Tumor growth inhibition and regression induced by photothermal vascular targeting and angiogenesis inhibitor retinoic acid

Cancer Letters 137 (1999) 35±44

Tumor growth inhibition and regression induced by photothermal vascular targeting and angiogenesis inhibitor retinoic acid Kathleen McMillan*, Ilya Perepelitsyn, Zhi Wang, Stanley M. Shapshay Otolaryngology Research Center, Department of Otolaryngology, Head & Neck Surgery, New England Medical Center, NEMC No. 187, 750 Washington Street, Boston, MA 02111, USA Received 10 September 1998; received in revised form 26 October 1998; accepted 29 October 1998

Abstract The effect of photothermal vascular targeting, alone and in combination with antiangiogenic therapy, was evaluated using tumors produced in mice by transplantation of KB cells. Tumor growth inhibition and regression followed vascular damage produced by pulsed dye laser (PDL) radiation. Administration of the antiangiogenic agent all-trans-retinoic acid (RA) was associated with smaller average tumor volumes in the presence and absence of PDL irradiation, but this effect was not statistically signi®cant. The ability of PDL photothermal vascular targeting to cause regression of tumors without harming normal tissue may be a consequence of preferential damage to supplying vessels at the tumor periphery. q 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Squamous cell cancer; Microvasculature, Pulsed dye laser

1. Introduction Cancer treatments designed to limit or eradicate tumor vascular supply are a promising alternative to traditional strategies aimed at directly damaging tumor cells. Angiogenesis inhibitors, for example angiostatin and endostatin, have raised hopes due to their ability to induce dormancy of tumors in mice [1]. Vascular targeting, de®ned as a strategy to destroy existing tumor vasculature [2], is distinct from antiangiogenic therapy in its approach and may provide an alternative or adjunct to the systemic administration of angiogenesis inhibitors. The challenge in implementing vascular targeting is to eradicate * Corresponding author. Tel.: 1 1-617-636-1686; fax: 1 1-617636-1690; e-mail: [email protected].

tumor vasculature while sparing vasculature critical to normal tissue; in the laboratory this has been achieved using tumor necrosis factor-a (TNF-a) gene therapy targeted by ionizing radiation [3] and antibody-directed targeting of vascular endothelial cells [4]. Clinical use of vascular targeting for cancer treatment is currently limited to TNF-a therapy with isolated limb or organ perfusion [5]. Photothermal techniques are well established for the selective destruction of abnormal blood vessels in benign cutaneous vascular lesions such as portwine stains (PWS). The basis of these techniques is the absorption of radiation by the chromophore oxyhemoglobin in blood with subsequent conductive heating of the vessel wall. With optimal choice of treatment parameters (wavelength, pulse duration, irradiance and spot size) the temperature rise causing irreversible

0304-3835/99/$ - see front matter q 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0304-383 5(98)00339-5

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thermal damage to the vessel wall occurs with minimal diffusion of heat to surrounding tissue and without irreversible damage to other tissue structures containing competing chromophores [6]. The pulsed dye laser (PDL) was the ®rst laser developed for selective photothermal eradication of lesional vasculature, and with its very low risk of scarring [7,8] remains the treatment of choice for many vascular lesions including PWS in infants and children. In the experiments reported here, the 585 nm PDL is used to target the vascular supply of malignant tumors induced in mice by intradermal inoculation with a human oral cavity squamous cell cancer (SCC) line. The most common malignancy in otolaryngology, SCC is associated with exposure of the epithelium of the oral cavity and upper aerodigestive tract (UADT) to environmental and endogenous mutagens and mitogens [9]. Because of the super®cial origin of SCC of the UADT, the vasculature of dysplastic lesions and early tumors may be accessible to laser radiation. Our experiments also evaluate the combination of photothermal vascular targeting with inhibition of neovascularization, using all-trans-retinoic acid (RA) as the antiangiogenic agent. RA has been shown to inhibit SCC angiogenesis by inhibiting the migration of endothelial cells [10] and inducing tumor cells to secrete angiogenesis inhibitors [11]. By combining the angiogenesis inhibitor with vascular targeting it may be possible to suppress vessel regrowth and enhance treatment effect. The objective of this work is the development of a highly selective treatment for cancer, with potential for preservation of normal tissue.

