Tumor vasculature is targeted by the combination of combretastatin A-4 and hyperthermia

Radiotherapy and Oncology 61 (2001) 313–320 www.elsevier.com/locate/radonline

Tumor vasculature is targeted by the combination of combretastatin A-4 and hyperthermia q Hans Petter Eikesdal a,*, Rolf Bjerkvig b, James A. Raleigh c, Olav Mella a, Olav Dahl a a

Department of Oncology, University of Bergen, Haukeland University Hospital, 5021 Bergen, Norway Department of Anatomy and Cell Biology, University of Bergen, Aarstadveien 19, 5009 Bergen, Norway c Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA b

Received 30 January 2001; received in revised form 25 June 2001; accepted 17 September 2001

Abstract Background and purpose: Combretastatin A-4 disodium phosphate (CA-4) enhances thermal damage in s.c. BT4An rat gliomas. We currently investigated how CA-4 and hyperthermia affect the tumor microenvironment and neovasculature to disclose how the two treatment modalities interact to produce tumor response. Methods: By confocal microscopy and immunostaining for von Willebrand factor, we examined the extent of vascular damage subsequent to CA-4 (50 mg/kg) and hyperthermia (waterbath 448C, 60 min). The influence on tumor oxygenation was assessed using interstitial pO2probes (Licox system) and by immunostaining for pimonidazole. We examined the direct effect of CA-4 on the tumor cell population by flow cytometry (cell cycle distribution) and immunostaining for b-tubulin (cytoskeletal damage). Results: Whereas slight vascular damage was produced by CA-4 in the BT4An tumors, local hyperthermia exhibited moderate antivascular activity. In tumors exposed to CA-4 3 h before hyperthermia, massive vascular damage ensued. CA-4 reduced the pO2 from 36.1 to 17.6 mmHg (P ¼ 0:01) in the tumor base, and tumor hypoxia increased slightly in the tumor center (pimonidazole staining). Extensive tumor hypoxia developed subsequent to hyperthermia or combination therapy. Despite a profound influence on b-tubulin organization in vitro, CA4 had no significant effect on the cell cycle distribution in vivo. Conclusion: Our results indicate that the anti-vascular activity exhibited by local hyperthermia can be augmented by previous exposure to CA-4. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Combretastatin A-4 disodium phosphate; Hyperthermia; Tumor oxygenation; Confocal microscopy; Pimonidazole

1. Introduction A considerable amount of research has recently focused on the interactions between neoplastic cells and the host microenvironment, and new therapeutic strategies have emerged based upon manipulation of the tumor stroma. In this context, targeting the tumor neovascularization process has provided promising results where tumor dormancy has been obtained during continuous anti-angiogenic therapy [22,29,30]. Disruption of established tumor blood vessels is also feasible, using various treatment modalities that are cytocidal to the tumor endothelium [8]. Such anti-vascular therapy has been successfully applied in a number of tumor models, and tubulin disrupting drugs, in particular, display a potent toxicity towards the tumor vasculature [7,17]. Addi-

q Results presented in part at the 8th International Congress of Hyperthermic Oncology, Kyongju, Korea, 26–29 April, 2000. * Corresponding author.

tionally, hyperthermia exhibits anti-vascular activity if high thermal doses are deposited in the tumor tissue [8,39]. We have previously demonstrated that the tubulin disrupting drug CA-4 enhances thermal damage in the BT4An rat glioma, and the tumor response was optimized if we administered CA-4 3–6 h before local hyperthermia [12,13]. CA-4 induced mild endothelial edema and stasis within the tumor blood vessels, indicating a possible antivascular mechanism of action [12]. Tumor blood flow was transiently reduced 3–6 h after drug delivery [12,13], and the tumor temperatures were significantly increased if hyperthermia was applied at this time point [13]. Instead of selective targeting of the tumor vasculature, the enhancement of thermal damage could therefore be caused by a general vasoactive response to CA-4, and this theory is strengthened by the observed transient hypertension occurring after drug administration [13]. The purpose of the current study was twofold. Firstly, we wanted to elucidate how CA-4 and local hyperthermia each

