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Cancer Research 67, 5076-5082, June 1, 2007. doi: 10.1158/0008-5472.CAN-06-3664
© 2007 American Association for Cancer Research
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Human Tumor Xenografts Recurring after Radiotherapy Are More Sensitive to Anti–Vascular Endothelial Growth Factor Receptor-2 Treatment than Treatment-Naive Tumors

Sergey V. Kozin1, Frank Winkler1, Igor Garkavtsev1, Daniel J. Hicklin2, Rakesh K. Jain1 and Yves Boucher1

1 Edwin L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts and 2 ImClone Systems, Inc., New York, New York

Requests for reprints: Rakesh K. Jain, Edwin L. Steele Laboratory for Tumor Biology Radiation Oncology, Cox 7 Massachusetts General Hospital, 100 Blossom Street, Boston, MA 02114. Phone: 617-726-4086; Fax: 617-724-1819; E-mail: jain{at}steele.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The effects of antiangiogenic therapy on tumors relapsing after irradiation are not known. To this end, we irradiated human tumors growing s.c. in nude mice with a single dose of 20 or 30 Gy. Compared with primary (treatment-naive) xenografts, the growth rate of recurrent tumors was 1.6-fold slower, which is consistent with the known "tumor bed effect." For similar size tumors, recurrences had fewer functional vessels, a reduced vessel coverage by perivascular cells, and were more necrotic. Placenta growth factor concentration was significantly lower in relapses, whereas vascular endothelial growth factor (VEGF) levels were similar between primary and recurrent tumors. On the other hand, fibrillar collagen deposition was significantly increased in recurrent tumors. This radiation-induced fibrosis was partially responsible for the slower growth of recurrences; the i.t. injection of collagenase increased the growth rate of tumor relapses without affecting primary tumor growth. The mouse-specific VEGF receptor 2–blocking antibody DC101 induced a 2.2-fold longer growth delay in recurrent tumors compared with treatment-naive tumors. DC101 significantly decreased the interstitial fluid pressure and did not change the functional vessel density and perivascular cell coverage in both tumor variants. Interestingly, DC101 induced a rapid (2 days after treatment initiation) and significant decrease in tumor cell proliferation in recurrent but not in primary tumors. Thus, our results show that the stromal compartment and the response to antiangionenic therapy of primary and in-field recurrent tumors are significantly different. Our findings suggest that antiangiogenic agents could be effective in the treatment of patients with relapses after radiotherapy. [Cancer Res 2007;67(11):5076–82]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The treatment of tumor recurrences after radiotherapy is a challenging problem. Although extensively studied in animals with untreated tumors, the effect of antiangiogenic agents on recurrent tumors following irradiation has not been evaluated in preclinical studies. To fill this gap in our knowledge, "primary" (treatment-naive) and "recurrent/relapsed" (previously irradiated) human tumor xenografts were treated with anti–vascular endothelial growth factor receptor-2 (VEGFR2) therapy. We hypothesized that tumors relapsing after irradiation may respond better to antiangiogenic therapy because (a) the slow growth observed in recurrent tumors is typically associated with angiogenesis defects (1–4), and (b) slowly growing, poorly vascularized tumors are potentially more susceptible to antiangiogenic therapy (5, 6). Other studies have generally used tumors produced by the implantation of nonirradiated cancer cells into preirradiated normal tissue as a model of recurrent tumor growth; our model is a closer representation of clinical recurrences because we irradiated neoplastic cells, tumor vessels, and other stromal components. We show that the blockade of mouse VEGFR2 with the DC101 monoclonal antibody is therapeutically more effective against radiation recurrences; this is presumably related to significant differences between the vasculature and microenvironment of recurrent versus treatment-naive tumors.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Tumor implantation and irradiation. As in our previous study (7), the human lung tumor 54A was implanted s.c. into the calf area of the right hind leg of athymic NCr/Sed nude (nu/nu) mice. At a size of 6 mm in diameter, tumors were locally {gamma}-irradiated with a single dose of 20 or 30 Gy (at a dose rate of ~5 Gy/min), whereas tumors in the control group were not irradiated. A schematic representation of the experimental protocol is represented in Fig. 1A .


