The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model.

Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1ß1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1ß1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.

Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.

Application of Volumetric Modulated Arc Therapy and Simultaneous Integrated Boost Techniques to Prepare "Safe Margin" in the Rabbit VX2 Limb Tumor Model.

BACKGROUND: In this study, we aimed to establish the rabbit VX2 limb tumor model, and then prepare a "necrotic zone" as a safe margin by volumetric modulated arc therapy and simultaneous integrated boost (VMAT-SIB) technique applied in the areas where the tumor is located adjacent to the bone (GTVboost area). MATERIAL AND METHODS: Rabbits in the control group (n=10) were not treated, while those in the test group (n=10) were treated with the SIB schedule delivering a dose of 40Gy, 35Gy, 30Gy, and 25Gy to the GTVboost, GTV (gross tumor volume), CTV (clinical target volume), and PTV (planning target volume) in 10 fractions. Magnetic resonance diffusion-weighted imaging (MRDWI), 3-dimensional power Doppler angiography (3D-PDA), and histological changes were observed after radiotherapy. RESULTS: After radiotherapy, the two groups showed a significant difference in the GTVboost area. In the test group, the tumor necrosis showed a significantly low signal in DWI and high signal in apparent diffusion coefficient (ADC) maps. The 3D-PDA observation showed that tumor vascular structures decreased significantly. Histological analysis demonstrated that a necrotic zone could be generated in the GTVboost area, and microscopic examination observed cell necrosis and fibroplasia. CONCLUSIONS: This studies demonstrated the feasibility of using VMAT-SIB technique in the rabbit VX2 limb tumor model. The formation of a necrotic zone can be effectively defined as safe margin in the GTVboost area. showing potential clinical applicability.

Support of a free radical mechanism for enhanced antitumor efficacy of the microtubule disruptor OXi4503.

Unlike normal blood vessels, the unique characteristics of an expanding, disorganized and leaky tumor vascular network can be targeted for therapeutic gain by vascular disrupting agents (VDAs), which promote rapid and selective collapse of tumor vessels, causing extensive secondary cancer cell death. A hallmark observation following VDA treatment is the survival of neoplastic cells at the tumor periphery. However, comparative studies with the second generation tubulin-binding VDA OXi4503 indicate that the viable rim of tumor tissue remaining following treatment with this agent is significantly smaller than that seen for the lead VDA, combretastatin. OXi4503 is the cis-isomer of CA1P and it has been speculated that this agent's increased antitumor efficacy may be due to its reported metabolism to orthoquinone intermediates leading to the formation of cytotoxic free radicals. To examine this possibility in situ, KHT sarcoma-bearing mice were treated with either the cis- or trans-isomer of CA1P. Since both isomers can form quinone intermediates but only the cis-isomer binds tubulin, such a comparison allows the effects of vascular collapse to be evaluated independently from those caused by the reactive hydroxyl groups. The results showed that the cis-isomer (OXi4503) significantly impaired tumor blood flow leading to secondary tumor cell death and >95% tumor necrosis 24h post drug exposure. Treatment with the trans-isomer had no effect on these parameters. However, the combination of the trans-isomer with combretastatin increased the antitumor efficacy of the latter agent to near that of OXi4503. These findings indicate that while the predominant in vivo effect of OXi4503 is clearly due to microtubule collapse and vascular shut-down, the formation of toxic free radicals likely contributes to its enhanced potency.

The antitumor function of tumor necrosis factor (TNF) II. Analysis of the role of endogenous TNF in endotoxin-induced hemorrhagic necrosis and regression of an established sarcoma.

