Dorsal Skinfold Window Chamber Research Articles

Microvascular Diameter Responses to the Controlled Release of Platelet Derived Growth Factor‐BB (PDGF‐BB) and Transforming Growth Factor‐β1 (TGF‐β1)

PDGF-BB, a smooth muscle cell (SMC) chemoattractant and mitogen, and TGF-β1, a SMC differentiation factor, may have utility as therapeutic arteriogenesis factors. However, few studies have examined whether the controlled release of PDGF-BB or TGF-β1 can amplify arteriogenesis. To this end, we utilized the mouse dorsal skinfold window chamber model, which allows for repeated vessel measurements over time and naturally exhibits arteriolar and venular structural lumen expansion. An 85:15 poly(DL-lactide-co-glycolide) microsphere containing either 0.2μg/mg PDGF-BB, 0.008μg/mg TGF-β1 or 4μg/mg BSA was inserted on Day 7. Microvascular diameter measurements were made at Days 7, 10 and 14 on individual vessels within the release profile of the microsphere (4mm) during topical superfusion of adenosine (10−4 M). Interestingly, PDGF-BB treatment elicited no change in arteriolar structural diameter [PDGF-BB: =25.0μm (48.0%) vs. BSA: =23.1μm (44.2%)], but a significant increase in venular structural diameter [PDGF-BB: =48.9μm (61.9%) vs. BSA: =14.2μm (18.2%)]. TGF-β1 treatment significantly attenuated normal arteriolar [TGFβ1: =7.8μm (17.1%) vs. BSA: =23.1μm (44.2%)] and venular [TGF-β1: =−14.4μm (−1.2%) vs. BSA: =14.2μm (18.2%)] structural lumen expansion. Because PDGF-BB elicited no further increase in arteriolar diameter and TGF-β1 caused vessel constriction, our results do not support the therapeutic potential of these factors. However, the mechanisms of these diameter responses remain unknown, and alterations in growth factor concentration and/or release kinetics may be needed to show efficacy. Supported by NIH HL65958.

Preferential extravasation and accumulation of liposomal vincristine in tumor comparing to normal tissue enhances antitumor activity

To quantitatively evaluate the extravasation, accumulation and selectivity to tumor tissues of liposomal vincristine (LV), dorsal skin-fold window chambers on athymic mice with or without LX-1, a human small cell lung cancer, xenograft implants and fluorescent intravital microscopy imaging were used. In vitro studies show that minimal loss of fluorescence marker DiI from liposomes occurs after 4 days of inoculation in murine plasma, and the release profiles of DiI-LV and LV were essentially the same with approximately 40% of the encapsulated vincristine sulfate (VCR) released after 26 h. Significantly faster extravasation of DiI-LV from tumor vessels was shown compared to non-tumor tissue after single dose i.v. administration. The relative interstitial amounts at 60 min (RIA(60)) for tumor and non-tumor tissues were 0.837+/-0.314 and 0.012+/-0.091, respectively (P=0.01). DiI-LV accumulation was significantly higher in tumor than in normal tissue, which continued beyond 48 h. Both DiI-LV and LV showed significant antitumor effects in window chambers and in flank tumors, compared with controls and VLS alone. The preferential extravasation of DiI-LV from tumor vasculature as well as its differential retention in tumor tissue provides the basis for the enhancement in antitumor activity of LV over VCR.