Figure 1
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  		Figure 1. Sluggish regrowth and increased sensitivity to DC101 of recurrent tumors. A, diagram of the primary and recurrent tumor models, treatment, and evaluation of response. Tumor xenografts of 6-mm diameter were irradiated with a single dose of 20 or 30 Gy, which induced a prolonged growth suppression followed by recurrence. When both recurrent and primary tumors reached 8 mm in diameter, in ~3 or 5 wk and in 4 to 5 d, respectively (everywhere referred to as day 0), half of the mice were treated with the VEGFR2-blocking antibody DC101 (three i.p. injections on days 0, 3, and 6; 27 mg/kg per injection). Changes induced in tumor tissue by radiation (assessed on day 0) and DC101 (on days 2 and 8) were correlated with tumor growth retardations. B, the dynamics of primary and recurrent tumor growth, with and without DC101, relative to initial tumor volumes Vo on day 0 (~260 mm3, corresponding to ~8 mm in diameter); n = 19 to 23 per group. C, individual values of time taken for a tumor to double its initial volume Vo; two recurrent tumors were locally controlled (LC) for 120 d after DC101 therapy. Filled symbols, median values in groups.

 
DC101 and collagenase treatments and evaluation of tumor response to therapy. Once treatment-naive and recurrent tumors reached a diameter of 8 mm (the corresponding volume ~260 mm3 is designated as Vo), they were treated with the monoclonal antibody DC101 or collagenase. Three i.p. injections of DC101 (ImClone Systems, Inc.) were given every 3 days at 27 mg/kg (Fig. 1A). DC101 specifically blocks the signaling of mouse VEGFR2/Flk-1 and does not cross-react with human VEGFR2/KDR or mouse and human VEGFR1 (8). Collagenase (clostridiopeptidase A, Sigma) was administered i.t. (50 µg/mL in PBS, two injections of 20 µL each per tumor) thrice every 2 days. Tumor size was measured thrice per week. The time taken for a tumor to double or triple its initial volume Vo was determined.

Tumor histology and immunohistochemistry. To identify perfuse vessels, 0.1 mL of biotinylated tomato lectin (Vector Laboratories) was injected i.v. and 5 min later the mice were sacrificed. The excised tumors were formalin fixed, embedded in paraffin, and 5-µm sections were cut. To colocalize the biotinylated lectin and perivascular cells in the same section, the staining was done with a streptavidin-conjugated fluorochrome (Alexa 647, Molecular Probes, Inc.) and a Cy3-conjugated mouse monoclonal antibody against {alpha}-smooth muscle actin ({alpha}SMA; clone 1A4, Sigma). Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining for apoptosis and Ki67 for tumor cell proliferation were done as previously described (9). To determine the collagen content in tumors, sections were stained with Masson trichrome. Images were captured using either an Olympus confocal or bright-field microscope and then processed with Adobe Photoshop 7.0 software (Adobe Systems, Inc.) and/or the NIH Image 1.63 software. A macro within the NIH Image 1.63 software was used to calculate the colocalization of {alpha}SMA-positive cells with perfused vessels. To quantify necrosis, we superimposed an electronic grid of small squares on randomly sampled images. The percentage of necrosis was obtained by dividing the number of squares associated with necrotic areas by the total number of squares.

Expression of angiogenesis-related molecules in tumors. Primary and recurrent tumors of 8 mm in diameter (n = 6) were snap frozen in liquid nitrogen and collected at –80°C. Tumors were grinded in liquid nitrogen and pooled aliquots of the samples were first used to measure mRNA expression (the remaining material was again stored at –80°C and used later for protein analysis). Total RNA was extracted with RNeasy Mini Kit (Qiagen, Inc.). To screen for relative gene expression, we used cDNA gene chips containing 96 human or mouse genes related to angiogenesis and vessel maturation (GEArray Q Series, SuperArray) and quantified chemiluminiscent spots on membranes by densitometry (FluoroChem 800 system, Alpha Innotech). The relative abundance of a transcript was normalized to ß-actin. Protein extraction from the stored material was done using NET buffer. Total protein concentrations were determined in individual samples by the Standard Lowry Method (Bio-Rad detergent-compatible protein assay). Human and mouse VEGF and human placental growth factor (PlGF) were measured by ELISA (the Quantikine Immunoassays, R&D Systems, Inc.).

Interstitial fluid pressure measurements. Mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.m.) and placed on a heating pad to maintain normal body temperature. Tumor interstitial fluid pressure (IFP) was measured with the wick-in-needle technique (10) before and on days 2 and 8 after the initiation of DC101 treatment.

