The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival. A number of clinical and experimental studies suggest that tumor-induced lymphangiogenesis driven by vascular endothelial growth factor (VEGF)-C- and/or VEGF-D-induced activation of VEGF receptor (VEGFR)-3 may promote metastasis to regional lymph nodes. Here we show that constitutive VEGF-C and VEGF-D expression by tumor cells of diverse origin grown in tissue culture does not correlate with metastatic potential in vivo. However, tumors derived from cell lines that do not constitutively express VEGF-C or VEGF-D in tissue culture can nevertheless express one or both of these factors. We demonstrate that both tumor and stromal cells can contribute to this expression, suggesting that tumor cell-host interactions determine tumor expression of VEGF-C and VEGF-D. Using immunocompetent rat mammary tumor models, we show in two ways that this expression can promote metastasis via the lymphatics. Firstly, ectopic expression of a soluble VEGFR-3 receptor globulin protein in MT-450 tumor cells that are highly metastatic via the lymphatics blocked VEGF-C and VEGF-D activity and suppressed metastasis formation in both the regional lymph nodes and the lungs. Secondly, ectopic expression in the weakly metastatic NM-081 cell line of a mutant form of VEGF-C that is only able to activate VEGFR-3 strongly promoted metastasis of these cells to the regional lymph nodes and lung. These data show that expression of VEGF-C and VEGF-D in tissue culture does not reflect expression in vivo and that activation of VEGFR-3 in the absence of VEGFR-2 activation is sufficient to promote tumor-induced lymphangiogenesis and metastasis, and they support the notion that blockade of VEGFR-3 activation will be useful as a novel form of cancer therapy.

https://cancerres.aacrjournals.org/content/canres/63/3/713.full.pdf

Differential in Vivo and in Vitro Expression of Vascular Endothelial Growth Factor (VEGF)-C and VEGF-D in Tumors and Its Relationship to Lymphatic Metastasis in Immunocompetent Rats

OBJECTIVE : Intracarotid infusion of the bradykinin analog, RMP-7, can increase permeability in brain tumor capillaries. This study sought to determine the following : 1) the unidirectional transport, K i , of radiolabeled [ 14 C]carboplatin into brain tumors with either intravenous or intracarotid RMP-7 infusions ; 2) the duration and extent of increased permeability in tumor capillaries during continuous RMP-7 infusions ; and 3) the effect on survival of carboplatin combined with RMP-7 treatment in rats with gliomas. METHODS : Wistar rats with RG2 gliomas were used, and a unidirectional transfer constant, K i , was determined using quantitative autoradiography. In the survival study, the rats were treated with intra-arterial carboplatin and RMP-7 at Days 5 and 7 after tumor implantation. RESULTS : Intracarotid infusion of RMP-7 for 15 minutes increased the transport of [ 14 C]carboplatin to tumors by 2.7-fold, as compared with saline infusion alone (P < 0.001). The transports of [ 14 C]dextran and [ 14 C]carboplatin into tumors were significantly higher with 15 minutes of intracarotid infusion of RMP-7 (0.1 μg/kg/min), compared to those with 10-, 30-, or 60-minute infusions (P < 0.01). Rats treated at Days 5 and 7 after tumor implantation with carboplatin alone (10 mg/kg) exhibited a modest increase in survival at 31 days (37%, compared to <10% of controls), while those given the combination of carboplatin with RMP-7 exhibited a significantly higher survival rate (74%). CONCLUSION : Intracarotid infusion of RMP-7 can selectively increase transport of carboplatin into brain tumors and results in higher survival in rats with gliomas. These findings support the use of intracarotid infusion of RMP-7 to enhance the delivery of carboplatin to patients with malignant brain tumors.

Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7.

The goal of this study was to determine the magnitude of "facilitated" amino acid transport across tumor and brain capillaries and to evaluate whether amino acid transporter expression is "upregulated" in tumor vessels compared to capillaries in contralateral brain tissue. Aminocyclopentane carboxylic acid (ACPC), a non-metabolized [14C]-labeled amino acid, and a reference molecule for passive vascular permeability, [67Ga]-gallium-diethylenetriaminepentaacetic acid (Ga-DTPA), were used in these studies. Two experimental rat gliomas were studied (C6 and RG2). Brain tissue was rapidly processed for double label quantitative autoradiography 10 minutes after intravenous injection of ACPC and Ga-DTPA. Parametric images of blood-to-brain transport (K1ACPC and K1Ga-DTPA, microL/min/g) produced from the autoradiograms and the histology were obtained from the same tissue section. These three images were registered in an image array processor; regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images to obtain mean values. The facilitated component of ACPC transport (deltaK1ACPC) was calculated from the K1ACPC and K1Ga-DTPA data, and paired comparisons between tumor and contralateral brain were performed. ACPC flux, K1ACPC, across normal brain capillaries (22.6 +/- 8.1 microL/g/min) was >200-fold greater than that of Ga-DTPA (0.09 +/- 0.04 microL/g/min), and this difference was largely (approximately 90%) due to facilitated ACPC transport. Substantially higher K1ACPC values compared to corresponding K1DTPA values were also measured in C6 and RG2 gliomas. The deltaK1ACPC values for C6 glioma were more than twice that of contralateral brain cortex. K1ACPC and deltaK1ACPC values for RG2 gliomas was not significantly higher than that of contralateral cortex, although a approximately 2-fold difference in facilitated transport is obtained after normalization for differences in capillary surface area between RG2 tumors and contralateral cortex. K1ACPC, deltaK1ACPC, and K DTPA were directly related to tumor cell density, were higher in regions of "impending" necrosis, and the tumor/contralateral brain ACPC radio-activity ratios (0 to 10 minutes) were very similar to that obtained with 0 to 60 minutes experiments. These results indicate that facilitated transport of ACPC is upregulated across C6 and RG2 glioma capillaries, and that tumors can induce upregulation of amino acid transporter expression in their supporting vasculature. They also suggest that early imaging (e.g., 0 to 20 minutes) with radiolabeled amino acids in a clinical setting may be optimal for defining brain tumors.