In agreement with the results of previous studies (1), it was shown that intravenous injection of endotoxin into mice bearing 9-d SA1 sarcoma resulted in a tumor hemorrhagic reaction that rapidly caused necrosis of most of the center of the tumor, and then the complete regression of the rim of living tumor tissue that survived the hemorrhagic reaction. The tumor hemorrhagic reaction was confined to the vascular bed of the tumor, and its rate and extent of development were measured in terms of the intratumor extravasation of 51Cr-labeled syngeneic red cells. The development of the hemorrhagic reaction was associated with the presence in the tumor over a 6-h period of endogenous TNF that was measured in terms of its capacity to kill L929B cells in vitro and identified by its susceptibility to neutralization with a monospecific, polyvalent anti-rTNF antibody. The same antibody was capable in vivo of inhibiting the endotoxin-induced tumor hemorrhagic reaction by only approximately 50%, even when present in the tumor in excess. However, it was capable when given in the same quantity of inhibiting the ability of endotoxin to cause complete tumor regression. The fact that TNF was generated in the tumor during the tumor hemorrhagic reaction, and that infusion of a sufficient quantity of anti-rTNF antibody severely interfered with hemorrhagic necrosis and prevented tumor regression represents convincing evidence that TNF is an essential participant in endotoxin-induced regression of an established SA1 sarcoma. Moreover, because tumor regression, as opposed to hemorrhagic necrosis, failed to occur if the tumor was growing in immunoincompetent mice, but did so if the mice were infused with tumor-sensitized T cells, it can be concluded that an adequate level of T cell-mediated immunity is also an essential requirement for endotoxin-induced tumor regression. The participation of other endotoxin-induced mediators in tumor regression cannot be ruled out.

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.

Nitric oxide synthase inhibition enhances the tumor vascular-damaging effects of combretastatin a-4 3-o-phosphate at clinically relevant doses.

PURPOSE: The therapeutic potential of combining the prototype tumor vascular-disrupting agent combretastatin A-4 3-O-phosphate (CA-4-P) with systemic nitric oxide synthase (NOS) inhibition was investigated preclinically. EXPERIMENTAL DESIGN: Vascular response (uptake of (125)I-labeled iodoantipyrine; laser Doppler flowmetry) and tumor response (histologic necrosis; cytotoxicity and growth delay) were determined. RESULTS: Inducible NOS selective inhibitors had no effect on blood flow in the P22 rat sarcoma. In contrast, the non-isoform-specific NOS inhibitor N(omega)-nitro- l-arginine (l-NNA; 1 and 10 mg/kg i.v. or chronic 0.1 or 0.3 mg/mL in drinking water) decreased the P22 blood flow rate selectively down to 36% of control at 1 hour but did not induce tumor necrosis at 24 hours. CA-4-P, at clinically relevant doses, decreased the P22 blood flow rate down to 6% of control at 1 hour for 3 mg/kg but with no necrosis induction. However, l-NNA administration enhanced both CA-4-P-induced tumor vascular resistance at 1 hour (chronic l-NNA administration) and necrosis at 24 hours, with 45% or 80% necrosis for 3 and 10 mg/kg CA-4-P, respectively. Bolus l-NNA given 3 hours after CA-4-P was the most effective cytotoxic schedule in the CaNT mouse mammary carcinoma, implicating a particular enhancement by l-NNA of the downstream consequences of CA-4-P treatment. Repeated dosing of l-NNA with CA-4-P produced enhanced growth delay over either treatment alone in P22, CaNT, and spontaneous T138 mouse mammary tumors, which represented a true therapeutic enhancement. CONCLUSIONS: The combination of NOS inhibition with CA-4-P is a promising approach for targeting tumor vasculature, with relevance for similar vascular-disrupting agents in development.

Synergistic antitumor effect of CXCL10 with hyperthermia.