A manganese porphyrin superoxide dismutase mimetic enhances tumor radioresponsiveness

To determine the effect of the superoxide dismutase mimetic Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) on tumor radioresponsiveness. Various rodent tumor (4T1, R3230, B16) and endothelial (SVEC) cell lines were exposed to MnTE-2-PyP(5+) and assayed for viability and radiosensitivity in vitro. Next, tumors were treated with radiation and MnTE-2-PyP(5+)in vivo, and the effects on tumor growth and vascularity were monitored. In vitro, MnTE-2-PyP(5+) was not significantly cytotoxic. However, at concentrations as low as 2 mumol/L it caused 100% inhibition of secretion by tumor cells of cytokines protective of irradiated endothelial cells. In vivo, combined treatment with radiation and MnTE-2-PyP(5+) achieved synergistic tumor devascularization, reducing vascular density by 78.7% within 72 h of radiotherapy (p < 0.05 vs. radiation or drug alone). Co-treatment of tumors also resulted in synergistic antitumor effects, extending tumor growth delay by 9 days (p < 0.01). These studies support the conclusion that MnTE-2-PyP(5+), which has been shown to protect normal tissues from radiation injury, can also improve tumor control through augmenting radiation-induced damage to the tumor vasculature.

Observation of Incipient Tumor Angiogenesis That Is Independent of Hypoxia and Hypoxia Inducible Factor-1 Activation

It is well established that hypoxia potently stimulates tumor angiogenesis by activating hypoxia inducible factor-1 (HIF-1)-induced proangiogenic factors, such as vascular endothelial growth factor. However, very little is known about the role of hypoxia in incipient angiogenesis in avascular tumors during their early stages of growth. To noninvasively investigate the functional significance of hypoxia and HIF-1 activation in incipient tumor angiogenesis, we genetically engineered HCT116 human colon carcinoma cells and 4T1 mouse mammary carcinoma cells with constitutively expressed red fluorescence protein as a tumor marker and green fluorescence protein (GFP) as a reporter for hypoxia and HIF-1 activation. The accuracy of GFP fluorescence in reporting hypoxia was confirmed by flow cytometry analysis and by immunohistochemical comparison with pimonidazole, a well-established hypoxia marker drug. Mouse dorsal skin-fold window chambers showed that incipient angiogenesis preceded a detectable level of hypoxia. The detectable levels of hypoxia were spatially and temporally related with more intensive secondary angiogenesis following the initial onset of new vessel formation. Selective killing of hypoxic cells by tirapazamine efficiently eliminated or delayed the detection of hypoxic cells, but it did not significantly delay the onset of incipient angiogenesis. These findings provide the first in vivo evidence that incipient tumor angiogenesis may not depend on hypoxia or HIF-1 activation. This is in contrast to the clear role of hypoxia in driving angiogenesis once initial tumor microvessel formation has occurred.

Study of non-uniform nanoparticle liposome extravasation in tumour

The effect of hyperthermia on the nanoparticle extravasation in different tumour regions was investigated in real time using confocal laser scanning microscopy. Murine mammary carcinoma 4T1 was implanted in the nude mice dorsal skin-fold window chamber. Tumour angiogenesis was observed through the window chamber on days 4, 7, 8 and 10 after the implantation. In 10 days, the tumour became 1–2 mm in diameter and 150 µm thick. Most vessels were found to be <15 µm in diameter. Histological examination showed that there were fewer vessels in a more ordered branching pattern inside the tumour than in the tumour periphery. After hyperthermia at 42°C for 1 h, numerous erythrocytes were found in the peripheral region. Extravasation of rhodamine-labelled 100 nm nanoparticles in different tumour regions under both normal and hyperthermic conditions (34 and 42°C) was quantified using confocal fluorescence microscopy. The relative fluorescence intensity hardly changed in tissue at 34°C, but increased by the local hyperthermia at 42°C. In particular, the relative intensity in the tumour periphery was more than 120 as compared to 40 in the tumour centre, after 1 h hyperthermia. Results showed that the thermally induced liposome nanoparticle extravasation was heterogeneous in tumour, owing to the non-uniform distribution of tumour vasculature. Further, the degree of vascular damage was found to be more severe in the tumour periphery, which is likely due to the high thermal sensitivity of newly formed tumour vessels in this region.