https://journals.sagepub.com/doi/pdf/10.1097/00004647-199805000-00005

'Facilitated' amino acid transport is upregulated in brain tumors

Nitric oxide (NO), a free radical gas implicated in a wide variety of biological reactions, is a novel signaling molecule that may regulate vasodilation, cerebral blood flow, and vascular permeability. This study was performed to determine whether NO mediates the selective increase in brain tumor microvessel permeability after intracarotid infusion of bradykinin in the RG2 rat glioma model. Intracarotid infusion of bradykinin selectively increased the transport of radiolabeled alpha-aminoisobutyric acid and dextran into brain tumors. Transport into normal brain was not increased. The administration of an NO synthase inhibitor, NG-nitro-L-arginine methyl ester, significantly inhibited the increased transport into tumors for both tracers. The inhibitory effect of NG-nitro-L-arginine methyl ester on the response to bradykinin was reversed by L-arginine. The expression of two NO synthase (NOS) isoforms in cultured RG2 glioma cell lines and intracerebral RG2 glioma was examined by immunohistochemistry and Western blot analysis. High levels of expression of neuronal NOS were detected in cultured and intracerebral RG2 cells but not in normal brain tissue, except in rare neuronal cells. The endothelial form of NOS was also expressed in cultured RG2 cells, but not as strongly as neuronal NOS expression. In intracerebral RG2 gliomas, expression of endothelial NOS in the tumor was detected at higher levels than in normal brain. These findings indicate that RG2 rat gliomas express high levels of NOS, which regulate the production of NO, compared with normal brain. We suggest that the selective permeability increase in brain tumor microvessels after bradykinin infusion is mediated by NO. Furthermore, the absence of high levels of NOS in normal brain may account for the attenuated permeability response to bradykinin in normal brain microvessels.

https://cancerres.aacrjournals.org/content/canres/56/17/4027.full.pdf

Increased Brain Tumor Microvessel Permeability after Intracarotid Bradykinin Infusion Is Mediated by Nitric Oxide

Two aspects of cytokine therapy of intracerebral tumors are considered in this study: modulation of tumor growth in vivo and central nervous system toxicity. Coimplantation of RG-2 glioma cells and retroviral vector producer cell lines was performed to provide a local source of interleukin-2 (IL-2) or IFN-gamma within the tumor and coinitiate an antitumor immune response. We demonstrated that local intratumoral production of IL-2 and IFN-gamma generates a cell-mediated antitumor response in vivo. This response was manifest as a diffuse infiltration of monocytes/macrophages, CD4+ and CD8+ T cells, and activation of microglial OX42+ cells in intracerebral RG2 tumors. The cell-mediated antitumor immune response resulted in the early suppression of intracranial and subcutaneous tumor growth, but the effect was not sustained and there were no tumor regressions. The absence of increased survival of animals with intracranial tumors is explained in part by the severe central nervous system toxicity caused by local production of IL-2 and IFN-gamma. Central nervous system toxicity induced blood-brain barrier disruption, vasogenic brain edema, and dislocation of the brain midline structures, as observed by dynamic magnetic resonance imaging and direct measurements of tissue water content. The clinical application of IL-2 and IFN-gamma gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with intracerebral production of these cytokines.

https://cancerres.aacrjournals.org/content/canres/55/9/1902.full.pdf

RG-2 Glioma Growth Attenuation and Severe Brain Edema Caused By Local Production of Interleukin-2 and Interferon-γ

The selective production of neurogenic tumors in rats by the resorptive carcinogens methyland ethylnitrosourea has become well established in experimental neurooncology [1]. Neurogenic transplantation tumors in inbred animals are useful for the investigation of a number of problems, e.g. histocompatibility or tumor-specific transplantation antigens. Previous isotransplantation experiments proved successful population with various degrees of differentiation and anaplasia. For isogenic transplantation, tumor tissue is inoculated subcutaneously either as minced tumorbrei or as small pieces (25--250 mg tissue/injection) into male and female rats of different age groups. Table t summarizes the percentage of acceptance of transplanted tumor samples and the rate of tumor growth in t h e two rat strains. CNS tumors have a lower rate of acceptance in the first passage than PNS tumors. The rate increases to about t00% after the second passage. Transplanted primary brain tumors are accepted more readily when injected into recipient hosts younger than one month, but age-dependent differences are less marked for PNS tumors. The mean growth rate for accepted CNS tumors in the first passage is generally slower than that for PNS tumors. Transplanted CNS tumors undergo marked sarcomatous changes, usually after the first passage, while the PNS tumors maintain their primary morphological characteristics for several passages. This study has shown that transplantable tumors of the central and peripheral nervous system can be established in inbred rat strains. The changes in morphology and growth behaviour of primary and transplanted tumors will be described in detail.

A. Gieseler

Papers

Isogenic transplantation of ethylnitrosourea-induced tumors of the central and peripheral nervous system in two different inbred rat strains.