PURPOSE: IFN-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) has been described as an antiangiogenic chemokine and displays a potent antitumor activity in vivo. In the present study, we try to investigate whether the combination therapy of hyperthermia, a physical antiangiogenic modality, with CXCL10 would completely eradicate the established solid tumors. METHODS: Immunocompetent BALB/c mice bearing Meth A fibrosarcoma were established. Mice were treated with either CXCL10 at 25 microg/kg once a day for 20 days, hyperthermia was given twice (at 42 degrees C for 1 h, on day 6 and 12 after the initiation of CXCL10), or together. Tumor volume and survival time were observed. The microvessel density was determined by CD31 immunofluorescence. Histologic analysis and assessment of apoptotic cells were also conducted in tumor tissues. RESULTS: The results showed that CXCL10 and hyperthermia inhibited the growth of Meth A fibrosarcoma and interestingly, the combination therapy enhanced the antiangiogenic effects and completely eradicated the established solid tumors. Histological examination revealed that CXCL10 + hyperthermia led to increased induction of apoptosis, tumor necrosis, and elevated lymphocyte infiltration compared with the controls. Moreover, the tumor eradicated animals developed a protective T-cell-dependent antitumor memory response against Meth A tumor cells rechallenge. CONCLUSIONS: Our finding is that the combination therapy can achieve a synergistic antitumor efficacy, supporting the idea that the combination of two antiangiogenic agents may lead to improved clinical outcome. These findings could open new perspectives in clinical antitumor therapy.

Segmentation of dynamic contrast enhanced magnetic resonance imaging data.

INTRODUCTION: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) allows in vivo characterization of tumour vasculature. As such, it is applicable for monitoring the effects of treatments targeting vasculature. The aims of this study were to evaluate the properties of tumour areas segmented-out by DCE-MRI parameters and to evaluate the changes induced by the vascular disrupting agent (VDA) combretastatin A-4 disodium phosphate (CA4DP), a leading VDA in clinical trials, in these areas. MATERIAL AND METHODS: Two tumour models previously shown to respond differently to CA4DP were chosen. The C3H mammary carcinoma and the KHT sarcoma were grown in the right rear foot of CDF(1) and C3H/km mice, respectively, and treated when at 200 or 800 mm(3) in size. DCE-MRI, using the contrast agent Gd-DTPA, was performed on a 7 T spectroscopy/imaging system before and 3 hours after i.p. CA4DP administration at a dose of 100 mg/kg. From the voxel concentration-time curves, the semiquantitative parameter of initial area under the curve (IAUC), the model parameters transfer constant K(trans), interstitial volume v(e), and blood plasma volume v(p), were calculated. Tumour images were segmented into three groups based on the DCE-MRI model parameters using the K-means algorithm, and the groups were ranked by IAUC. RESULTS: The resulting voxels of the tumour segments were mainly spatially connected structures. Initial DCE-MRI parameter values showed different dependencies on tumour model and size in the regions. For all regions in all tumour groups, the treatment reduced IAUC by 36-51%, whereas the model parameters showed more dependencies on tumour model and size. DISCUSSION: This segmentation technique identifies tumour regions with different microenvironmental characteristics responding differently to CA4DP and may be valuable in the optimization of combined VDA with radiotherapy or chemotherapy. The method may also prove useful for optimization and monitoring of local treatment such as radiotherapy.

The dual effects of a novel peptibody on angiogenesis inhibition and M2 macrophage polarization on sarcoma.

Inhibition of the VEGF/VEGF receptor (VEGFR) and angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway is a potential target for tumor angiogenesis. We previously showed that a peptide AS16 which dually inhibits VEGFR/Ang-2 could reduce the tumor growth and decrease the number of microvessels in tumor. However, its short circulating half-life in the serum limits its clinical applications. In this study, as an effort to prolong the short in vivo half-life of AS16, we designed a fusion protein containing peptide AS16 and an IgG Fc fragment. Pharmacokinetic study also revealed that AS16-Fc has a prolonged circulating half-life of about 231 min in rats. We examined the effects of treatment on the tumor vasculature and immune cell populations, tumor growth, in both the MCA-205 and S180 tumor models. We found that AS16-Fc dramatically reduced tumor volume, vascular density and tumor-associated macrophages. Macrophages were identified as potential novel targets following anti-angiogenic therapy, our findings imply a novel role for anti-angiogenic peptide AS16-Fc. These findings indicate that AS16-Fc could be more effective on inhibiting tumor growth angiogenesis and tumor immune microenvironment than that of peptide AS16.

Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma.

