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Initial Stages of Tumor Cell-Induced Angiogenesis: Evaluation Via Skin Window Chambers in Rodent Models

Chuan-Yuan Li, Siqing Shan, Qian Huang, Rod D. Braun, Jennifer Lanzen, Kang Hu, Pengnian Lin, Mark W. Dewhirst

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).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
A widely accepted theory is that tumor cells are genetically triggered during tumorigenesis to initiate angiogenesis (1-3). This phenomenon is elegantly demonstrated by previous reports (4,5). However, it is currently unclear as to what exactly happens when tumor cells are primed to initiate angiogenesis. This question is particularly relevant to tumor progression and to metastatic tumor growth, since the angiogenic trigger is presumably present in both cases. Understanding tumor growth at a very early stage can provide new insights into tumor metastasis and could facilitate the development of new antiangiogenic therapies. In previous experiments, it has also been observed that tumors can sustain growth without angiogenesis to sizes of several hundred microns to 1 mm in diameter or when the tumor mass contains roughly 105-106 cells (6). We sought to develop an approach that would allow a more direct and precise assessment of early stages of tumor growth and angiogenesis. Our goal was to determine the timing of the earliest stage of the initiation of angiogenesis induced by established (previously transformed cells but not primary tumors) tumor cells.

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).


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Establishing GFP-expressing tumor cell lines. To achieve individual tumor cell visibility in vivo, we transfected the R3230Ac cell line (14) (originally derived from a spontaneous mammary carcinoma of the Fischer 344 rat) and the 4T1 cell line (15) (originally derived from a spontaneous mammary carcinoma of the BALB/c mouse) with a plasmid, pEGFP-N1, that constitutively expresses an enhanced version of the GFP (Clontech Laboratories, Inc., Palo Alto, CA) (16,17). The liposome DMRIE from Life Technologies, Inc. (GIBCO BRL), Gaithersburg, MD, was used to aid the transfections. A protocol recommended by the manufacturer was followed. G418 selection at 400 µg/mL started 2 days after transfection. Two to 3 weeks later, colonies that emerged in Petri dish cultures were examined for GFP expression under a fluorescence microscope. Those colonies with robust GFP expression were picked, trypsinized, and expanded for further experiments.

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.


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Induction of Angiogenesis by Tumor Cells at the Earliest Stage of Tumor Growth

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. 1Go 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. 1Go (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. 1Go [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. 1Go [day-8 panel], as indicated by the circle), there was the first unequivocal evidence for neovasculature (Fig. 1Go [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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Fig. 1. Growth of a tumor from single 4T1 cells in a BALB/c mouse window chamber. Approximately 20 cells were injected in a BALB/c mouse window chamber, and their growth was followed serially after the initial implantation. Red arrow in the day-2 panel indicates an elongated cell. Red arrows in the day-6 panel indicate dilated host vessels compared with those seen in the day-4 panel. Arrows in the day-8 panel indicate new microvessels. Pink arrows point to tumor (localized in the marked circle)-associated microvessels, and red arrows beneath the circled area point to dilated and/or tumor-induced vasculature outside the tumor. Size bars in the day-1 to day-8 panels represent 200 µm; size bar in the day-20 panel represents 500 µm.

 
Morphologic and Spatial Adaptation of Tumor Cells Around Existing Host Vasculature and Tumor Neovasculature

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,Go 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. 1Go). This feature was also apparent in Fig. 2;Go 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;Go inset in the day-2 panel).



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Fig. 2. Chemotaxis-like tumor migration toward a host blood vessel. About 20 4T1 tumor cells expressing the green fluorescence protein (GFP) were injected into the window chamber on day 1. Red arrow in the day-2 panel indicates a cell that is elongated, while the inset shows a cell (middle) with apoptotic debris. The same area with the elongated cells (the one shown by the arrow) in the window chamber was followed at indicated time points. In panels representing days 1, 2, and 8 (lower right), fluorescein isothiocyanate (FITC)-filtered epifluorescence was used. In the other three panels, a combination of transmitted white light and epifluorescence was used. Size bars in all panels represent 200 µm.