2. Materials and methods 2.1. Animals and animal care Animal care was in accordance with institutional guidelines. Homozygous (nu/nu) mice aged 40±50 days were purchased from Charles River Laboratories, Inc. (Wilmington, MA). The mice were sterilely housed and allowed food and water ad libitum. Intradermal tumor cell inoculations were performed using an operating microscope with mice under general anesthesia (ether). Mice were brie¯y

restrained in a holder for intraperitoneal injections of RA or vehicle. 2.2. Cell line and culture All experiments used KB human carcinoma cells (CCL-17) purchased from ATCC (Rockville, MD). Cells were grown in monolayers at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air, in a growth medium prepared from 500 ml Minimal Essential Medium (Gibco, Grand Island, NY), 5 ml non-essential amino acid solution (Gibco), 50 ml fetal bovine serum (Hyclone, Logan, UT), and 5.5 ml penicillin± streptomycin (Gibco), in cell culture ¯asks with vented caps. Prior to inoculation, cells were harvested by washing with phosphate-buffered saline (PBS) lacking calcium and magnesium, followed by trypsinization, centrifugation at 1000 rev./min for 6 min, and resuspension in normal saline at 500 000 cells/ml. 2.3. Laser Laser treatments employed a pulsed dye laser (PDL) producing 300±500 ms duration (full width half-maximum) pulses at 585 nm (Model SPTL-1a, Candela Corporation, Wayland, MA). PDL irradiation was delivered using an optical ®ber with standard handpiece optics designed to produce a 5 mm diameter irradiated spot with homogeneous energy distribution. Recent experiments in our laboratory [12] have shown that when this irradiation is con®ned to the surface of the tumor only, with a template completely masking skin peripheral to the tumor, the reduction in tumor growth compared to controls is much less than when the periphery is also irradiated. Therefore, in the tumor growth experiments each tumor along with a 2 mm margin of normal-appearing peripheral tissue was treated with minimally overlapping single pulses from the laser at 10 J/cm 2 using an aluminum template to mask skin tissue outside the margin zone. In this way, the tumor periphery was irradiated but the extent of vascular damage in the tumor margin area was independent of the size of the tumor relative to the irradiated spot size produced by the handpiece optics. 2.4. Retinoic acid All-trans retinoic acid (RA) was purchased from

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Fig. 1. (a) Limited ingrowth of vessels at tumor nodule (arrow) in mouse receiving RA injections. (b) Greater number of new vessels at circumference of tumor nodule (arrow) in control group mouse.

Sigma Chemical Co. (St. Louis, MO). A stock solution of 50 mg of RA in 2.5 ml dimethylsulfoxide

(DMSO, Sigma) was prepared and stored at 2208C in a light-protected container for the duration of each

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at each inoculation site. Three inoculation sites in the control group had no discernible tumor nodule; therefore, a total of nine inoculation sites in the control group and 12 in the treatment group were subjected to analysis. 2.6. Evaluation of PDL effect on vasculature

Fig. 2. Effect of all-trans retinoic acid on tumor-induced angiogenesis, as quanti®ed by the angiogenesis assay. Horizontal lines indicate (in increasing order): mean 2 2 £ SD, mean 2 2 £ SE, mean, mean 1 2 £ SE, mean 1 2 £ SD, where SD is standard deviation and SE is standard error.

experiment. For each procedure, 0.05 ml of this solution was diluted with 0.95 ml of PBS and injected intraperitoneally into mice in a daily dose of 5 mg/ kg body weight. This dosage of RA has been shown by Majewski and co-workers [13] to decrease angiogenesis at sites of inoculation in mice with HeLa and Skv-e2 cells harboring HPV DNA. 2.5. Evaluation of RA effect on vessel growth The effect of RA was determined using the angiogenesis assay ®rst described by Sidky and Auerbach [14] and more recently applied to the evaluation of antiangiogenic agents [13,15]. Four mice were inoculated intradermally in the ¯ank with the KB cell suspension, using a 30 gauge hypodermic needle. Each mouse received three inoculations in each ¯ank. Fifty thousand cells per single injection in 0.1 ml saline were used, with a drop of 0.4% Trypan Blue (Sigma) for better visualization of the injection site. The mice were then randomly assigned to control and treatment groups of two mice each. RA was administered starting on the day of inoculation. Treatment group mice received daily intraperitoneal injections of RA, and control mice received daily doses of vehicle only. After 3 days the mice were euthanized and the inner surface of their skin was assessed using a dissecting microscope for the number of newly formed blood vessels growing into the tumor nodule