Statistical analysis. Differences between group mean values were evaluated by t tests and considered significant at P < 0.05. The comparison of a collection of several groups for one tumor type with a collection of the corresponding groups for another tumor type was carried out using t test for linear contrasts in one-way ANOVA (11). Unless indicated otherwise, all error values represent the SE.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Recurrent tumors are more sensitive to DC101 than treatment-naive tumors. Following a minor increase in size over the first days after irradiation at 6 mm, apparent tumor dimensions remained almost unchanged after 20 Gy and slowly decreased by 1 to 2 mm after 30 Gy. Regardless of the dose, when the tumors relapsed, they reached 8 mm in diameter (the point of DC101 treatment initiation) at a stabilized growth rate. The mean time (± SD) between tumor irradiation and regrowth to 8 mm was 20.3 ± 4.2 and 35.9 ± 6.1 days following 20 and 30 Gy, respectively; treatment-naive tumors grew from 6 to 8 mm typically in 4 to 5 days (Fig. 1A).

Figure 1B shows that after 20 Gy, the time needed to double and triple tumor volume Vo increased 1.6- and 1.8-fold, respectively. The same sluggish growth was observed for tumors recurring after 30 Gy (n = 9; data not shown). Our tumor growth results are similar to the experimental findings of other studies and consistent with the classic tumor bed effect, which usually reaches saturation at ~20 Gy (2). The doses and schedules of fractionated irradiation commonly used in the clinic may be as effective in inducing the tumor bed effect as the 20-Gy single irradiation dose used in the present study (2).

During DC101 therapy, the growth of primary tumors was only modestly inhibited whereas it was completely arrested in most tumors recurring after 20 Gy, with an occasional decrease in size (Fig. 1B). After DC101 treatment, primary tumors immediately started to grow at a rate indistinguishable from nonirradiated controls. In contrast, several recurrent tumors remained dormant for a certain period of time following the last antibody injection, although they eventually regrew at approximately the same rate as control relapses. The tumor growth delay induced by DC101 (the difference between DC101-treated and untreated groups) was significantly longer in recurrent than primary tumors at both 2Vo (P < 0.001) and 3Vo (P < 0.01). Because primary and recurrent tumors grew at different rates, we also calculated the mean specific tumor growth delay induced by DC101 (normalized to the tumor doubling times of the corresponding controls), which was 40% greater in recurrences. Figure 1C shows the individual tumor volume doubling times for the four experimental groups. For the 19 recurrent tumors treated with DC101, 3 tumors were relatively resistant and 6 tumors were especially sensitive (including 2 tumors that were locally controlled for 120 days). The individual variability within groups was greater for the relapsed tumors, especially in the case of DC101 treatment (increased intertumor variabilities of certain growth parameters were also noticed for tumors implanted in preirradiated sites; refs. 2, 12). The effects of DC101 on tumor growth parameters were similar when the 30-Gy dose was used for tumor irradiation instead of 20 Gy (n = 8; data not shown).

Collectively, our results indicate that DC101 induces a greater growth retardation in recurrent than primary 54A tumor xenografts. Although antiangiogenic agents are often used in combination with radiation, the present data are the first evidence of highly effective antiangiogenic therapy of post-radiation recurrences. Probably closest to our findings is the recent study of Zips et al. (13); they showed that tumors implanted in normal tissue 10 days after its irradiation were sensitive to the VEGFR2 inhibitor PTK787/ZK222584 at an initial stage of growth, whereas control tumors growing in nonirradiated tissue were not. The greater antitumor effect of DC101 on radiation recurrences occurred after a relatively short period of antibody administration. Therefore, greater benefits may be expected from a longer administration of antiangiogenic agents. In fact, we have recently shown in a phase 2 study that AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, tended to increase median overall survival time in comparison with a historical database and significantly alleviated edema in recurrent glioblastoma patients (14), although no comparison with AZD2171 effects in newly diagnosed glioblastomas was studied.

The decrease in functional vascular density in recurrent tumors is associated with less mature vessels and a reduction in PlGF concentration. Presumably distinctive, the characteristics of tumors regrowing after irradiation have been virtually unexplored. Table 1 shows that the percentage of necrotic area was significantly increased in relapsed versus primary 54A tumors, whereas the fractions of proliferating and apoptotic tumor cells were not significantly different. A strong trend for a lower functional vessel density was also found in recurrent tumors. Our findings are similar to results obtained for tumors growing in preirradiated normal tissue, wherein a slower growth rate has been associated with a reduced vascular density and increased necrotic loss of nonirradiated cancer cells at a constant proliferation rate (4, 12, 15, 16).


