Affiliations of authors: E.-G. Achilles, Department of Surgery, Division of Surgical Research, Children's Hospital, Harvard Medical School, Boston, MA, and Department of Hepato-Biliary Surgery, University of Hamburg, University Hospital Hamburg, Germany; A. Fernandez, T. Udagawa, E. Flynn, J. Folkman, Department of Surgery, Division of Surgical Research, Children's Hospital, Harvard Medical School; E. N. Allred, Department of Surgery, Division of Surgical Research, Children's Hospital, and Neuroepidemiology Unit, Harvard Medical School; O. Kisker, Department of Surgery, Division of Surgical Research, Children's Hospital, Harvard Medical School, and Department of General Surgery, University Hospital Marburg, Philipps University Marburg, Germany; W.-D. Beecken, Department of Surgery, Division of Surgical Research, Children's Hospital, Harvard Medical School, and Clinic for Urology and Pediatric Urology, J. W. Goethe University, University Hospital Frankfurt, Germany.
Correspondence to: Judah Folkman, M.D., Department of Surgery, Division of Surgical Research, Children's Hospital, Harvard Medical School, 300 Longwood Ave., Boston, MA 02115.
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ABSTRACT |
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INTRODUCTION |
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Furthermore, we have shown previously that, in tumors arising spontaneously in transgenic mice, where all of the beta cells in the pancreatic islets uniformly express the large T-antigen oncogene (7), the individual islets can be considered as a single tumor, but disseminated throughout the pancreas as subclones. This configuration makes it possible to visualize and to quantify the angiogenic switch. Only 1% of the tumors in that reported study became angiogenic at 67 weeks of age, and 4% of the tumors were angiogenic at 13 weeks of age when the mice were dying of tumor burden. Thus, approximately 95%96% of tumors remained nonangiogenic and did not grow beyond a volume of about 0.60.8 mm3. Taken together, these findings suggest that subpopulations of cells within a given tumor may be heterogeneous in their angiogenic activity. On the basis of these results, we asked whether an established human tumor could contain subpopulations of tumor cells that were not angiogenic or had varying degrees of angiogenic activity. If so, this might clarify certain unexplained observations, such as "no take" of human tumors transplanted into immunodeficient mice (811).
To address this question, we chose a human liposarcoma (SW-872) cell line that grows reproducibly in severe combined immunodeficient (SCID) mice. Tumor growth was recorded for up to one fourth the life span of the animal. (The life span of SCID mice is approximately 2 years.)
Microvessel density, tumor cell apoptosis, and tumor cell proliferation rates were quantified on histology sections. We determined whether a single human tumor type contained subpopulations of tumor cells that were heterogeneous in angiogenic activity. We define the variableangiogenic activityas the total output of positive and negative regulators of angiogenesis by the tumor cells. We measured the angiogenic activity indirectly by quantifying the microvessel density in the areas of intensive neovascularization (hot spots) and by analysis of apoptosis and proliferation throughout the tumor. Other factors or sources contributing to the angiogenic heterogeneity, such as tumor site (12,13) or genetic background (14), were unchanged in this study.
We attempted to elucidate whether angiogenic activity correlated with tumor growth rate, tumor cell apoptosis, and tumor cell proliferation. We hypothesized that the growth rate (increased tumor volume) is controlled mainly by the angiogenic activity because tumor cell apoptosis rate rises in direct proportion to inhibition of angiogenesis (15).
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MATERIALS AND METHODS |
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The human liposarcoma cell line SW-872 (passage 13) was purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified essential medium (DMEM) (JRH Co., Lenexa, KS) supplemented with 5% heat-inactivated fetal calf serum (Intergen, Purchase, NY) in 75-cm2 Falcon tissue-culture flasks (Becton Dickinson, Franklin Lakes, NJ) and a humidified atmosphere containing 10% CO2 at 37 °C. Subclones were established and selected according to high, intermediate, or low proliferation rates in vitro. These subclones were prepared as follows: Monolayers of cells from the parental line were trypsinized, appropriately washed, and suspended at one cell/mL concentration. The cells were then plated (200 µL/well) in 96-well plates (Corning Costar Corp., Cambridge, MA). After the cells became confluent (approximately 1 week), they were transferred to 24-well plates and then to T-75 flasks. After the cells were propagated for 6 weeks in DMEM, six of 11 clones (of either slow, intermediate, or fast proliferation rate in vitro) were randomly chosen for investigations in vivo.