Photothermal damage to vasculature was assessed histologically. Two mice were inoculated intradermally at three sites per mouse with the KB suspension, as described above. Seven days after inoculation, tumors had reached 3±5 mm in diameter and were irradiated with single laser pulses at 10 J/cm 2. In addition, three normal skin sites 5 mm away from the tumor sites were treated with the same laser parameters in each of the two mice. The ®rst and second mice were sacri®ced 0.5 h and 3 days after irradiation, respectively. Biopsy specimens of irradiated sites were ®xed in formalin and processed for light microscopic examination. 2.7. Tumor growth experiments Mice were inoculated with KB cell suspension as described above, at three sites on each ¯ank. Five days after inoculation, at which time most tumors were palpable, the mice were randomly assigned to two groups of four each, one of which received daily injections of RA for a total of 12 days, and the other daily injections of vehicle only for the same period of time. All eight mice received a single treatment with the PDL on tumor inoculation sites on the left ¯ank only. RA and vehicle injections began on the day of the laser treatment (day 5 after inoculation). Tumors were measured using a sliding caliper at 2±4-day intervals, and volume calculated using the formula V ˆ abc…p=6†, where a, b and c are the three orthogonal diameters. Mice were weighed each day that tumors were measured, and were euthanized 20 days after inoculation. 2.8. Statistical analysis The numbers of new blood vessels in RA treatment and control groups in the angiogenesis assay experiment were compared using Student's t-test. In the tumor growth experiments, tumor volumes were compared using the non-parametric Kruskal±

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Fig. 3. (a) Normal mouse skin immediately after PDL irradiation. Arrows indicate damaged microvessels (H&E, £ 40). (b) Normal mouse skin 3 days after PDL irradiation. Arrows indicate damaged vessels with surrounding in¯ammation (H&E, £ 40).

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Fig. 4. Measured tumor volume as a function of time after inoculation, for the four experimental groups.

Wallis statistic and Dunn's test. P values larger than 0.05 are considered insigni®cant; all P values are two-tailed.

3. Results 3.1. Evaluation of RA effect on vessel growth Under the dissecting microscope, the effect of RA on tumor-induced angiogenesis is readily observed (Fig. 1). Compared with control mice, mice receiving RA had signi®cantly fewer new vessels growing into the tumor nodules at inoculation sites (mean number for control and treatment mice is 24.0 and 15.8, respectively, P , 0:005, Fig. 2).

3.2. Evaluation of PDL effect on vasculature Macroscopically, both normal mouse skin and tumor area responded with a mild purpuric response that appeared within 10 min after irradiation. Within a few days of irradiation normal skin returned to its usual appearance while tumors typically appeared to develop regions of necrosis. Fig. 3 shows the microscopic response of normal mouse skin to PDL irradiation. Immediately after irradiation, visible vessels (larger than approximately 25± 30 mm in diameter) are intact but appear packed with erythrocytes (Fig. 3a, arrows). There is no apparent damage to other tissue structures including epidermis. Three days after irradiation (Fig. 3b), some in¯ammation is seen in the vicinity of the damaged vessel. The lower of the two vessels marked with an arrow is

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Table 1 Results of Dunn's test for pairwise comparisons of experimental groups a

RA2/PDL2

RA2/PDL1

RA2/PDL1

RA1/PDL2

RA1/PDL1

Day 7: ns Day 10: ns Day 13: P , 0.05 Day 17: P , 0.05

Day 7: ns Day 10: ns Day 13: ns Day 17: ns Day 7: P , 0.01 Day 10: P , 0.01 Day 13: P , 0.05 Day 17: ns

Day 7: ns Day 10: ns Day 13: P , 0.05 Day 17: P , 0.05 Day 7: ns Day 10: ns Day 13: ns Day 17: ns Day 7: P , 0.05 Day 10: P , 0.01 Day 13: P , 0.01 Day 17: P , 0.05

RA1/PDL2

a

ns, not signi®cant.