0167-8140/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0167-814 0(01)00450-9

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affected the tumor parenchyma to disclose why thermal damage was enhanced by the drug. Secondly, we examined how CA-4 and hyperthermia jointly affected the tumor vasculature and oxygen status to disclose possible reasons why the combination therapy prolonged tumor response. The anti-vascular activity of CA-4 and hyperthermia was assessed by studying the morphology of the BT4An neovasculature after treatment, and the influence of CA-4 on tumor oxygenation and cell cycle kinetics was examined to disclose other possible reasons why the drug enhanced hyperthermic cytotoxicity. Effects on the tumor cell cytoskeleton were studied in vitro, to further assess the direct cytotoxicity exhibited by CA-4 towards the malignant cell population. 2. Materials and methods 2.1. Animals and tumor type Rats of both sexes of the inbred strain BD IX/HanFoss, mean weight 206 g (SD 39), were used. They were given water and standard pelleted diet ad libitum and caged at 228C in a night/day cycle 12:12 h. The BT4An rat glioma was implanted s.c. on the right hind foot dorsum according to an established procedure [13]. The animals were treated 9–11 days after transplantation at a mean tumor volume of 117 mm 3 (SD 37). 2.2. Drug administration During interventional procedures and hyperthermia, the animals were kept anesthetized with s.c. injections of midazolam (1.25 mg/kg)/fentanyl (0.05 mg/kg)/fluanisone (2.5 mg/kg) [14]. CA-4 was kindly supplied by Oxigene Inc. (Lund, Sweden). It was dissolved in 0.9% NaCl to a final concentration of 50 mg/ml and administered i.p. at a maximum tolerated dose of 50 mg/kg [13]. Pimonidazole hydrochloride (NPI Inc., Belmont, MA) was dissolved in 0.9% NaCl to a final concentration of 60 mg/ml and administered i.p. at 60 mg/kg. 2.3. Flow cytometry Tumors were taken out three hours after injecting CA-4 (n ¼ 6) or an equivalent volume of 0.9% NaCl (n ¼ 6), and prepared for flow cytometry according to established methods [44]. The nuclei were stained with propidium iodide and the DNA content was measured using a FACSort flow cytometer (Becton Dickinson, Palo Alto, CA). Cell cycle kinetics were assessed by gating a two-parameter fluorescence width/fluorescence area cytogram into a one-parameter DNA histogram. We also compared the sub-G0/G1 peaks of non-gated DNA histograms to assess the amount of apoptosis after CA-4 treatment. The mean coefficient of variation for the G0/G1 peak was less than 6.0 in all cases, and a minimum of 50 000 cells were counted for each