 
The second important observation was the pattern of tumor cell growth around newly formed microvessels. This pattern was clearly demonstrated for the rat R3230Ac cells in Fig. 3Go (panels a and b) and for the mouse 4T1 cells in Fig. 3Go (panels c and d). In both cases, tumor cells appeared to be closely associated with the newly formed microvessels and aligned themselves longitudinally along the tumor vessels (see arrows). Tumor cells did not grow randomly at the edge. Rather they seemed to associate tightly with the in-growing network of tumor microvessels. Again, this phenomenon suggests active "cross-talk" during the early stages of tumor growth.



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Fig. 3. Interactions of tumor cells and neovasculature. Panel a: micrograph showing the outer edge of an R3230Ac tumor grown in the window chamber. Notice the tumor cells (green) that grew along and around the microvessels. Both transmitted light and fluorescein isothiocyanate (FITC)-filtered epifluorescence light were used for observation. Panel b: higher magnification view of one of the areas at the edge of the tumor shown in panel a. Some tumor cells appeared to be growing along the vessels longitudinally. Both transmitted white light and FITC-filtered epifluorescence light were used for observation. Panel c: 4T1 tumor cell cluster grown in the window chamber. Red arrows point to new microvessels. FITC-filtered epifluorescence was used for observation. Notice the spindle-shaped, fibroblast-like morphology of the cells, which are quite different in shape in comparison to the parent cells grown in vitro. Panel d: higher magnification view of one area at the edge of the tumor cell cluster shown in panel c. Red arrows indicate the presence of microvessels. Size bars in panel a and panel c represent 100 µm; size bar in panel b represents 50 µm; size bar in panel d represent 25 µm.

 
Inhibition of Tumor Growth at the Earliest Stage by Truncated Soluble VEGF Receptor Protein

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)Go. 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. 4Go). 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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Fig. 4. Inhibition of tumor growth at the earliest stage by an antiangiogenic recombinant protein, ex-flk1 (see "Materials and Methods" section for details). About 20-50 green fluorescence protein (GFP)-labeled 4T1 tumor cells were implanted into the window chamber at the same time with 12 µg of ex-flk1 or control bovine serum albumin protein on day 1, when the surgical procedure for making the window chamber was carried out. Panels a, b, and c: tumor window chamber injected with bovine serum albumin. Panels d, e, and f: tumor window chamber injected with ex-flk1 protein. Size bars in all panels represent 400 µm.

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The timing of the initiation of angiogenesis for disseminated tumor cells is a very important issue, since it will lead to a better understanding of the role the process of angiogenesis plays in tumor metastasis. The current thinking is that angiogenesis starts only when the tumor mass reaches 1 mm in diameter and when hypoxia occurs. This theory is largely based on the data obtained in transgenic murine primary tumor models. However, the actual angiogenesis initiation in metastatic tumor cells may be very different from that in primary tumors. Our study is an attempt to address this very important issue using GFP-labeled tumor cells and rodent dorsal skinfold window chamber models. Such a combination allowed tumor growth and angiogenesis to be observed simultaneously and serially in vivo for up to 4 weeks. Both processes were visible at the approximately 20- to 50-cell stage of tumor growth. Very importantly, the results indicate that angiogenesis can start at the earliest stage of tumor growth when the tumor mass has approximately 100 cells. This result is in variance to previous reports indicating that tumors can grow to diameters from several hundred microns to 1 mm without inducing angiogenesis (26-28). A possible explanation for the discrepancy is the resolution of the experimental systems. Specifically, our GFP-labeled tumor cells in combination with the dorsal skinfold window chamber provides unprecedented clarity that allows the observation of tumor growth from individual tumor cells, while earlier studies depended mostly on immunohistochemistry of well-established tumors that does not allow similar observations of earlier angiogenic activities at equivalent spatial and temporal resolutions.

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.


    NOTES
 
C.-Y. Li, S. Shan, and Q. Huang contributed equally to this work.

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.


    REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received August 4, 1999; revised October 28, 1999; accepted November 18, 1999.


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