Hyperspectral imaging of hemoglobin saturation in tumor microvasculature and tumor hypoxia development

Tumor hypoxia has been shown to have prognostic value in clinical trials involving radiation, chemotherapy, and surgery. Tumor oxygenation studies at microvascular levels can provide understanding of oxygen transport on scales at which oxygen transfer to tissue occurs. To fully grasp the significance of blood oxygen delivery and hypoxia at microvascular levels during tumor growth and angiogenesis, the spatial and temporal relationship of the data must be preserved and mapped. Using tumors grown in window chamber models, hyperspectral imaging can provide serial spatial maps of blood oxygenation in terms of hemoglobin saturation at the microvascular level. We describe our application of hyperspectral imaging for in vivo microvascular tumor oxygen transport studies using red fluorescent protein (RFP) to identify all tumor cells, and hypoxia-driven green fluorescent protein (GFP) to identify the hypoxic fraction. 4T1 mouse mammary carcinoma cells, stably transfected with both reporter genes, are grown in dorsal skin-fold window chambers. Hyperspectral imaging is used to create image maps of hemoglobin saturation, and classify image pixels where RFP alone is present (tumor cells), or both RFP and GFP are present (hypoxic tumor cells). In this work, in vivo calibration of the imaging system is described and in vivo results are shown.

Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution

BackgroundElectroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal.MethodsIntravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP) and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared.ResultsElectroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses.ConclusionsIntravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous distribution of gene expression in the syngeneic P22 rat tumor model.

Responses of vascular endothelial cells to angiogenic signaling are important for tumor cell survival.

Neoplastic cells overexpress several angiogenic cytokines, which stimulate neovascularization. Whether the responses of the host endothelial cells to these signaling molecules affect tumor cells during early tumorigenesis has not been investigated. We investigated pre-angiogenic tumor cell survival and angiogenesis initiation by two murine tumor lines (4T1 mammary carcinoma and B16 melanoma), which constitutively expressed GFP, in dorsal skin-fold window chambers of mice treated with extracellular domain of Tie-2 (ExTek) or bFGF. ExTek reduced tumor cell survival, retarded tumor growth, and inhibited angiogenesis onset compared with controls. bFGF increased tumor cell survival and promoted earlier angiogenesis and tumor growth. Neither bFGF nor ExTek affected cell proliferation in vitro. RT-PCR showed mRNA expression of bFGF receptor 2 (FGFR2) IIIb, which does not bind bFGF efficiently, by 4T1 cells and B16 cells express FGFR1 but not FGFR2. B16 cells expressed angiopoietin (Ang) 2, but neither cell line expresses Ang1. Both tumor lines express VEGF. These findings suggested that effects of bFGF and ExTek on tumor cell survival and angiogenesis were not due to direct action but were instead a result of paracrine factors secreted by endothelial cells. These subsequent signals from endothelial cells promote early survival and proliferation of disseminated tumor cells before onset of angiogenesis.

Selectin-dependent rolling and adhesion of leukocytes in nicotine-exposed microvessels of lung allografts.

The interaction of circulating leukocytes with lung microvessels is a critical event in the recruitment of effector cells into the interstitial tissue during episodes of inflammation, including smoking-induced chronic airway disease. In the present study, murine lung tissue transplanted into a dorsal skinfold window chamber in nude mice was used as a model system to study nicotine-induced leukocyte trafficking in vivo. The revascularized lung microvessels were determined to be of pulmonary origin based on their ability to constrict in response to hypoxia. We demonstrated that nicotine significantly enhanced rolling and adhesion of leukocytes within lung microvessels comprising arterioles and postcapillary venules in a dose-dependent manner, but failed to induce leukocyte emigration. Nicotine-induced rolling and adhesion was significantly higher in venules than in arterioles. Treatment of mice with monoclonal antibodies (MAbs) against L-, E-, or P-selectin after exposure of lung allografts to nicotine resulted in variable but significant inhibition of nicotine-induced rolling, whereas nicotine-induced subsequent adhesion was inhibited by MAbs against L- and P-selectin but not E-selectin. Exposure of lung allografts to nicotine along with PD-98059, a mitogen-activated protein kinase (MAPK)-specific inhibitor, resulted in significant inhibition of nicotine-induced rolling and adhesion. In vitro, exposure of murine lung endothelial cells to nicotine resulted in increased phosphorylation of mitogen-activated/extracellular signal-regulated protein kinase 1/2, which could be blocked by PD-98059. Overall, these results suggest that nicotine-induced inflammation in the airways could potentially be due to MAPK-mediated, selectin-dependent leukocyte-endothelial cell interactions in the lung microcirculation.