Fibrin is deposited on the endothelial cell surface in the vasculature of murine methylcholanthrene A-induced sarcomas after injection of tumor necrosis factor (TNF). Capillary endothelial cells of the tumor vascular bed become positive for tissue factor after TNF injection, based on immunocytochemistry and in situ hybridization. Intravascular clot formation was not dependent on tissue factor derived from tumor cells, since in vessels of tumors not expressing tissue factor, TNF also induced fibrin/fibrinogen deposition. However, the time course of fibrin/fibrinogen deposition after TNF differed in tumors expressing no, little, or greater amounts of tissue factor. Fibrin/fibrinogen deposition was more rapid in tumors in which the neoplastic cells expressed tissue factor than in tumors not expressing tissue factor. In the tumors not expressing tissue factor, activation of coagulation was dependent on TNF-induced synthesis of tissue factor by host cells, i.e., endothelium or monocytes/macrophages. Intravenous somatic gene transfer with tissue factor cDNA in the antisense orientation (but not sense or vector alone) reduced intravascular fibrin/fibrinogen deposition and restored blood flow to the tumor, showing that de novo tissue factor expression is central in TNF-induced activation of the coagulation mechanism.

Characterizing the tumor response to treatment with combretastatin A4 phosphate.

PURPOSE: To examine the pathophysiologic impact of treatment with combretastatin A4 phosphate (CA4P) in regions of tumors that ultimately either necrose or survive treatment with this agent. METHODS AND MATERIALS: Proliferation, perfusion, vessel density, and expression of vascular endothelial growth factor (VEGF) were analyzed in the KHT tumor model after treatment with CA4P. Analyses were conducted in the whole tumor and the tumor periphery. RESULTS: Perfusion in the tumor periphery decreased 4 h after treatment, but returned to baseline 20 h later. Whole-tumor perfusion also decreased 4 h after treatment, but did not return to baseline. Vessel density decreased in the tumor as a whole, but not in the tumor periphery. No significant effect on the expression of VEGF was observed, but a decrease in proliferation in the whole tumor and the periphery was noted. CONCLUSIONS: The present study shows that those areas of a tumor that survive treatment with CA4P are affected by CA4P exposure, though only transiently. The decrease in perfusion could negatively affect therapies utilizing the combination of CA4P and conventional anticancer agents by decreasing drug delivery and tissue oxygenation. These findings suggest that the timing of CA4P treatments when used in conjunction with conventional anticancer therapies should be considered carefully.

Effect of the second-generation vascular disrupting agent OXi4503 on tumor vascularity.

PURPOSE: As first-generation small-molecule vascular disrupting agents (VDA) have begun to enter clinical trials, second-generation agents are under active development. One such agent is the combretastatin A4 disodium phosphate (CA4P) analogue OXi4503 (CA1P). EXPERIMENTAL DESIGN: C3H/HeJ mice bearing KHT sarcomas were treated with CA4P and OXi4503 and the effect on tumor vasculature was determined by evaluating the extent of vascular shutdown (Hoechst-33342 vessel staining) and tumor perfusion inhibition (dynamic contrast-enhanced magnetic resonance imaging). Dynamic contrast-enhanced magnetic resonance imaging and tumor necrosis end points also were used to examine the pathophysiologic tumor effects following repeated exposures to these agents. RESULTS: Single doses of either agent (CA4P, 100 mg/kg; OXi4503, 25 mg/kg) resulted in an 80% to 90% reduction in tumor perfusion 4 hours after treatment. Whereas recovery in tumor perfusion was observed 48 hours posttreatment, this recovery was significantly slower in mice treated with OXi4503. Tumors re-treated with either VDA 72 hours after the first drug exposure showed a similar reduction and recovery in tumor perfusion. Histologic evidence showed the presence of a smaller viable rim after exposure to OXi4503 than that observed after CA4P treatment. Furthermore, the extent of recovery of tumor necrosis 72 hours after drug treatment was less for OXi4053. CONCLUSIONS: The present studies show that the second-generation VDA OXi4503 possesses significant antivascular effects in solid tumors. Importantly, the vasculature of tumors of mice that had received an initial dose this agent was as responsive to a subsequent treatment.

