutralized the cytotoxic action of rTNF in vitro.

Serum pseudocholinesterase levels in murine C3H mammary adenocarcinoma.

Serum pseudocholinesterase (PSC) levels may be depressed in persons with malignancy. Deficiency of this enzyme can lead to prolonged apnea in patients who receive succinylcholine. An animal model was developed to study this phenomenon in a controlled setting. C3H/HeJ mice inoculated subcutaneously with C3H mammary adenocarcinoma demonstrated lowering of their PSC levels. This decrease was attenuated by chemotherapy with intraperitoneal cyclophosphamide which also prolonged survival. Non-tumor bearing control animals identically treated with cyclophosphamide experienced a transient drop in PSC on the 29th day which reverted to control values by the 36th day. No gradient of PSC could be demonstrated across a tumor's vascular bed. The effect of other chemotherapy agents on PSC is unknown. A possible role for PSC as a non-specific marker for malignancy is worthy of further study.

Validation of the fluorescent dye Hoechst 33342 as a vascular space marker in tumours.

The DNA-binding fluorescent dye Hoechst 33342 (H33342) has been used in a series of investigations of the vascular parameters of two murine tumours. This dye has been shown, to have a short half-life in the circulation (T1/2 less than 2 min), but is stably bound for at least 2 h after it enters cells. It can be used in morphometric studies on frozen sections to determine the effective vascular volume, the capillary fraction and the size distribution of blood vessels in each tumour. These latter two parameters cannot be deduced from the less labour intensive techniques using radioactive isotopes. The effective vascular volume perfused in 1 min by H33342 was compared with the volume perfused in 30 min with 51Cr labelled erythrocytes. Similar volumes were estimated with the two techniques in a murine carcinoma and in a sarcoma. Both techniques showed that the vascular volume decreased in larger tumours. The H33342 analysis of vessel size showed the decrease in capillary vessels in the carcinomas was even greater, falling from 70% in small tumours to 20% in larger tumours. The deteriorating vascular network in larger tumours is associated with an increasing fraction of necrotic tissue. Experiments in which the isotopes and dye were co-injected suggest that at 40 mgkg-1 the dye may rapidly lead to a partial shutdown of the tumour vascular bed. This is less marked with 20 mg kg-1. In spite of this effect there is in general a close correlation between the volumes perfused by labelled red blood cells and the fluorescent dye.

A dog model using an implanted system for protracted hepatic arterial chemotherapy

A model for hepatic arterial chemotherapy studies using large dogs and an implantable infusion pump has been developed. Using this technique near complete perfusion (>90%) of the liver can be achieved in vivo as determined by hepatic arterial perfusion scintigraphy with technitium 99m macroaggregated albumin. The system is reliable and has been in use for a total of 1353 days (mean of 104 days, range 52–239) in 13 dogs. Pump implantation causes no apparent acute liver damage based on pre- and post-operative alkaline phosphatase and serum glutamic-pyruvic transaminase determinations and does not affect the general mobility or behavior of the animals. Careful placement of the catheter and attention to the physicochemical properties of the solutions loaded are factors contributing to the success of the model. The model permits comprehensive preclinical pharmacokinetic and toxicologic studies of new or preexistent chemotherapeutic agents in the same device that will be used for later administration in human subjects. By providing the means to examine and develop new treatment modalities, it enables the design of even more potent cytotoxic therapy directed into the tumor vascular bed.

Tumour microcirculation as a target for hyperthermia.

A great number of investigators have, independently, shown that tumour blood flow is affected by a hyperthermic treatment to a larger extent than normal tissue blood flow. While the majority of the studies on experimental tumours show a decrease and even a lapse in blood flow within the microcirculation during or after hyperthermia, the data on human tumours are less conclusive. Some of the investigators do not find a decrease in circulation, while others do. Obviously, this is an important field of investigation in the clinical application of hyperthermia because a shut down of the circulation would not only facilitate tumour heating (by reducing venous outflow, this reducing the 'heat clearance' from the tumour), but would also facilitate tumour cell destruction. The same holds for alterations that occur subsequently to the circulatory changes, like a heat-induced decrease of tissue pO2 and pH. If the frequently reported circulatory collapse of the tumour circulation could selectively be stimulated by, e.g. acidification or by vasoactive agents, hyperthermic treatment of patients would possibly be greatly facilitated and intensified. In hyperthermic tumour therapy a number of complex processes and interactions takes place, especially when the treatment is performed in combination with radiation therapy. One of them represents the group of processes related to the random probability of cell sterilization of individual tumour cells resulting in exponential survival curves which are typically evaluated with e.g. cell survival assays. This aspect has not been the issue of this paper. The other group of processes deals with the heat-induced changes in the micro-physiology of tumours and normal tissues which, as discussed before, may not only enhance the exponential cell kill, but which may also culminate in vascular collapse with the ensuing necrosis of the tumour tissue in the areas affected. If this takes place, a process of bulk killing of tumour cells results, rather than the random type of cell sterilization. At present it is not clear to what extent the various separate mechanisms contribute to the total effect of tumour control. With all these considerations in mind, one should be aware of the fact that effects, secondary to heat-induced vascular stasis alone will never be efficient enough to eliminate all tumour cells, even though a heat reservoir is created. This is so because some malignant cells will inevitably have already infiltrated normal, surrounding structures and will therefore not be affected by changes in the tumour vascular bed.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of noradrenaline on interstitial fluid pressure in induced rat mammary tumours