approximately 0.75 mm below the tissue surface. Examination of tumor tissue indicated similar speci®c effect of PDL irradiation on relatively large caliber microvessels; however, these vessels were only observed in the region of the tumor periphery. 3.3. Tumor growth experiments The ®rst tumor volume measurements were made on day 5 after inoculation, at the time treatments commenced. Because the vascularity of tumors and hence their response to treatment is expected to depend on tumor size, tumors with volumes greater than 1 standard deviation from the mean volume were excluded from further analysis [16]. Of the original 48 inoculation sites, ranging in size from 3.4 to 19.8 mm 3, 37 remained for analysis using this criterion. Tumor volume is shown in Fig. 4 for each of the four experimental groups as a function of time after inoculation. At day 17 the average tumor volume for the control group (RA2/PDL2), and three treatment groups (RA2/PDL1), (RA1/PDL2), (RA1/PDL1) is 198.7, 64.1, 124.1 and 34.7 mm 3, respectively. Because PDL treatment produced complete response in some tumors with the remainder showing continued growth, non-parametric statistics were used to determine the signi®cance of differences between the groups. Calculating the Kruskal±Wallis test statistic for each point in time the tumors were measured; it is

found that, as expected, there is no signi®cant difference at day 5, and signi®cant effects of treatment at days 7 (P , 0:005), 10 (P , 0:005), 13 (P , 0:001) and 17 (P , 0:001). The result of Dunn's test for pairwise comparison of the experimental groups at days 7 through 10 is shown in the Table 1. It can be seen that pairs, in which one group received PDL treatment and the other did not, show signi®cant difference, regardless of whether RA was administered. The P value decreases or shows no trend with time, with the exception of the pair (RA2/ PDL1):(RA1/PDL2) in which the RA and PDL administration is expected to have an opposing effect on tumor growth. The number of complete remissions per total number of tumors at day 17 in each group was 0/7 (RA2/PDL2), 4/10 (RA2/PDL1), 0/9 (RA1/ PDL2) and 4/11 (RA1/PDL1). By day 20, 3 days after RA/vehicle administration ceased, two of the regressed tumors in the RA2/PDL1 group had recurred, while none of the tumors in the RA1/ PDL1 group had shown regrowth. The four mice receiving RA injections showed a total weight gain of 1.1 g, which compares closely with the increase in total tumor burden between days 5 and 17, 1.3 g (assuming tumor density of 1.0 g/cm 3). The four mice receiving vehicle injections gained a total of 11.5 g, while total tumor burden increase was 4.2 g. The lack of increase in nontumor body weight in mice receiving RA is the only indication of possible toxicity seen in this experiment.

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4. Discussion By manipulating tumor blood supply, it is apparent that the growth and viability of tumors can be affected. In these experiments it has been shown that a single PDL treatment producing selective thermal damage to vasculature leads to growth inhibition and regression of tumors created in mice by transplantation of malignant human cells. Effective treatment of tumors is achieved using irradiation parameters that produce no apparent lasting or deleterious effect on normal mouse skin. These results support and extend our recent work using the 7,12-dimethylbenz[a]anthracene (DMBA)induced hamster cheek pouch tumor model [17]. Those experiments demonstrated that PDL irradiation causes regression of tumors induced by the carcinogen and furthermore that by irradiating dysplastic lesions, tumorigenesis can be inhibited. The hamster DMBA model has the important advantage of closely simulating the time-dependent sequence of changes from dysplasia to carcinoma in situ and microinvasive carcinoma seen in human SCC of the UADT [18]. However, that model also has the limitation that when the cheek pouch has developed malignant tumors, it is also likely to display a range of other chemically induced lesions including benign papilloma. The murine model used here allowed experiments to be performed on de®nitively malignant tumors. To our knowledge, these are the ®rst reports of effective treatment of cancer using selective photothermal vascular targeting. Chong et al. [19] demonstrated tumor growth inhibition in an experimental intraocular melanoma model using 810 nm diode laser irradiation following intravenous indocyanine green (ICG) administration as an exogenous chromophore. The 0.2 s exposure time of the diode laser as used by those authors signi®cantly exceeded the thermal relaxation time of the tumor microvasculature and widespread coagulative necrosis occurred with some thermal damage extending to nearby ocular structures. Karrer and co-workers [20] recently reported successful palliative treatment of metastatic skin tumors using ICG-enhanced diode laser irradiation in a single patient. A continuous wave laser source was used and diffuse thermal damage resulted in some scar formation. The only experience with