Genetic analysis of high-metastatic clone of RCT sarcoma in mice, and its growth regression in vivo in response to angiogenesis inhibitor TNP-470.

Genetic analysis of a high-metastatic clone of RCT sarcoma (HM-RCT) was the aim of the study. HM-RCT was developed by the lung passage as well as limiting dilution method from the original RCT sarcoma, in which a tumor was spontaneously developed in a C3H/He mouse. HM-RCT expressed enhanced POU domain (class 2, associating factor 1), adenylate cyclase 7, procollagen type III (alpha), A kinase anchor protein 4 and Ehm (expressed on high-metastatic cells) and 11 expressed sequence tags (ESTs). compared with the original clone of RCT. Eighteen specific genes and 14 ESTs were underexpressed in HM-RCT. We investigated the effects of angiogenesis inhibitor TNP-470 on tumor growth and metastasis of this HM-RCT in vivo. In an experimental group, mice received TNP-470 (30 mg/kg) intraperitoneally every other day. After 5 weeks, the growth of the TNP-470-treated tumor was significantly suppressed in vivo, but did not affect the metastasis. The proportion of positive PCNA-stained cells and cellular telomerase activity was significantly low in response to TNP-470.

Targeting neurokinin-3 receptor: a novel anti-angiogenesis strategy for cancer treatment.

Angiogenesis is essential for tumor growth and metastasis, controlling angiogenesis is a promising strategy in cancer treatment. However, thus farther severe side effects of anti-angiogenic drugs have been rather demonstrated, stimulating interest in seeking novel targets of anti-angiogenesis. Neurokinin receptors, also known as tachykinin receptors, are usually considered as drug targets due to diverse physiological functions and their tractability. Although Neurokinin B, the selective natural agonist of neurokinin-3 receptor, have been shown to exhibit anti-angiogenesis activity, the effect and mechanism of neurokinin-3 receptor-mediated angiogenesis still remains unclear. In the present study, we demonstrated that [Mephe7]NKB, an analogue of NKB, possess significant anti-angiogenic effect on CAM. Furthermore, by introducing the tumor angiogenesis homing sequence (NGR), we designed and synthesized two novel agonist analogues of NK3R, NK3R-A1 and NK3R-A2. Both of the two analogues exhibit more efficient anti-migration effect on HUVECs by activating NK3R in vitro, and showed potent antitumor activities with no significant side effects in vivo. Taken together, our results illuminated that NK3R might be a potential novel target for the anti-angiogenesis therapy. Notably, NK3R-A1 might be used as a template for the development of the anti-tumor drugs on the basis of the anti-angiogenesis strategy.

Tissue perfusion inhomogeneity during early tumor growth in rats.

Tissue perfusion in BA 1112 sarcomas of WAG inbred Rijswijk rats was determined from in vivo measurements of capillary density, length, and erythrocyte velocity in modified Algire chamber preparations. Studies were done with the use of television techniques in situ during a period of 26 days, both in control chambers and after implantation of a 0.1-mm3 piece of tumor tissue. Perfusion in control areas void of tumor tissue. Perfusion in control areas void of tumor was approximately 8-10 ml/minute/100 g of tissue. Flow in active tumor growth regions on the outward side of the tumor edge was through undifferentiated channels and had characteristics of flow through a porous medium. Despite enhanced arterial supply, the stabilized tumor microcirculation at the inward side of the growing tumor retained its perfusion rate constant (15-18 ml/min/100 g). Perfusion in central portions of the tumor was about 2-4 ml/minute/100 g during 12 days, whereas the tumor doubled in diameter. Our findings support the concept of temporal and functional blood flow inhomogeneity in the microcirculation of spreading tumors.

Morphologic and DNA autoradiographic studies of the microcirculation in a transplantable rat fibrosarcoma: growth phase.