The proliferation rate was determined in vitro by plating 2500 cells/well in 24-well plates. Cell counts in each well were performed by use of a hemacytometer (Hausser Scientific, Horsham, PA) every 24 hours during the following 7-day period.
For xenotransplantation, confluent tumor cells were rinsed in phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO), briefly trypsinized, and suspended in DMEM, and cell suspensions were adjusted to a density of 25 x 106/mL. The cells were centrifuged at 300g for 1 minute at room temperature (20 °C) and finally suspended at a density of 25 x 106 cells/mL in DMEM without serum. From this suspension, 200 µL was immediately injected subcutaneously with a 30-gauge needle into the anterior flank or into the dorsum at the midline of male 6-week-old SCID mice.
Animal Studies
In this study, the following terms are used to distinguish different growth rates of tumors. The term "dormant" is used to describe a microscopic tumor that is not expanding, "stable disease" describes a macroscopic tumor that is not visibly expanding, "slowly growing" describes a tumor that is expanding slowly over months, and "fast growing or aggressive" describes a tumor that is expanding over a few weeks. The growth rates (95% confidence intervals) in log10 mm3/day for different tumors were as follows: dormant tumors, 0.029 (-0.033 to -0.026); stable tumors, -0.009 (-0.014 to -0.004); slow-growing tumors, -0.003 (-0.009 to 0.003); and aggressive tumors, 0.028 (0.022-0.035).
The negative values observed for the dormant, stable, and slow-growing tumors were due to the initial decrease in volume following wound edema immediately after transplantation. In initial experiments, 6- to 8-week-old male SCID mice (obtained from Massachusetts General Hospital, Boston, MA) kept in microisolator cages (four mice/cage) were inoculated subcutaneously in their anterior flanks with SW-872 tumor cells (5 x 106 cells in 200 µL/injection).
In the next set of experiments, tumor-bearing mice (mean tumor volume, 853 mm3; range, 123302 mm3) were killed with methoxyflurane, and the lesions were removed. Tumor tissues were rinsed in DMEM, and macroscopically viable areas were cut with a scalpel into small pieces of similar size in each experiment (mean size, 17 mm3; range, 0.566 mm3). The reason for varying the size of the transplanted tumor tissue was to demonstrate or exclude any relation of the resulting tumor phenotype and the tissue size transplanted initially.
To transplant these tumor implants, we made a 5-mm-long horizontal incision in the midline dorsum 1 cm from the base of the tail of SCID mice after they were anesthetized by administering Avertin (40 µg/kg) intraperitoneally. Through the incision, a 1-cm subcutaneous tunnel was made cephled (toward the head of the animal) by use of fine forceps. The tumor implant was inserted into this tunnel so that it was positioned 1 cm from the skin incision. The wounds were closed with fine sutures (PDS 4.0; Ethicon, Somerville, NJ). All procedures were done under aseptic conditions. In further studies, the parental cell line, or clones derived from cell culture of the parental line, were injected (5 x 106 cells in 200 µL/injection) or transplanted as tissue pieces. Tumors were measured every 34 days, and volumes (in millimeters cubed) were calculated by use of the formula (width [mm]2 x length [mm] x 0.52). All of the experiments were carried out in accordance with the Animal Research Regulations at Children's Hospital, Boston, MA. The animals were observed daily, and those bearing big tumors were monitored carefully for any signs of discomfort.