selective photothermal targeting for treatment of malignancy is reported by Tappero et al. [21] who found signi®cant but temporary lesional regression following treatment of patients with Kaposi's sarcoma with the PDL. Kaposi's sarcomas can extend to subcutaneous tissue layers, considerably deeper than the depth to which the 585 nm PDL can affect vasculature. The potential advantage of selective photothermal vascular targeting over conventional anticancer strategies is the preservation of normal tissue surrounding the tumor. In the case of standard photothermal treatment of PWS lesions, lesional eradication is also achievable without damage to normal skin although therapeutic ¯uences should be below the threshold ¯uences for damage to the melanin-containing epidermis. The basis of the ability of photothermal techniques to discriminate between lesion and normal skin may be differences in size between pathologic and normal cutaneous vasculature; it is known that for effective and selective photothermal injury to the vessel the pulse duration of the laser should match the thermal relaxation time of the targeted vessel [22]. For a cylindrical vessel, the thermal relaxation time is given by the expression tr ˆ d 2 =16x, where x is the thermal diffusivity of blood [22]. The pulse duration of the 585 nm PDL used in these experiments corresponds to a blood vessel with diameter of approximately 30 mm. When vessels of smaller diameter are irradiated heating will be inef®cient, as heat will diffuse out of the vessel during the laser pulse. Most blood vessels in the papillary dermis of normal human skin have outer diameters of 17±22 mm [23] and in a recent experimental study it was shown that only a small fraction of blood vessels 20 mm or smaller can be damaged by this laser [24]. The important consequences of vessels size considerations in the treatment of PWS are that microvessels in normal skin can be irradiated with little irreversible damage, and that the small vessels that replace the abnormal ectatic vessels of the PWS lesions are unaffected by subsequent retreatments. Those PWS lesions nonresponsive to PDL therapy may consist of relatively small diameter vessels [24,25]. In the experiments reported here, it is signi®cant that irradiation of a small margin of skin at the tumor periphery is required for tumor regression. This peripheral region is seen to be the location of relatively large caliber vessels that

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supply and drain the tumor microvasculature. Successful tumor treatment may be the result of selective damage to the arterioles and venules at the periphery, rather than direct damage to the much smaller microvessels within the tumor itself. The existence of large caliber feeding vessels as an integral part of the tumor microvascular architecture has been documented in corrosion cast studies of human colorectal carcinomas [26], cutaneous basal cell tumors [27] and xenotransplanted human tumors grown in mice [28]. Other factors may be at work, for example the relative fragility of tumor microvasculature and the closer dependence of proliferating tumor cells on their vascular supply. Further study is needed to elucidate the extent and nature of vascular photothermal damage that is associated with tumor regression; however, if ectatic vessels originating in normal host tissue but serving to supply the tumor can serve as the treatment target, photothermal techniques may be expected to selectively affect tumor tissue and spare normal tissue, as was the case in this work. The experiments presented here also tested the concept of increasing the effectiveness of photothermal vascular targeting by use of an antiangiogenic agent to inhibit vessel regrowth after irradiation. Our data demonstrate the reduction in tumor-induced angiogenesis associated with RA administration. RA reduced the number of new vessels developing over the ®rst 3 days of tumor growth by 34%, and reduced ®nal average tumor volumes by 37 and 46%, respectively, in the absence and presence of PDL irradiation. The statistical signi®cance of the effectiveness of RA therapy on tumor growth could not be demonstrated in these experiments, however, due to the dispersion in tumor volumes. RA has shown ef®cacy in preventing second malignancies in patients with SCC of the head and neck but with some toxicity concerns [29]. Further work is needed to demonstrate the bene®t of combining antiangiogenic therapy with photothermal vascular targeting. References [1] M.S. O'Reilly, T. Boehm, Y. Shing, N. Fukai, G. Vasios, W. S. Lane, E. Flynn, J.R. Birkhead, B R. Olsen, J. Folkman, Endostatin: an endogenous inhibitor of angiogenesis and tumor growth, Cell 88 (1997) 277±285. [2] J. Denekamp, Angiogenesis, neovascular proliferation, and

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