Affiliation of authors: Department of Radiation Oncology, Duke University Medical Center, Durham, NC.
Correspondence to: Chuan-Yuan Li, Ph.D., Box 3455, Duke University Medical Center, Durham, NC 27710 (e-mail: cyli{at}radonc.duke.edu).
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ABSTRACT |
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INTRODUCTION |
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In this study, we attempted to use tumor cells labeled with the green fluorescence protein (GFP) (7-9) in rodent dorsal skinfold window chamber models to develop approaches by which noninvasive and serial observation (up to 4 weeks) of tumor formation could be carried out. For this purpose, a dorsal skinfold window chamber model (10,11) created in the rat or mouse was used to monitor tumor formation. The skin flap window chamber model has been in use in our laboratory since 1982 (12,13). Using this approach, we serially followed tumor formation from about 20-50 cells to tumors reaching 4-7 mm in diameters (containing roughly a few million cells).
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MATERIALS AND METHODS |
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Preparation of window chambers and tumor cell injections. The preparation of the dorsal skinfold window chamber has been described elsewhere (11). All procedures employed in this study were approved by the Duke University Institutional Animal Use and Care Committee. Briefly, a 1-cm-diameter flap of skin was dissected away from opposing surfaces of the dorsal skin flap of anesthetized Fischer 344 rats or BALB/c mice, leaving a fascial plane with associated vasculature. The hole was held vertically away from the body with a anodized aluminum saddle that was sutured to both sides of the flap. Glass windows were attached to the center of the saddle to cover the surgical site. Tumor cells were usually injected into the window chamber at that time. About 20-50 cells suspended in 10-15 µL of Opti-MEM medium (serum-free medium from Life Technologies, Inc.) were injected by use of 30-gauge needles. To achieve desired cell numbers in defined volumes, we quantified the cells by use of a Coulter Z2 Counter (Beckman Coulter, Fullerton, CA). After being counted, the cells were diluted with Opti-MEM medium to the desired concentrations. The cell counts were further confirmed by enumerating the number of fluorescent cells at the injection site under a fluorescent microscope. After the surgery was completed and after the tumor cells were implanted by injection, the skin surfaces adjacent to the window assembly were covered with neosporin ointment (Warner-Lambert Co., Morris Plains, NJ). The chamber design for mice was similar to the one used for rats, except that the chamber diameter was 5 mm instead of 1 cm and the chambers were made of titanium instead of aluminum. Both Fischer 344 rats and BALB/c mice were obtained from Charles River, Raleigh, NC.
Fluorescence microscopy. Observations of the window chambers were carried out on a Zeiss Axioscope equipped with a Scion frame grabber (Scion, Gaithersburg). See references (12,13,18,19) for more detailed setup of the videomicroscopy. A xenon-arc lamp was used as the source for generating epifluorescence. This setup allowed us to capture microscopic images online. Fluorescein isothiocyanate-filtered epifluorescence or a combination of transmitted white light and epifluorescence was used. Observations were made daily up to a week and every other day thereafter. Ketamine/xylazine was used to anesthetize the animals and was given at a dose of 50 mg of ketamine/7 mg of xylazine per kg body weight for the rats and 175 mg of ketamine/10 mg of xylazine per kg body weight for mice before each observation.
Administration of ex-flk1 protein into the skin window chambers. A recombinant protein, ex-flk1, which is a truncated, extracellular domain of the vascular endothelial growth factor (VEGF) receptor flk1 (also known as KDR), was used to examine the potential antitumor effects of ex-flk1 on the growth of 4T1 cells in window chambers. The truncated protein ex-flk1 was made as a free protein in the Baculovirus system of insect cells. As shown previously (20), this protein inhibits tumor growth and angiogenesis. Twenty microliters of phosphate-buffered saline solution containing 12 µg of the ex-flk1 protein was injected into the window chamber together with tumor cells. As controls, equal amounts of bovine serum albumin or no protein was injected.
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RESULTS |
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To observe tumor formation from a very early stage, two series of experiments were done with the use of two different tumor cell lines. In the first series, about 20-50 GFP-labeled 4T1 cells suspended in a small volume (10-15 µL) of Opti-MEM medium were injected into the fascia of BALB/c mice right after the surgery for the skinfold window. By this approach, tumor growth from such a small number of cells was monitored. The growth of tumors from these GFP-labeled 4T1 cells was followed and was found to be reproducible in replicate experiments.