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  		Table 1. Comparison of molecular, structural, and functional characteristics of 8-mm primary versus recurrent (following 20 Gy) 54A tumors

 
Although the perfused vessel density was approximately proportional to the viable tumor parenchyma in both tumor types, in recurrent tumors vessel coverage by perivascular cells significantly decreased by 44% (Table 1). This novel finding for radiation recurrences may be consistent with a decrease in vessel maturation observed in tumor xenografts shortly before their apparent regrowth following radioimmunotherapy (17).

To determine if differences in the vasculature of primary and recurrent tumors were related to the expression of angiogenesis-related genes, we used human- and mouse-specific angiogenic gene arrays. Except for human PlGF expression, which was down-regulated by 2.7-fold in relapsed xenografts, the expression of other genes did not change by more than 2-fold. In agreement with gene array results, human and mouse VEGF protein levels were not different between primary and recurrent tumors whereas the human PlGF concentration was 1.64-fold lower in recurrences (Table 1). The reduction in PlGF concentration could affect the density and maturation of blood vessels and decrease the growth rate of radiation recurrences. PlGF, a specific ligand of VEGFR1, can potentiate the angiogenic effects of VEGF, enhance the survival of tumor-associated endothelial cells and monocytes/macrophages, and stimulate the recruitment of perivascular cells and vessel stabilization (18–20).

Collagenase accelerates the growth of recurrent tumors. We have shown that the tumor collagen content (collagen type I) significantly increased at an early time point (5 days) after irradiation doses of 10 and 30 Gy (21). In the present study, the quantitative evaluation of the Masson staining revealed a 3.3-fold higher fraction of tissue occupied by collagen in recurrent than primary tumors of the same size (Fig. 2A ; Table 1). The increased collagen content observed in recurrent tumors is also consistent with normal tissue fibrosis after radiotherapy (22, 23); however, to our knowledge, collagen levels have not been previously reported for recurrent tumors or tumors growing in preirradiated normal tissues.


Figure 2
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  		Figure 2. Fibrillar collagen accumulation and growth retardation in 54A tumors recurring after irradiation. A, Masson staining for fibrillar collagen (blue) in recurrent versus primary xenografts. B, i.t. injection of bacterial collagenase (on days 0, 2, and 4) partially restored the growth rate of relapsing tumors while not affecting the growth of primary xenografts; n = 9 per group.

 
Based on in vitro studies showing that collagen inhibits the growth of tumor cells and spheroids (24, 25), we assumed that the increased collagen deposition in recurrent tumors retarded their growth. To test this hypothesis, similar to our previous studies, we injected bacterial collagenase i.t. to degrade collagen fibers (26, 27). Collagenase did not change the growth of primary tumors but the growth rate of recurrent tumors was partially restored (Fig. 2B). Thus, radiation-induced accumulation of collagen affected the growth rate of recurrent tumors without altering tumor cell proliferation (Table 1).

DC101 does not affect the vessel density or perivascular cell coverage but reduces the tumor IFP in both primary and recurrent tumors. The analysis of the vasculature showed that, in both tumor types, the DC101-induced growth inhibition was not associated with parallel changes in the density of perfused vessels (Fig. 3A ) or vessel perimeter covered by {alpha}SMA-positive perivascular cells (Fig. 3B). At the early time points studied (2 and 8 days after the initiation of DC101 treatment), the vasculature of primary and even recurrent (with less vessel coverage by perivascular cells) tumors was relatively more resistant to DC101 than the vasculature of other tumor types treated with similar or higher doses of DC101 (28–30). However, consistent with our findings, in a model of colon carcinomatosis, Shaheen et al. (31) found no change in total vessel density after multiple injections of DC101 over a period of 2 weeks, but the number of vessels decreased at later time points. Our findings also provide an additional example of the lack of correlation between changes in microvessel density during antiangiogenic treatment and alterations in tumor growth (6, 29).


Figure 3
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  		Figure 3. DC101 does not affect the functional vessel density and coverage by perivascular cells but significantly decreased the IFP in both primary and recurrent 54A tumors. A and B, density of lectin-perfused vessels and their coverage with {alpha}SMA-stained perivascular cells in DC101-untreated tumors (day 0) and during DC101 therapy (days 2 and 8); n = 6 per group. *, P < 0.05, significant difference between primary and recurrent tumors for a particular time. When all three pairs of values were analyzed collectively, the intermodel difference was highly significant (P < 0.01) for both parameters. C and D, serial measurements of IFP in primary (n = 16) and recurrent (n = 14) tumors shortly before (day 0) and on days 2 and 8 of DC101 therapy and the kinetics of growth of these xenografts. Of note, for both tumor variants, the multiple IFP measurements did not modify the early tumor size kinetics (D) or the DC101-induced growth delay, as compared with tumors without IFP measurements (Fig. 1B).