sample. The cell cycle distribution was determined as described elsewhere [2]. 2.4. Scanning confocal microscopy For the immunohistochemical studies described below, fluorescent images were made using a Leica TCS NT confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) attached to an upright Leica DM RXA microscope. The optical sections were recorded with the Leica software and stored in the native file format. Each section was recorded as an average of eight pictures to improve the image quality, and eight serial sections along the z-axis were obtained from each tissue location according to an established set-up [47]. For the b-tubulin study, the Leica native image files were merged using Leica software to produce three-dimensional (3D) image reconstructions of the cells. For 3D reconstructions of the tumor tissue, the native image files were transferred to a Silicon Graphics Octane workstation, and the serial confocal sections were combined using AVS/Express software (Advanced Visual Systems, Waltham, MA) [32]. 2.5. Immunostaining of b -tubulin in vitro We used the BT4Cn cell line which was originally developed from an ethylnitrosourea induced rat glioma [28]. The cells were cultured as described previously [13], and exponentially growing monolayers were exposed to 1.0 nM CA4 for 60 min. The concentration was chosen based on a previous MTT-assay demonstrating low in vitro cytotoxicity at this CA-4 level [13], and this dose level is also far below the reported murine plasma concentration of 10 mM measured 6 h after CA-4 100 mg/kg [46]. After drug exposure, immunostaining for b-tubulin was performed according to an established procedure [24]. The cells were incubated for 60 min with a mouse monoclonal antibody against rat b-tubulin (Sigma, St. Louis, MO), diluted 1:200 in Dulbecco’s phosphate-buffered saline (DPBS, Sigma). A FITC-conjugated goat anti-mouse secondary antibody (Sigma), diluted 1:30 in DPBS, was then applied for 60 min and the monolayers were examined using confocal microscopy. 2.6. Immunostaining of tumor vasculature The animals were stratified after tumor size and randomized to the following treatment groups (n ¼ 6 in all groups): controls, CA-4 alone, hyperthermia alone, CA-4 immediately before hyperthermia (C0HT), and CA-4 3 h before hyperthermia (C3HT). The tumors were removed 15 h after completion of therapy, coded as to the therapy they had received and frozen in N2. Six serial transverse cryosections (60 mm) were prepared from the mid portion of each tumor before fixation in acetone (48C) for 15 min. The tumor vasculature was stained using a rabbit antihuman polyclonal antibody against von Willebrand factor

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(Dako, Glostrup, Denmark), diluted 1:100 in DPBS, and a FITC-conjugated secondary antibody (Dako), diluted 1:30 in DPBS. Both antibodies were applied for 60 min. Propidium iodide was used for general staining of cell nuclei in tumor sections. 2.7. Immunostaining for tumor hypoxia Tumor hypoxia was demonstrated using the pimonidazole technique [33]. Briefly, six rats from each of the treatment groups mentioned above were given pimonidazole (NPI) 2 h after the therapy was completed, and the tumors were excised 1 h later. Transverse cryosections (20 mm) were collected from the mid portion of the tumors, and every first section of each series of five was mounted on gelatin-coated slides (six sections per tumor). After fixation in methanol (2208C) for 20 min, immunostaining for pimonidazole adducts was performed according to a standardized protocol [33]. Briefly, the sections were incubated with a mouse monoclonal antibody (clone 4.3.11.3, NPI) (a noncommercial polyclonal antibody was used for the first series of ten tumors), diluted 1:100 in DPBS, before adding a FITC-conjugated F(ab 0 )2 fragment secondary antibody (Dako), diluted 1:30 in DPBS. 2.8. Quantification of vascular damage and extent of tumor hypoxia The influence of anti-angiogenic agents on the tumor vasculature is commonly quantified by vascular length density or microvessel density assays, where vascular lengths are measured in skin window chambers [26] or blood vessels are counted in vascular hot-spots [48]. Antivascular therapy induces vascular shutdown in patchy tumor areas, and typically, some tumor areas remain unaffected [8]. The assessment of blood vessels in vascular hot-spots would therefore ignore the present vascular damage, as long as some viable tumor areas remain [36]. As shown previously [7,15], morphological alterations, such as the extent of hemorrhagic necrosis, can be assessed microscopically, and quantified as a percentage of the section area. We used the same kind of method to quantify vascular damage and tumor hypoxia; Two independent observers, blinded as to treatment group, assessed the extent of vascular damage and tumor hypoxia in the tumor sections at low magnification using the Leica DM RXA microscope and fluorescence microscopy. Vascular damage was defined as a reduction in vascular profiles, and the tumor sections were categorized as having no vascular damage (0%), 1–10, 10–25, 25–50, 50–75 or . 75% vascular damage, compared with sham-treated tumors. Vascular profiles were defined as distinct fluorescent dots and stripes that could be clearly visualized at low magnification, and which stained positively for von Willebrand factor [48]. In separate sections and tumors, the total hypoxic area was categorized as being , 10, 10–25, 25– 50, 50–75 or . 75% of the tumor section. The observers