Interstitial fluid pressure was measured by the wick-in-needle technique in DMBA-induced rat mammary tumours during resting conditions and during noradrenaline infusion. The rate of infusion was chosen to increase the systemic blood pressure by 30–40 mmHg, known to result in a markedly increased vascular resistance of these tumours. During resting conditions tumour interstitial fluid pressure was 10.3 ± 1.3 mmHg ( n = 13) but fell to 7.0 ± 1.1 mmHg ( n = 13) during noradrenaline infusion. The result suggests that noradrenaline is likely to affect the pre-capillary resistance vessels of the tumour vascular bed. This result, together with earlier perfusion studies, indicates that noradrenaline is unsuitable for increasing tumour cell perfusion as an adjunctive in radiation and cytotoxic drug therapy.

Vasoactive drugs and tumor blood flow.

Different observations on the reactivity of tumor vessels to vasoactive drugs have suggested a decreased, a similar or an increased reactivity to vasoactive stimuli in the vascular bed of tumors as compared to normal tissues. No adrenergic innervation of newly developed tumor vessels has been found, while preexisting normal vessels incorporated during tumor growth may retain some innervation. In transplantable rat tumors, contractile cells, including smooth muscle cells, have been seen in tumor vessels. From recent experimental studies, it was concluded that the tumor's vascular bed is probably in a state of maximal dilatation and therefore sensitive to vasoconstriction, but less sensitive to pharmacological dilatation. These observations may correspond to regional tumor hypoxia and progressive development of tumor necrosis during tumor growth. The results of experimental tumor studies might question the reliability of diagnostic and therapeutic procedures in clinical oncology, which are based on differences in the reactivity to vasoactive drugs between normal and malignant tissues.

Influence of vasoactive drugs on the intratumor distribution of blood flow and vascular volume in a transplantable rat fibrosarcoma.

The intratumor distribution of plasma volume and of blood flow was studied simultaneously by radioisotope techniques in a transplantable rat fibrosarcoma. Intravenous administration of norepinephrine changed the distribution of both plasma volume and blood flow to produce low values in tumor and in normal muscle. Intravenous administration of papaverine was surprisingly followed by similar effects, probably due to a direct influence of papaverine on cardiac function. Intraaortal administration of papaverine into the studied tissue area did not influence tumor blood flow or plasma volume but increased normal muscle plasma volume. The results might support a previously suggested possibility that one can directly influence the tumor vascular bed with vasoconstricting drugs, while tumor vessels are more resistant to pharmacologic dilatation probably because of their normal state of close to maximal dilatation.

Influence of vasoactive drugs on local tumor blood flow

The influence of vasoactive drugs, including noradrenaline, isoprenaline and papaverine, on the local blood flow of a transplantable rat sarcoma and of normal subcutaneous tissue of the rat was studied by local [ 133 xenon] clearance technique. The microvascular bed of the tumour was studied by transmission electron microscopy (TEM). Vessels with one layer of contractile cells were found in the tumour vascular bed by TEM. A significant reduction of both tumour and normal subcutaneous tissue blood flow was found after administration of noradrenaline added to the injected xenon solution. In a dose—response experiment the tumour and subcutaneous vascular beds seemed to be equally sensitive to noradrenaline. Isoprenaline did not influence the blood flow in either subcutaneous tissue or tumour, while papaverine increased subcutaneous tissue blood flow significantly but did not influence tumour blood flow. The results of the present blood flow studies might suggest that the tumour vascular bed is normally in a state close to maximal dilatation.

Chemoembolization: transcatheter management of neoplasms

"Chemoembolization," the combination of intra-arterial infusion of a chemotherapeutic agent and arterial embolization of the vascular supply to a neoplasm as proposed by Kato et al elsewhere in this issue (p 1123), presents an interesting concept with considerable potential. In addition to the direct effect of ischemia on the neoplasm by temporary occlusion, the emboli decrease the transit time through the tumor vascular bed, theoretically increasing the contact time of the chemotherapeutic agent with the neoplastic cell, increasing local drug concentration, and probably increasing tissue permeability because of the anoxia. The cytotoxic effect is not only on the neoplasm, but on the vessel embolized and infused, producing vasculitis and occlusion. The systemic toxic effect may be reduced by metabolism of the drug on its first passage through the infused organ, thereby confining the higher concentration of the chemotherapeutic agent to the target organ. There is considerable clinical support for each

Chemoembolization: Transcatheter Management of Neoplasms

"Chemoembolization," the combination of intra-arterial infusion of a chemotherapeutic agent and arterial embolization of the vascular supply to a neoplasm as proposed by Kato et al elsewhere in this issue (p 1123), presents an interesting concept with considerable potential. In addition to the direct effect of ischemia on the neoplasm by temporary occlusion, the emboli decrease the transit time through the tumor vascular bed, theoretically increasing the contact time of the chemotherapeutic agent with the neoplastic cell, increasing local drug concentration, and probably increasing tissue permeability because of the anoxia. The cytotoxic effect is not only on the neoplasm, but on the vessel embolized and infused, producing vasculitis and occlusion. The systemic toxic effect may be reduced by metabolism of the drug on its first passage through the infused organ, thereby confining the higher concentration of the chemotherapeutic agent to the target organ. There is considerable clinical support for each