 
Although there was no change in the functional vascular density in both tumor models (Fig. 3A), it is quite possible that DC101 affected angiogenesis preferentially in recurrent tumors. DC101 immediately arrested the growth of relapses (Figs. 1B and 3D), suggesting that their neovascularization was almost completely inhibited. In contrast, primary tumors continued to grow, only slower (Fig. 1B), which implies that the formation of new vessels was still active.

To determine if DC101 could affect the vascular function in 54A tumor xenografts, we measured the tumor IFP, a reliable surrogate marker of changes in tumor vascular permeability induced by the inhibition of VEGF signaling. We have shown that DC101 reduces the tumor vascular permeability and the transfer of plasma proteins to the interstitial space, thus reducing fluid accumulation and the tumor IFP (32). To our knowledge, the IFP has not been compared between primary and recurrent tumors. The mean baseline IFP was virtually identical in the two tumor variants (Table 1; Fig. 3C). DC101 induced a significant reduction in IFP in both primary and recurrent 54A tumors (Fig. 3C), thus suggesting a reduction of their vessel permeability as well.

DC101 selectively reduces the tumor cell proliferation in recurrent tumors. We also related the effects of DC101 on tumor growth to secondary effects on tumor cells (DC101 is highly specific for mouse VEGFR2; ref. 8). Compared with the corresponding controls (day 0), DC101 increased necrosis in primary and recurrent tumors (Fig. 4A ) whereas neoplastic cell apoptosis was not significantly changed in both tumor models (data not shown). Figure 4B shows that DC101 had no more than a tendency to decrease cancer cell proliferation in the viable areas of primary tumors. In contrast, in recurrent tumors, DC101 induced a 55% reduction in tumor cell proliferation on day 2 versus day 0 (P < 0.05). In addition, there were significantly less proliferating cells in recurrent than primary tumors on days 2 and 8 (Fig. 4B). The rapid decrease in proliferation (after a single injection of DC101) in recurrent tumors is intriguing, given that in the present study and other studies (29, 31), the treatment of primary tumors with DC101 for 1 to 2 weeks did not affect neoplastic cell proliferation. Thus, neoplastic cells in both tumor variants responded to therapy, but in recurrent tumors they were more sensitive to the antiproliferative stimuli triggered by the VEGFR2 blockade. In addition to changes in the tumor vasculature and collagen content in recurrent tumors (Table 1), radiation could induce in tumors some other thus far unidentified alterations, which stimulated the antiproliferative effects of DC101 therapy. These changes in tumor cell proliferation may be mediated through specific paracrine interactions between tumor and endothelial cells or other stromal cells in the irradiated site. As shown in coculture, endothelial cells or sprouts can stimulate tumor cell proliferation (33). Such paracrine interactions are likely distinct in previously irradiated tumors but have not been specifically studied yet. The mechanisms explaining why DC101 preferentially affected tumor cell proliferation in radiation recurrences need to be further explored.


Figure 4
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  		Figure 4. DC101 progressively increases tissue necrosis in both primary and recurrent tumors and selectively decreases tumor cell proliferation in recurrent tumors. Tumor tissue necrosis (A) and cancer cell proliferation (B) were assessed before (day 0) and during DC101 therapy (days 2 and 8); n = 5 to 6 per group. *, P < 0.05, significant difference between primary and recurrent tumors for a particular time point.

 
Conclusion. We uncovered several characteristics of experimental recurrences following irradiation that distinguish them from primary tumors of the same origin and size and affect their growth and response to antiangiogenic therapy. Post-radiation recurrences had fewer functional vessels and a lower vessel coverage by perivascular cells. The vascular abnormalities, together with the elevated collagen content, could inhibit the growth of recurrent tumors. We also show that anti-VEGFR2 treatment has greater inhibitory effects on tumor cell proliferation and growth in previously irradiated than treatment-naive tumors.


   Acknowledgments
 
Grant support: National Cancer Institute grants PO1-CA80124 and RO1-CA115767 (R.K. Jain) and grant RO1-CA98706 (Y. Boucher).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Leo E. Gerweck, Dan Duda, Kevin Kozak, and Dai Fukumura for helpful discussions.

Received 10/ 3/06. Revised 3/ 7/07. Accepted 4/10/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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