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studied one coded slide per tumor, and there were six different tumors per treatment group. Each coded slide was then inspected jointly by the two observers, and joint conclusions were made. In cases where different percentage categories had been assigned to the same slide, this tumor was given the lowest of the two values. The immunostaining and quantification procedures were repeated three times per tumor (three different slides) on 3 different days to assure reliable categorization for each tumor. Finally, the mean percentage category for each tumor and each treatment group was calculated. 2.9. Tumor oxygen tension Tumor oxygenation was measured polarographically using a flexible Clark electrode (diameter , 0:5 mm), connected to a LICOX computer (GMS, Kiel-Mielkendorf, Germany). The electrode has a pO2-sensitive area of 5 mm, and depending upon the surrounding tissue destruction, the tissue oxygenation is measured from an area of 7.1–15 mm 2 [11]. The LICOX system, as compared to the Eppendorf system, permits continuous recordings of tissue oxygenation without moving the probe [45]. The electrode was calibrated with room air and an oxygen free solution and corrections were made for temperature and barometric pressure. An i.v. and i.a. line was established for drug administration and continuous recordings of the mean arterial blood pressure (MAP) and heart rate. The animals were tracheostomized in order to ensure adequate, spontaneous breathing, and the body temperature was kept stable between 36 and 388C using a heating pad (Harvard Apparatus 50-7061, Kent, UK). The pO2-probe was inserted into the tumor base, and the measurements commenced once constant baseline readings for MAP and pO2 had been obtained for at least 15 min. Tumor pO2 was monitored continuously for 3 h after the injection of CA-4 (n ¼ 7) or saline (sham treatment, n ¼ 6). Simultaneously, normal tissue oxygenation was assessed by insertion of a pO2-probe subcutaneously into the right flank. The measured pO2 values were averaged over a period of 1 min, and expressed as one data-set every 10 min. We analyzed arterial blood gases (AVL Opti CCA, AVL Scientific Corp., Roswell, GA) before and after each experiment to assure that the animals had proper spontaneous ventilation. 2.10. Statistics We used the nonpaired or paired Student’s t-test to compare data from the flow cytometry- and tumor pO2 studies. 2.11. Ethics The experiments and procedures described were approved by the local responsible laboratory animal science specialist under the surveillance of the Norwegian Animal Research Authority. The experiments were therefore

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Table 1 Flow cytometric cell cycle distribution of BT4An tumors exposed to CA-4 or sham treatment 3 h previously Group a

G0/G1 (%)

S (%)

G2/M (%)

CV (%) b

Control c CA-4 c

75.2 ^ 1.1 71.7 ^ 3.7

10.6 ^ 0.6 12.4 ^ 1.1

14.1 ^ 0.7 15.9 ^ 3.1

4.6 ^ 0.6 4.7 ^ 0.9

a b c

CA-4: 50 mg/kg i.p.; control: 0.9% NaCl, 1 ml/kg i.p. CV, coefficient of variation. Mean values ^ SD of six tumors (in vivo data).