Histology and Immunohistochemistry
Mice were killed at intervals during a period of 26 months after transplantation. All of the animals underwent a thorough autopsy to search for hidden secondary tumors, which could possibly suppress the subcutaneous tumor. Such a tumor was found in only one animal, thus eliminating the complication of a hidden primary tumor in the other animals (15,16). Representative tumor tissues were harvested and fixed in 10% neutral buffered formalin at 4 °C for 24 hours. All tissues were paraffin embedded. Sections (10 µm thick) were first stained with hematoxylineosin to evaluate tissue viability and quality. The microvessel density was determined after immunocytochemical staining by use of the Vectastain avidinbiotin detection system (Vector Laboratories, Inc., Burlingame, CA) with anti-CD 31 monoclonal antibody (dilution: 1 : 250; Pharmingen, San Diego, CA) according to the manufacturer's protocol (CD 31 is also known as PECAM [platelet/endothelial cell adhesion molecule] located on the cell surface). With the use of the method of Weidner et al. (3), the regions of highest vessel density ("hot-spot" regions) were scanned at low magnification (x40 to x100) and then counted at a x200 magnification (0.738-mm2 field) by an observer who was blinded to the code for the tumor source. At least five fields were counted in a representative tumor section, and the highest count was taken as described previously (3). Tumor sections were stained with anti-PCNA (proliferating cell nuclear antigen) monoclonal antibody (dilution: 1 : 50; Signet, Dedham, MA) to assess cell proliferation and with terminal deoxynucleotide transferase (Apotag; Intergen) to assess the degree of apoptosis. The sections were evaluated at low magnification (x40 to x100) so that cell counts were not done in necrotic areas. PCNA-positive cells (red stain) and apoptotic tumor cells (brown stain) were counted at x400 magnification and were expressed as the percentage of the total cells in the field. At least 1000 cells were counted in three to five random fields.
Statistical Methods
Because the measurements of tumor volume, cell apoptosis, microvessel density, and proliferation rate were not normally distributed, we evaluated relationships among them with nonparametric regression models (17). This evaluation was achieved by employing standard least-squares regression methods on the ranks of the data values (Stata, version 6; Stata Corp., College Station, TX). We also examined some relationships with least-squares regression after log10 transformation of the data. All statistical tests were two-sided.
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RESULTS |
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All mice were healthy and gained weight normally throughout the experiments. Xenotransplantations were performed by injections of tissue culture-derived tumor cell suspensions (series 1) (Fig. 1, A) or were performed via tumor tissue transplants (series 26) derived from a carrier mouse with a known phenotype after a follow-up of 16 months (Fig. 1
, B). The results are summarized in Table 1
. We numbered the series to clarify separate experiments with different tumor volumes and different tumor phenotypes.
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In series 2, five mice received tissue transplants of an approximate volume of 0.5 mm3 from an aggressive tumor derived from the parental cell line. Three of the five mice had early-onset aggressive tumor growth, and two showed a slowly growing phenotype.
In series 3, five mice received tissue transplants of an approximate volume of 4 mm3 taken from a stable tumor produced by the parental cell line. Only one tumor showed aggressive growth, whereas three tumors remained stable and one showed very slow growth.
In series 4, 10 mice received tissue transplants (approximate volume, 15 mm3) derived from a stable tumor produced by the parental cell line. The majority (seven of 10 animals) of the tumors showed long-term stable or very slow growth, whereas aggressive growth was observed less frequently (three of 10 animals). Only one tumor of series 24 underwent spontaneous regression. This means that one tumor in a total of 20 mice of series 24 underwent spontaneous regression.
In series 5, each mouse (total, 10 animals) received a tissue transplant of an approximate volume of 62 mm3 taken from an aggressively growing tumor derived from the parental cell line. The majority (eight of 10 animals) of tumors grew aggressively but at slightly different growth rates, whereas only two (two of 10 animals) showed a phenotype with stable growth.
In series 6, pieces (approximate volume, 0.514 mm3) of tumors with a known phenotype were taken from the carrier mice. The tumors were derived from the parental cell line as well as from the clones (clones 1, 8, 9, and 17), which produced macroscopically measurable stable or aggressive tumors. The pieces taken from tumors of each phenotype were retransplanted subcutaneously in five animals for a given phenotype, and tumor growth was followed up to a period of 6 months.