Shown in Fig. 1 is a representative experiment with 4T1 cells. On day
1, all of the cells appeared rounded, which is typical of cells that had just been trypsinized. On day
2, the cells assumed a fibroblast-like morphology; one cell of such morphology is clearly indicated
by the red arrow in Fig. 1
(day-2 panel). On day 4, about 20 progeny cells
(cell number estimated from counting under a higher magnification) derived from the three or four
originally implanted cells with the fibroblast-like morphology were visible. It is clear that they
preferentially proliferated and migrated toward and around the existing host blood vessels. Most
of the other cells that did not assume the fibroblast shape had disappeared by that time. This
phenomenon is similar to metastasis, where a very low fraction of extravasated tumor cells
eventually succeeds in establishing tumors and such tumors often have clonal origin (21-23). By day 6, proliferation had continued, and it was estimated that there were
approximately 60-80 cells in the cluster. We estimated the cell number by counting the cells under
a higher magnification. At that time, host vessel dilation and increased tortuosity were obvious
(Fig. 1
[day-6 panel]; dilated vessels indicated by red arrows).
The increased dilation and tortuosity were not observed in window chambers in which no tumor
cells were implanted (data not shown), indicating that these effects are tumor cell specific. By day
8, when there could be no more than 300-400 cells (estimated from the in vitro doubling
time of 25 hours for these cells) in the tumor cell cluster (Fig. 1
[day-8 panel], as indicated by the circle), there was the first unequivocal evidence for
neovasculature (Fig. 1
[day-8 panel], pink arrows). The
microvessels observed at that stage were already fully functional, and red blood cells were clearly
visible on recorded video images. Thus, vascular tube formation must have occurred at earlier
times (on day 6 or day 7), when there were approximately 100-300 cells present. Indeed, it was
obvious that, on day 6, there was already clear evidence of tumor-induced host vessel dilation
(examples are indicated by red arrows) and increased tortuosity, which were even more apparent
on day 8 (examples indicated by the red arrows). By day 20, the tumor was filled with a plexus of
newly formed vasculature that exhibited the hallmarks of tumor microvessels, including high
tortuosity, low vascular density, and regurgitant and intermittent flow. These observations were
confirmed in more than 10 similar experiments involving GFP-transduced 4T1cells. Similar
observations were also documented in the series of experiments by monitoring tumor formation
from GFP-transduced R3230Ac mammary adenocarcinoma cells (data not shown).
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In addition to the documentation of the timing of early tumor
angiogenesis, there are two other important observations concerning the
growth pattern of tumor cells. The first is the chemotaxis-like
movement of the tumor cells toward specific host blood vessels. The
second is the intimate relationship between the tumor cells and tumor
neovasculature. As shown in Fig. 1, only those tumor cells that were
elongated and had assumed fibroblast-like morphology eventually
proliferated and grew into tumors. A striking feature was that all of
the cells appeared to orient themselves in the same direction, relative
to the orientation of surrounding vasculature (Fig. 1
). This feature
was also apparent in Fig. 2;
e.g., when cells divided
and migrated toward an existing vessel, all the progeny cells seemed to
align themselves in the same direction until they reached their target
blood vessel. Thus, cell division was unidirectional instead of
multidirectional. This observation was reproducible in more than 10
independent experiments. The fibroblast shape of the tumor cells and
their orientation contrasted sharply with the behavior of the same
cells in tissue culture, where the cells did not assume fibroblast-like
shape and cell division did not follow any one spatial pattern. In some
cases, apoptosis-like cell debris of such cells was observed (Fig. 2;
inset in the day-2 panel).