conducted in accordance with the laws and regulations controlling experiments on live animals in Norway. 3. Results 3.1. Flow cytometry The cell cycle distribution was not significantly altered 3 h after exposing the tumors to CA-4 (Table 1). In particular, we found no accumulation of cells in the G2/M-phase. The apoptotic fraction was also unaffected by CA-4 3 h after drug administration (data not shown). 3.2. Immunostaining of b -tubulin in vitro Immunofluorescence of sham treated BT4Cn cells, using anti-b-tubulin, revealed a dense network of microtubules in the cytoplasm with perinuclear condensation (Fig. 1a). Exposure to CA-4 disrupted the microtubular network, and led to contraction of the tumor cells (Fig. 1b). Diffuse overall fluorescence could be seen in the cytoplasm, and cellular blebbing suggested that the drug initiated apoptosis in some tumor cells. 3.3. Immunostaining of tumor vasculature The BT4An tumors had a rich and tortuous network of tumor blood vessels, and a similar vascular morphology was found in central and peripheral tumor parts (Fig. 1c). CA-4 alone did not reduce the number of blood vessels in the peripheral tumor areas (Table 2), but they were dilated with diffuse perivascular fluorescence (Fig. 1d). In 4 out of 6 tumors CA-4 induced a minor central necrosis, indicated by the disappearance of vascular profiles and a diffuse fluorescent blur in central tumor parts. Hyperthermia alone had moderate anti-vascular activity, and CA-4 administered immediately prior to the heating session did not enhance vascular damage (Fig. 1e). The number of visible blood vessels were reduced by 25–50% in the tumors, and there were no distinct difference between tumor periphery and tumor center (Table 2). Thus, the tumors exhibited heterogeneous vascular damage, and the remaining blood vessels were shortened or had perivascular fluorescence, indicating a partial vascular disruption. Tumors exposed to CA-4 3 h before heating exhibited massive vascular damage (Table

Fig. 1. (a,b) Scanning confocal laser micrographs of subconfluent BT4Cn monolayers after immunostaining for b-tubulin. Oil immersion, 40£ objective. (a) Control. (b) BT4Cn cells exposed to CA-4 1 nM for 60 min, showing a contracted morphology and cellular blebbing. Scale bar: 100 mm. (c–h) Scanning confocal laser micrographs of BT4An tumors after 3D reconstruction using AVS/Express software. Each picture was taken from a representative section chosen among six different tumors from the respective treatment group, and the areas with maximum vascular damage or hypoxia are shown. (c–f) Immunostaining for von Willebrand factor 15 h after treatment. Water immersion, 16£ objective. Scale bar: 250 mm. Red fluorescence (nuclear staining) was omitted from the images to reveal the vascular morphology better. (c) Control. (d) CA-4 alone; tumor blood vessels appear dilated. (e) CA-4 immediately before hyperthermia; moderate vascular damage. (f) CA-4 3 h before hyperthermia; extensive vascular damage. Arrow points at an area of total vascular destruction. (g,h) Immunostaining for tumor hypoxia 3 h after treatment using the pimonidazole technique. 5£ objective. Scale bar: 1000 mm. (g) CA-4 alone; small, scattered areas of hypoxia in the tumor. Arrowhead points to an area without hypoxia. (h) Hyperthermia alone; larger, confluencing areas of hypoxia. CA-4: 50 mg/kg i.p. Hyperthermia: 448C, 60 min.

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Table 2 Quantitative assessment of tumor hypoxia 3 h after treatment and vascular damage 15 h after treatment with CA-4 1 hyperthermia Group a

Control CA-4 HT C0HT C3HT

Tumor hypoxia b,c (%)

, 10 10–25 25–50 25–50 25–50

Vascular damage b,d (%) Tumor periphery

Tumor center

0 0 25–50 25–50 50–75

0 25–50 25–50 25–50 50–75

a Control: 0.9% NaCl, 1 ml/kg i.p.; CA-4: CA-4 50 mg/kg i.p. alone; HT: hyperthermia alone, 448C/60 min; C0HT: CA-4 immediately before hyperthermia; C3HT: CA-4 3 h before hyperthermia. b Each value indicates the mean percentage of the section area for six different tumors. Two independent observers, blinded as to treatment group, assessed the extent of hypoxia and vascular damage at low magnification using fluorescence microscopy. The coded tumor sections were categorized as having no vascular damage (0%), 1–10, 10–25, 25–50, 50–75 or . 75% vascular damage, compared with sham treated tumors. In separate sections and tumors, the total hypoxic area was categorized as being , 10; 10–25, 25–50, 50–75 or . 75% of the tumor section. Joint conclusions were made subsequently (see text). Tumor periphery was defined as the outer 50% of the tissue section radius. c Immunostaining for pimonidazole adducts. d Immunostaining for von Willebrand factor.