All xenotransplants of tissue pieces from five experiments (series 26) by use of a total of 55 animals generally resulted in tumors (54 of 55 animals) (Fig. 1, B), with a phenotype similar to the one from which they were derived (45 of 55 tumors) (Table 1
, B). In addition, they retained the same phenotype as those primarily derived from cell suspension. Slowly growing tumors when subsequently transplanted tended to maintain the same stable phenotype as their parental tumor (clones 1, 8, and 17; Fig. 1
, Bgroup b). Similarly, those tumors (parental cell line and clone 9) with an original aggressively growing phenotype generally retained the same phenotype as their parent after further transplantation (Fig. 1
, A and Bgroup a). Several of the long-term stable tumors eventually began to grow near the end of a follow-up period of up to 6 months. The median time (range) in days when tumors were measured was 95 (29173). The median times (range) when types of tumors were measured were as follows: parental cell line, 61 (50173); clone 1, 96 (74154); clone 4, 95 (9595); clone 5, 117 (102140); clone 8, 96 (74133); clone 9, 78 (29102); and clone 17, 96 (74133). The median (range) of the tumor volume (in mm3) derived from parental cells and the different clones were as follows: parental, 641 (85608); clone 1, 7 (131); clone 4, 1 (11); clone 5, 1 (1948); clone 8, 1 (11315); clone 9, 2812 (8955680); and clone 17, 22 (1205).
In mice bearing small or microscopic tumors, no lung or other organ metastases could be detected macroscopically. In some animals with highly aggressive lesions, a few (<10) small lung metastases could be observed.
Histology and Immunohistochemistry
Of a total of 83 tumors in the different experiments, 18 specimens (with stable [n = 3], slowly progressive [n = 6], and aggressive [n = 9] phenotypes) suitable for immunocytochemical investigation were selected randomly. Statistical analysis was conducted by comparing the tumor group whose tissues were studied by immunohistochemistry with the other group whose tissues were not studied by immunohistochemistry. The median values (and interquartile range, i.e., the 25th to 75th percentile values) of the post-transplantation day when the tumor tissue was suitable for analysis and the tumor volumes on that same day in both groups showed no statistically significant differences (median value of 95 days for mice whose tumors were not studied by immunohistochemistry versus 102 days for mice whose tumors were studied by immunohistochemistry; P = .35; Wilcoxon rank sum test). Although, the median tumor volume was slightly higher in those tumors selected for immunohistochemical analysis, this difference did not reach statistical significance (the median tumor volume [range] in mm3 for those selected for immunochemical analysis was 229 [35870] versus 41 [02153] for those not selected; P = .23; Wilcoxon rank sum test).
Seventeen of the remaining 65 tumors were used for retransplantation. Nineteen of the dormant or stable tumors could not be fully investigated by immunocytochemistry because of the limited amount of the tissue. All tumors showed variable areas of viable neoplastic tissue consistent with a pleomorphic or undifferentiated sarcoma in slides stained by hematoxylineosin. Statistical analysis revealed that the tumor volume was positively associated with microvessel density and inversely associated with tumor cell apoptosis (Spearman correlation coefficient [r] = .89 [P.0001] and r = .68 [P = .002], respectively). Therefore, the microvessel density was highest in the largest tumors with an aggressive phenotype in vivo (Fig. 2
, A), whereas the apoptotic rate was highest in the smallest tumors with a slowly progressive or stable phenotype in vivo (Fig. 2
, B). The proliferation rate of tumor cells was less strongly, but positively, correlated with tumor volume (Spearman r = .55; P = .02) (Fig. 2
, C). Representative specimens stained for the different antigens are shown in Fig. 3
.
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The median of tumor cell proliferation (percent of proliferating cells/high-power field) in aggressive tumors was 48 (range, 2966), which is slightly higher than in the stable or slowly progressive tumors (median, 35; range, 1847) (P = .02). The median of tumor cell apoptosis (percent apoptotic cells/high-power field) was lower in aggressive tumors (median, 0.69; range, 0.570.95) compared with 1.65 (range, 0.563.69) in stable or slowly progressive tumors (P = .007).