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To determine when the process of angiogenesis becomes essential for
tumor survival and growth, we used a recombinant truncated soluble VEGF
receptor, ex-flk1, to probe its effect on early tumor growth in the
window chamber (Fig. 4). This protein has been shown
to be nontoxic to tumor cells, yet it has a potent antiproliferative
activity against endothelial cells and has proven antitumor and
antiangiogenic efficacy (20,24). Free ex-flk1 protein was
administered by injection into the window chambers together with a
small number (i.e., 40-50) of GFP-transduced 4T1 tumor cells. Tumor
growth was then monitored continuously. In four of six window chambers,
the ex-flk1 protein effectively inhibited tumor cell proliferation
right from the beginning. All tumor cells disappeared within the first
5 days after implantation, without evidence of neovessel formation that
was very striking in the earlier experiments. In the two window
chambers where tumor growth was not completely inhibited, tumor growth
was slowed down substantially in one case and was reversed in the
other. In contrast, tumors formed readily in two control window
chambers that had been treated with bovine serum albumin and in 10
experiments where no exogenous protein was added at the time of surgery
(Fig. 4
). These findings are crucial because ex-flk1 protein was
effective at the earliest stage of tumor growth when there were no
visible signs of angiogenic activity. It, therefore, demonstrates the
dependence of individual tumor cells on early angiogenic activities for
both survival and proliferation in vivo. This result may
explain why, in many cases, angiogenesis inhibitors alone can suppress
tumor growth completely while they do not possess any antitumor
activity in vitro(24,25).
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DISCUSSION |
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The second important observation from this study is the chemotaxis-like movement of the tumor cells toward the host blood vessels prior to any evidence of tumor angiogenesis. It strongly suggests that the host "target" vessels are secreting a signal or signals that are chemotactic for the tumor cells. It is unclear at this point what these signals are. Potential candidates include oxygen or other nutrient gradients, growth factors, or other cytokines. This observation is consistent with a recent observation by Holash et al. (29), who suggested that established tumor cells can coopt (i.e., migrate toward) existing host vasculature. However, their conclusion was inferred from the fact that tumor cells tended to proliferate around host vasculature. Because the animals were killed to obtain the information, the researchers could not distinguish the phenomenon of preferential proliferation near host vessels (as might occur because of nutrient gradients) from the actual tumor cell migration. To our knowledge, our data, therefore, provide the first unequivocal evidence that tumor cells can migrate toward host vasculature before the onset of angiogenesis. In addition, this observation is consistent with the "two-compartment" theory proposed by Folkman et al. (30) that tumor and endothelial cells secrete chemotactic signals that attract each other. It is also apparent that the ability to respond to these yet unidentified signal(s) was important for tumor cells, since those cells that did not adapt (to the signal) disappeared gradually. Furthermore, two related observations from this study are 1) the intimate association of tumor cells with neovasculature and 2) the morphologic adaptation of the tumor cells; both of these observations are consistent with those of earlier studies. For example, earlier reports (31,32) have demonstrated the fusion of the basal lamina of tumor cells and endothelial cells in an in vitro rat aortic ring model. There are also observations of tumor cell elongation during the process of extravasation (33,34).
Taken together, our results suggests that blocking the early "communication" between tumor cells and host vessels may stop or even kill tumor cells at the earliest stage of tumor growth that is prior to the onset of visible angiogenesis. Indeed, the effective inhibition of early tumor growth in the window chamber by a soluble VEGF receptor clearly demonstrated this point. This study, therefore, raises the possibility that targeting angiogenesis may stop tumor cell growth, especially metastatic tumor growth at an even earlier stage than was previously thought.
In summary, our results provided new insights into the complex interplay between tumor cells and host vasculature at these earliest stages of tumor growth. Four stages of early angiogenesis were seen to occur: 1) The initial orchestration of tumor angiogenesis involved migration of tumor cells toward existing vasculature before neovascularization. 2) Changes in surrounding microvessel structure, such as vasodilation and increased tortuosity, were seen at the approximately 60- to 80-cell stage of tumor growth. 3) Clear demonstration of new vessel formation was seen at the approximately 100- to 300-cell stage of tumor growth. 4) Both tumor cell lines developed intimate contact with the developing neovasculature as the tumor continued to expand into surrounding normal tissue. In addition, our data indicate that angiogenesis inhibitors can prevent tumor growth at these earliest stages of tumor growth.
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NOTES |
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Supported by a startup grant provided by the Department of Radiation Oncology, Duke University Medical Center; by a grant from the Duke University SPORE in Breast Cancer (to C.-Y. Li); and by Public Health Service (PHS) grants CA40355 and CA42745 (to M. W. Dewhirst) and PHS grant CA81512 (to C.-Y. Li) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Q. Huang is a Raycem/Duane/Roger/John Morris Fellow.
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Manuscript received August 4, 1999; revised October 28, 1999; accepted November 18, 1999.
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