2), and there were large avascular areas in the tumor interior (Fig. 1f). Yet, small areas typically remained in the tumor periphery where intact blood vessels could be found. 3.4. Immunostaining for tumor hypoxia

Fig. 2. Time-related changes in tumor pO2 after injecting CA-4 (50 mg/kg i.p.) or 0.9% NaCl (1 ml/kg i.p., sham treatment). Values are mean ^ SEM.

those in the normal host tissue (Table 3). Continuous measurements in control animals demonstrated a minor and not significant decline in tumor pO2 from a mean of 30.2 to a mean of 23.7 mmHg over 3 h (Fig. 2). CA-4 decreased tumor pO2 significantly from a mean of 36.1 to a mean of 17.6 mmHg over a similar time span (P ¼ 0:01), but tumor oxygenation was not significantly different in the two groups 3 h postinjection. Arterial blood oxygenation was not significantly altered during anesthesia (Table 3).

4. Discussion

Whereas tumor hypoxia could not be detected in sham treated tumors, small, spotted areas of tumor hypoxia were seen 3 h after CA-4 exposure (Fig. 1g). Tumors exposed to hyperthermia alone exhibited more tumor hypoxia (Fig. 1h), with pimonidazole staining in 25–50% of the section area (Table 2). Pretreating the animals with CA-4 before hyperthermia yielded, however, no additional increase in tumor hypoxia when assessed 3 h after the heating session. 3.5. Tumor oxygen tension The pO2-values in the BT4An tumor base approached

Various treatment modalities exert part of their therapeutic effect through anti-vascular mechanisms [8], and by altering the dose and schedule of common chemotherapeutic drugs, one can achieve selective targeting of the endothelial cell population [4,22]. The tubulin disrupting agent, CA4, facilitated vascular damage in xenotransplanted as well as spontaneous murine tumors [7,15,19], and the drug is currently in phase I clinical trials [34]. However, the tumor growth delay induced by CA-4 is short or absent in most tumor models [5,15,16,20,23]. Combination strategies are therefore warranted to obtain prolonged tumor regres-

Table 3 Tissue oxygenation and arterial blood gases before and 3 h after CA-4 or sham treatment Group a

Control (0) Control (13 h) CA-4 (0) CA-4 (13 h) a b c d

BT4An tumor base b

Subcutis b

Mean pO2

Median pO2

Mean pO2

Median pO2

Mean pO2

Mean pCO2

Mean pH

30 ^ 3.6 24 ^ 3.3 36 ^ 5.4 18 ^ 1.9 d

29 25 38 17 d

37 ^ 5.3 38 ^ 3.5 42 ^ 2.1 39 ^ 4.2

34 38 44 37

8.0 ^ 0.6 7.8 ^ 0.5 9.8 ^ 0.9 9.0 ^ 0.2

6.1 ^ 0.4 6.4 ^ 0.2 6.0 ^ 0.4 5.6 ^ 0.2

7.4 ^ 0.0 7.3 ^ 0.0 7.4 ^ 0.0 7.4 ^ 0.0

CA-4: 50 mg/kg i.p.; control: 0.9% NaCl 1 ml/kg i.p. Mean ^ SEM and median pO2 (mmHg) of six animals. Mean pO2 (kPa) ^ SEM of six animals. P ¼ 0:01.