The morphology and tritiated thymidine uptake of the vascular channels in a transplantable W rat fibrosarcoma sampled at various times during growth are documented. New vessels originated primarily from normal venules in the subcutaneous tissue surrounding the neoplastic implant and grew at least twofold faster than did wound-induced vessels. During the 4-week observation period, partial maturation of vascular channels newly induced by the neoplasm was seen. This partial maturation was evidenced by an increase in the concentration of micropinocytic vesicles, a reduction in concentration and localization of intraluminal processes at or near interendothelial cell junctions, changes in the endothelial cell nuclei, and partial deposition of basement membrane material. The development of smooth muscle and nerve tissue was not seen. The proportion (13%) of labeled endothelial cells in normal subcutaneous connective tissue surrounding the 3-day-old fibrosarcoma implant was significantly higher than that seen in controls, as was the labeling index (14-25%) for endothelial cells in the fibrosarcoma up to 2 weeks after implantation. Vascular channels in the established neoplasm were examined by scanning and transmission electron microscopy and freeze-fracture techniques, and a resemblance to venular morphology was detected.

Passive local anaphylaxis: demonstration of antitumor activity and complementation of intratumor BCG.

When an extracellular dye, Lissamine green, or 51Cr-labeled spleen cells were injected iv into C3H mice bearing small, partially necrotic 3-methylcholanthrene-induced transplantable fibrosarcomas (McC3), the tumor content of these circulating elements per unit weight was substantially lower than that of other selected organs. The level of these blood-borne materials was, however, significantly augmented by the intratumor induction of passive local anaphylaxis (PLA). The PLA-induced augmentation was inhibited by administration of the histamine and serotonin antagonist cyproheptadine; comparable increases were also induced by the intratumor injection of a histamine and serotonin mixture or BCG. The weekly intratumor induction of PLA in McC3 tumors resulted in the complete regression of a significant number of the tumors, and this therapeutic effect was eliminated by cyproheptadine treatment. The intratumor injection of BCG induced the regression of approximately 50% of injected tumors, and the combination of this immunostimulant treatment with the generation of PLA was more therapeutically effective than either treatment alone. PLA in the vicinity of solid tumors may, by increasing vascular permeability, potentiate antitumor effector mechanisms, particularly when these are BCG-stimulated. Despite this demonstration of a possible role of anaphylactic reactions in tumor immunity, no definitive evidence was found that active reagin-mediated local anaphylaxis occurred in C3H mice bearing the McC3 tumor, whether or not they were treated with immunostimulants.

Effect of hyperglycemia on blood flow, pH, and response to hyperthermia (42 degrees) of the Yoshida sarcoma in the rat.

Hyperglycemia (blood glucose, > 20 mmol/liter) caused a 90 to 100% inhibition of blood flow in the solid Yoshida sarcoma of rat feet, as measured by the fractional distribution of 86Rb and 133Xe clearance. Blood flow through the normal gastrocnemius muscle was increased by 50%, while liver blood flow remained unaltered. Hyperglycemia abrogated the temperature differential (approximately 1 degree) between the heating bath and the tumor, promoting more uniform tumor heating. During the period of reduced blood flow, the pH of the tumor extracellular fluid, measured by miniature glass electrode, declined from 7.19 to 6.63 due to decreased efflux of lactate from the tumor. Tumor intracellular pH, measured by partitioning of dimethyloxazolidinedione across the cell membrane, increased from 7.21 to 7.36. At a very high blood glucose concentration (50 mmol/liter), the tumor was isolated from the host, with almost total blockade of water, chloride, glucose, lactate, and dimethyloxazolidinedione exchange between the tumor and the blood. Hyperglycemia therefore represents a convenient means of isolating the Yoshida sarcoma from the host blood supply to enable more selective treatment with hyperthermia and possibly other modalities.

Angiogenesis and prediction of sarcoma formation by plastic.

The capacity to induce new formation of capillaries was tested in cells attached to plastics. It is known that large plastic coverslips implanted s.c. in CBA mice produce sarcomas more rapidly and in a greater number than do small coverslips. We observed that within a few weeks after implantation the cells attached to the large coverslips showed an angiogenic capacity about 5-fold greater than that of the cells attached to the small coverslips. Months before a sarcoma was evident, angiogenesis induced by the cells attached to the large coverslips predicted the high risk of neoplastic transformation by large coverslips.