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DISCUSSION |
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These results also show that tumor cell apoptosis is inversely proportional to angiogenic activity. This suggests that the angiogenic regulation of tumor growth operates mainly by its effect on tumor apoptosis but to a lesser extent on tumor cell proliferation. The observation that the microvessel density is inversely proportional to tumor cell apoptosis but generally independent or, to a lesser extent, proportional to tumor cell proliferation was also reported in part by others (15,16,1820). In addition, the inverse relationship between apoptosis and tumor progression has been demonstrated in a murine model (18) and, more recently, as an inverse relation between apoptosis and microvessel density in human gastric carcinoma (19). However there is no previous report, to our knowledge, of the heterogeneity of angiogenic activity expressed by subpopulations of tumor cells isolated from a single human tumor or tumor cell line.
It has been demonstrated that, in human prostate cancer, there is no statistically significant difference in proliferation rate between indolent, slow-growing primary tumors and rapidly growing metastatic tumors, even though there is 10-fold difference in doubling time (21). Nevertheless, in this study, there was an inverse relationship between apoptosis and rate of tumor growth.
Our results were generated from 83 different tumor implants, all taken from one human tumor type. Therefore, it seems likely that these findings may be similar in other tumors; however, this remains to be demonstrated.
While the literature contains many references describing the heterogeneity of different tumor phenotypes, such as tumor cell proliferation rate, enzymatic activity (2224), and adhesion (25,26), we found only two previous reports of angiogenic heterogeneity in different human tumors (27,28).
It could be argued that the heterogeneity of tumor growth rate and angiogenesis in our study is governed by the initial size of the implanted tissues. Against this is the demonstration that, when a rapidly growing tumor derived from the original primary tumor was itself divided into pieces of different sizes (range, 0.562 mm3, a volume difference of 124-fold), the resulting tumors grew at similarly aggressive rates.
Our results suggest that a small subpopulation of tumor cells in a heterogeneous mixture is capable of inducing an aggressive, angiogenic phenotype and that this phenotype is generally stable during further transplantation. Similarly, in a transgenic model of spontaneous tumor growth, only a minority (4%) of transformed pancreatic islets had angiogenesis early and gave rise to aggressive tumors (7). The essential role of angiogenesis as the early-onset event in neoplastic progression has also been demonstrated in human preneoplastic breast lesions that showed a range of high and low angiogenic activities in the rabbit cornea, despite the fact that there was no difference in their morphology (28).
There are several clinical implications of this work. Our studies provide a possible model for the clinical observation that some tumors, such as breast cancer, may recur as metastases of the original tumor, as late as 1020 years after removal of the primary tumor. The long-term dormant or stable tumor implants in our study that were nonangiogenic and that generally did not expand their tumor volume during at least 140 days would be equivalent to human tumors that had remained dormant for up to 13.4 years (based on the equivalence of 1 mouse-day to 35 human days).
Our findings also propose a mechanism for the common observation that certain kinds of human tumors transplanted into immunodeficient mice have a poor rate of "take" (811). This has been ascribed until now to immune differences, to the transplantation of necrotic pieces, or to the observation that human tumors may be heavily infiltrated by fibroblast and collagen deposits. However, we show that tumors that may have formerly been labeled as "no take" in fact are viable, dormant, or stable and of small size due to lack of angiogenic activity. Furthermore, our study shows that a subpopulation of these dormant or stable tiny tumors eventually become angiogenic and grow rapidly into large visible tumors. The fact that this angiogenic switch can take as long as 40160 days may explain why tumors that were previously "no take" were thought to be dead.
The importance of understanding and evaluating angiogenesis in the early phase of tumor growth is underscored by numerous reports of the high incidence of occult dormant microscopic tumors in the breast (29), the thyroid (30), and the prostate (31). The lack of suitable models to fully understand the problem of tumor dormancy has recently been emphasized (32).
Our results may also explain why, in some patients with retroperitoneal liposarcoma, the tumor remains stable or "indolent" for decades while, in other patients, the same tumor type follows a highly aggressive course over a single year (33).
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NOTES |
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We thank Lori DeSantis (Photography) and Steven Moskowitz (Graphics) for their help.
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