Arterial blood gases c

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sions, and cytotoxic drugs, as well as radiation and hyperthermia, augment the anti-tumor activity of CA-4 [5,13,16,20,25]. In the s.c. BT4An rat glioma, tumor growth was not affected by CA-4 (50 mg/kg) [13], but 3 h after drug administration, histology revealed vascular stasis, edema in the tumor endothelium and leukocytes surrounding the blood vessels [12]. These are indications of an anti-vascular effect mediated by the drug, although less potent than in murine tumor models [7,19]. CA-4 has a wide therapeutic window in mice with an estimated maximum tolerated dose (MTD) above 1000 mg/kg i.p. [7]. The toxicity profile is less favorable in rats, where serious side effects develop at doses above 50 mg/kg i.p. [13,23]. Thus, the lower dosage may explain the inefficiency of CA-4 in BT4An rat tumors. Using confocal microscopy, we found slight, architectural changes in the tumor vasculature subsequent to CA-4 exposure. The blood vessels were dilated with perivascular glow, indicating vascular damage (Fig. 1d). Furthermore, four out of six tumors had a minor central area where the vasculature was totally disrupted. This suggests that CA-4 exerted a slight anti-vascular action in these tumors. The effect of local hyperthermia is highly dependent on the temperatures obtained, and at tumor temperatures above 42–438C (30–60 min), vascular damage generally develops [39]. In the BT4An tumors, hyperthermia alone (448C, 60 min) yielded only moderate vascular damage (Table 2) and a brief growth delay [13]. Large temperature variations exist within waterbath-heated tumors, and regrowth commonly occurs from the tumor base where minimal temperatures are found [18,27]. Temperatures of 41.2–41.88C were measured in the BT4An tumor base when hyperthermia (448C, 60 min) was administered alone [13], and thus insufficient tumor heating was achieved. CA-4 reduced tumor blood flow by 50% in the BT4An tumors, and the tumor temperatures were elevated to 43.1–43.38C when CA-4 was injected 3 h before heating [13]. This combination therapy caused extensive hemorrhagic necrosis and a prolonged growth delay in the BT4An neoplasms [12], and using confocal microscopy, we found massive vascular damage in the C3HT-tumors (Fig. 1f and Table 2). Lower tumor temperatures [13] and less vascular damage (Fig. 1e and Table 2) was inflicted if CA-4 and hyperthermia was coadministered (C0HT-group). CA-4 thus increased the antivascular activity of local hyperthermia by facilitating higher tumor temperatures in the C3HT-group. Based on mode of action, CA-4 could also have enhanced thermal damage in the C3HT-group by inducing a timedependent deterioration of the tumor microenvironment. It is well known that tumor hypoxia and acute lowering of the intracellular pH increase the efficiency of hyperthermia [40]. CA-4 facilitated such alterations of the internal milieu in two rodent tumor models [3,19]. Tumor oxygenation, estimated by interstitial oxygen probes, was reduced by CA-4 in the BT4An tumor base (Fig. 2), but the mean pO2 readings remained above values generally considered hypoxic [41].