A Murine Model to Study Leukocyte Rolling and Intravascular Trafficking in Lung Microvessels

The cascade of leukocyte interactions under conditions of blood flow is well established in the systemic microcirculation, but not in lung microcirculation. We have developed a murine model to study lung microcirculation by transplanting lung tissue into dorsal skin-fold window chambers in nude mice and examining the ability of leukocytes to traffic within revascularized lung microvessels by intravital microscopy. The revascularized lung allograft demonstrated a network of arterioles, capillaries, and postcapillary venules with continuous blood flow. Stimulation of the lung allograft with TNF-alpha induced leukocyte rolling and adhesion in both arterioles and venules. Treatment with function-blocking anti-selectin mAb revealed that P- and L-selectin are the predominant rolling receptors in the lung microvessels, with E-selectin strengthening P-selectin-dependent interactions. Intravital microscopic studies also demonstrated that during their transit in capillaries, some leukocytes undergo shape change and continue to roll as elongated cells in postcapillary venules. Furthermore, the revascularized microvessels demonstrated the ability to undergo vasoconstriction in response to superfusion with endothelin-1. Overall, these studies demonstrate that the revascularized lung allograft is responsive to various external stimuli such as cytokines and vaso-active mediators and serves as a model to evaluate the interaction of leukocytes with the vascular endothelium in the lung microcirculation under acute as well as chronic experimental conditions.

A novel rodent mammary window of orthotopic breast cancer for intravital microscopy

Orthotopic and ectopic organ environments differentially influence tumor growth, metastasis, and sensitivity to therapy. In this study we present a novel rodent mammary window of orthotopic breast cancer, which is amenable to study of microvascular function and angiogenesis in this orthotopic site. The skin around the nipple of selected mammary glands of female Fischer 344 rats was removed and the nipple was cut at its base. R3230Ac tumor fragments or cells in Gelfoam were aseptically implanted into the nipple sinus. An acrylic disk was placed on top of the implant and was sutured in place. Histology showed that tumors were well established within 5 days. Similar techniques were also applied to BALB/c mice transplanted with 4T1 murine mammary carcinoma cells. With GFP-expressing tumor cells and serial observations, we demonstrated unique patterns of tumor cell proliferation and vascularization in both tumor models. The images obtained were comparable to those from the dorsal skinfold window chambers. This model will allow for study of tumor microcirculatory function, angiogenesis, tumor cell–host interactions, and evaluation of effects of various treatments.

Drug targeting using thermally responsive polymers and local hyperthermia

We report a new thermal targeting method in which a thermally responsive drug carrier selectively accumulates in a solid tumor that is maintained above physiological temperature by externally applied, focused hyperthermia. We synthesized two thermally responsive polymers that were designed to exhibit a lower critical solution temperature (LCST) transition slightly above physiological temperature: (1) a genetically engineered elastin-like polypeptide (ELP) and (2) a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). The delivery of systemically injected polymer–rhodamine conjugates to solid tumors was investigated by in vivo fluorescence video microscopy of ovarian tumors implanted in dorsal skin fold window chambers in nude mice, with and without local hyperthermia. When tumors were heated to 42°C, the accumulation of a thermally responsive ELP with a LCST of 40°C was approximately twofold greater than the concentration of the same polymer in tumors that were not heated. Similar results were also obtained for a thermally responsive poly(NIPAAM–co-AAm), though the enhanced accumulation of this carrier in heated tumors was lower than that observed for the thermally responsive ELP. These results suggest that enhanced delivery of drugs to solid tumors can be achieved by conjugation to thermally responsive polymers combined with local heating of tumors.

Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models.

There is a paucity of information about events that follow immediately after tumor cells are triggered to initiate the process of angiogenesis (the formation of new blood vessels). Such information is relevant to the issue of when micrometastases vascularize and has implications for the accessibility of micrometastases to various treatments. In this study, we attempted to monitor events at the initiation of angiogenesis at the earliest possible stage of tumor growth in vivo. Two different rodent mammary tumor cell lines, R3230Ac from the Fischer 344 rat and 4T1 from the BALB/c mouse, were stably transfected with a gene that encodes an enhanced version of green fluorescence protein (GFP). GFP-labeled R3230Ac or 4T1 cells (about 20-50 cells) were implanted into dorsal skinfold window chambers of Fischer 344 rats or BALB/c mice, respectively. Tumor angiogenesis was then monitored serially and noninvasively for up to 4 weeks. Clear evidence of modification of the host vasculature was observed when tumor mass reached approximately 60-80 cells, and functional new blood vessels were seen when tumor mass reached roughly 100-300 cells. Individual tumor cells exhibited a chemotaxis-like growth pattern toward the pre-existing host vasculature. When ex-flk1 (a soluble, truncated vascular endothelial cell growth factor receptor protein known to be antiangiogenic) was injected with the tumor cells, the initial angiogenic and tumor growth activities were inhibited considerably, indicating that angiogenesis inhibitors may halt tumor growth even before the onset of angiogenesis. Angiogenesis induced by tumor cells after implantation in the host begins at a very early stage, i.e., when the tumor mass contains roughly 100-300 cells. Identification of chemotactic signals that initiate tumor cell migration toward the existing vasculature may provide valuable targets for preventing tumor progression and/or metastases.

Inhibition of radiation-induced up-regulation of leukocyte adhesion to endothelial cells with the platelet-activating factor inhibitor, BN52021

Purpose : The inflammatory process is likely involved in normal tissue damage after radiation exposure, yet few studies have directly evaluated the factors that might be involved in the regulation of inflammation after irradiation in vivo. We tested the hypothesis that platelet-activating factor, a neutrophil agonist synthesized by endothelial cells, is involved in the upregulation of radiation-induced leukocyte-endothelial cell interactions by using an inhibitor of its receptor, BN52021. Methods and Materials : Fischer-344 rats with dorsal skin-fold window chambers were randomized to three experimental groups: control (sham irradiation); 6 Gy radiation; and 6 Gy + BN52021. BN52021 (0.5 mg/kg) was administered 5 min prior to 6 Gy radiation. Leukocytes were stained in vivo with i.v acridine orange for visualization with fluorescent microscopy. Venous vessel diameters were measured and numbers of rolling leukocytes were counted per 30-s period. The number of adhering leukocytes per unit surface area was also determined. Differences among that three experimental groups for rolling and adhering leukocytes were analyzed using a mixed-effects linear model with vessel shear rate used as a covariate. Results are reported as means ± standard errors. Results : Irradiation caused upregulation of leukocyte rolling, as compared with sham-treated controls ( p = 0.04): the BN compound in addition to radiation did not downregulate this effect. Irradiation also upregulated leukocyte adhesion ( p < 0.001), but the addition of BN52021 prior to irradiation blocked this effect. The drug did not affect heart rate or blood pressure. Conclusions : These results support the hypothesis that radiation-induced upregulation of leukocyte adhesion is mediated by platelet-activating factor. These results are consistent with prior reports that platelet-activating factor is not involved in leukocyte rolling, which involves separate families of adhesion molecules from those that regulate adhesion. BN52021, a ginkolide, or other related drugs might provide a useful pharmacological means to prevent or ameliorate inflammatory pathways that are invoked after radiation exposure.