Subcutaneous foot tumors are generally less hypoxic than tumors transplanted to other locations [1], and pO2-values comparable to ours were obtained in sham-treated DS-sarcomas implanted s.c. onto the foot dorsum [45]. The mean pO2 of 30.2–36.1 mmHg prior to therapy therefore suggests that the BT4An tumors are well oxygenated. Alternatively, the results may reflect that the Clark-probes were situated in close proximity to the normal tissue in the tumor bed. Using the pimonidazole technique, we examined the midtumor areas to assess whether CA-4 induced more hypoxia in this tumor segment. We found small, spotted areas of tumor hypoxia 3 h after CA-4 injection (Fig. 1g and Table 2), which indicates a minor and heterogeneous disturbance of tumor metabolism also in the tumor center. It therefore seems unlikely that CA-4 alone induced tumor hypoxia to such an extent that thermal damage was enhanced. The distribution of circulating hypoxia markers like pimonidazole could have been hampered by vascular damage induced by CA-4 and hyperthermia. Pimonidazole was therefore injected 2 h after concluding the allocated treatment to forestall evolving vascular shutdown in the tumor tissue, whereas the assessment of vascular damage was not undertaken until 15 h after therapy (Table 2). The strong and extensive pimonidazole-staining that was observed 3 h after hyperthermia or combination therapy (Fig. 1h) indicates that tumor perfusion was sufficient for distribution of the hypoxia marker. To test this hypothesis further, we examined the vasculature of the same tumors that were taken out in the pimonidazole study, and although the blood vessels looked dilated and less tortuous, vascular disruption was not evident 3 h after treatment (data not shown). It was also recently shown that the entrance of 99m Tc-labeled HL-91 (Prognox), another circulating hypoxia marker, was not precluded by a previous blood flow reduction induced by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) [38]. Secondary tumor cell death is a characteristic hallmark of anti-vascular therapy, as the induced vascular shutdown causes severe hypoxia and a deprived nutrient supply to the tumors [8,35]. When BT4An tumors were taken out after hyperthermia or combination therapy, they displayed large, confluent areas of hypoxia (Fig. 1h and Table 2), probably caused by an evolving vascular damage inflicted by the therapy. Tumor hypoxia was not enhanced by combining hyperthermia with CA-4, although the combination treatment yielded more vascular damage than heating alone (Table 2). This apparent discrepancy is probably due to the assessment of tumor oxygenation at an earlier time point (3 h) than the study of vascular morphology (15 h). However, the possible interference between vascular damage and pimonidazole-distribution precluded a simultaneous evaluation of these parameters after 15 h. It still seems reasonable to conclude that the induced wide-spread hypoxia in the tumor tissue after hyperthermia or combination therapy may have contributed to the observed tumor growth delays.

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The cytoskeleton of the endothelial or the malignant cell population is a potential joint target for hyperthermia and CA-4. Tubulin disrupting drugs, as well as hyperthermia, interfere with the microtubular assembly process [6,31], and the combination of the two treatment modalities has been suggested as a promising strategy [9]. By immunostaining for b-tubulin, it was demonstrated that CA-4 targets the cytoskeleton of endothelial cells [15], and we observed a disrupted microtubular network in BT4Cn cells subsequent to drug exposure in vitro (Fig. 1b). If a similar response was mediated in vivo, CA-4 could augment hyperthermic cytotoxicity, either through an enhanced disruption of the cytoskeleton or by halting tumor cells while in mitosis [10]. When using flow cytometry, we found no interference with the cell cycle distribution 3 h after injecting CA-4 (Table 1). Cytotoxic drugs targeting check-points within the cell cycle have the ability to accumulate tumor cells in different cell phases [37,42,49], but the clinical relevance of this has been debated [43]. The proliferation index of solid tumors is commonly below 10% [21,43], and few neoplastic cells are thus available for phase specific drugs like the tubulin disrupting agents during a 3-h interval. It therefore seems unlikely that displacement of the cell cycle distribution by CA-4 was of major importance for the interaction with hyperthermia. Nevertheless, a combined disruption of the cytoskeleton inflicted by the two treatment modalities cannot be excluded. We conclude that the anti-vascular activity of CA-4 alone was low in the current tumor model, and although the tumor cell cytoskeleton was targeted by the drug in vitro, the cell cycle distribution was not affected in vivo. CA-4 augmented the anti-vascular activity of local hyperthermia in BT4An neoplasms, and if an optimal timing of drug and heating was applied, massive vascular damage developed in the tumors. This investigation thus elucidates how CA-4 and hyperthermia interact to facilitate tumor response in a tumor resistant to the anti-vascular properties of CA-4 alone.

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Acknowledgements The authors gratefully acknowledge the technical assistance of F. Rykkja. We wish to express our gratitude to professor J. Saraste (Bergen, Norway) for important advice on immunostaining techniques, and to T. Froystein (Bergen, Norway) for expert assistance on 3D image reconstructions. This investigation was supported by Oxigene Inc. (Lund, Sweden), L. Meltzers Høyskolefond and grants from The Norwegian Cancer Society.

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