Molecular Cancer Therapeutics Molecular Diagnostics in Cancer Therapeutic Development: Fulfilling the Promise of Personalized Medicine Targeting the PI3-Kinase Pathway in Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Q.
Right arrow Articles by Yuan, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Q.
Right arrow Articles by Yuan, F.
Mol Cancer Ther. 2004;3:1311-1317
© 2004 American Association for Cancer Research

Targeting tumor microvessels using doxorubicin encapsulated in a novel thermosensitive liposome

Qing Chen1, Sheng Tong1, Mark W. Dewhirst2 and Fan Yuan1

Departments of 1 Biomedical Engineering and 2 Radiation Oncology, Duke University, Durham, North Carolina

Requests for reprints: Fan Yuan, Department of Biomedical Engineering, Duke University, Box 90281, Durham, NC 27708. Phone: 919-660-5411; Fax: 919-684-4488. E-mail: fyuan{at}duke.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Liposomal drugs accumulate only in perivascular regions in tumors after i.v. injection. Thus, they cannot kill tumor cells in deeper tissue layers. To circumvent this problem, we investigated effects of doxorubicin (DOX) encapsulated in a lysolecithin-containing thermosensitive liposome (LTSL) on tumor microcirculation because damaging microvessels would stop nutrient supply to deeper tumor cells. We used LTSL-DOX in combination with hyperthermia to treat a human squamous carcinoma xenograft (FaDu) implanted in dorsal skinfold chambers in nude mice. Before the treatment, the RBC velocity in tumors was 0.428 ± 0.037 mm/s and the microvascular density was 3.93 ± 0.44 mm/mm2. At 24 hours after the treatment, they were reduced to 0.003 ± 0.003 mm/s and 0.86 ± 0.27 mm/mm2, respectively. The same treatment, however, caused only 32% decrease in the RBC velocity and no apparent change in microvascular networks in normal s.c. tissues over the same period. LTSL and LTSL-DOX alone had no effect on tumor microcirculation, and LTSL plus hyperthermia caused only a transient decrease in the RBC velocity in tumors. At 24 hours after treatments, tumor microcirculation in all these control experiments was insignificantly different from that before the treatments. We also examined apoptosis of cells in tumors at different time points after LTSL-DOX plus hyperthermia treatment and observed few apoptotic cells in tumor microvessels. In conclusion, the rapid release of DOX during hyperthermia could make the drug to shutdown tumor blood flow while have only minor effects on normal microcirculation in s.c. tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Anticancer drugs encapsulated in liposomes accumulate preferentially in solid tumors due to the leakiness of tumor microvessels (1–7), and the release of the drugs from liposomes can be controlled either chemically or physically (8–10). Despite these advantages, liposomal drugs have not yet led to a significant improvement in the clinical outcome in cancer treatment (11–13). The lack of improvement is likely to be due to the low concentration of free drugs in tumor tissues, although the total concentration of drugs (i.e., free plus liposome-associated drugs) may not be low.

Liposomes are nanoparticles (~100 nm in diameter). They can accumulate only in perivascular regions in tumors after i.v. injection (7, 14, 15). The smaller drug molecules released from liposomes may penetrate into deeper tissue layers, but the penetration depth is often limited. This is because (a) the interstitial concentration gradient of free drugs is greater in the direction toward the microvessel wall than away from it and (b) many anticancer drugs bind strongly to tumor tissues. For example, >80% of doxorubicin (DOX) molecules in tumors are bound to proteins, membranes, and nucleic acids (16, 17). The binding hinders or even prevents interstitial transport of drugs.

To circumvent the penetration problem, we proposed to use liposomal drugs to target endothelial and tumor cells in perivascular regions because the damage of these cells may shutdown tumor blood flow, which in turn will kill deeper tumor cells through reducing the nutrient supply. To significantly damage perivascular cells, free drugs must be highly toxic and achieve an adequately high concentration in the vicinity of these cells. In general, the concentration depends on the difference between the rate of drug release from liposomes and the rate of drug clearance through the microcirculation. Thus, one can either reduce the clearance rate or increase the release rate to increase the local concentration of free drugs (8–10). The approach used in this study was to use a lysolecithin-containing thermosensitive liposome (LTSL) to rapidly release drugs in heated tumor tissues. LTSL can completely release its DOX content within 20 seconds at 42°C (16, 18).3 As a result, the peak concentration of free DOX in solid tumors can be ~30-fold higher than that in the same tissue after i.v. injection of free DOX (18) and at least 2-fold higher than the peak concentration after i.v. injection of DOX encapsulated in other thermosensitive liposomes (16). DOX at such a concentration is able to cure a mouse tumor in vivo, which cannot be eliminated by the same drug injected i.v. or delivered through other thermosensitive liposomes (16, 19).

To investigate the effects of DOX released from LTSL on tumor and normal microcirculation, we quantified RBC velocities, microvascular densities, and microvessel diameters in a human pharyngeal squamous carcinoma xenograft (FaDu) and the normal s.c. tissues in nude mice before and after the combined treatment with LTSL-DOX and hyperthermia. We also treated s.c. tissues with empty LTSL plus hyperthermia and FaDu tumors with either LTSL plus hyperthermia, LTSL without hyperthermia, or LTSL-DOX without hyperthermia. We observed that DOX released from LTSL during hyperthermia could shutdown blood flow in tumors within 24 hours after treatment but had only minor effects on the microcirculation in s.c. tissues. Other treatments caused at most a minor and transient decrease in the blood flow and the microvascular density in tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of LTSL-DOX
The LTSL was prepared using dipalmitoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a molar ratio of 90:10:4 (19). It was ~100 nm in diameter. DOX (Sigma Chemical Co., St. Louis, MO) was loaded into the liposome at a final concentration of 2 mg/mL using a pH gradient method (20).

Preparation of Fluorescently Labeled RBC
Mouse RBC were labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes, Eugene, OR) using a technique described previously (21, 22). Briefly, RBC were isolated from the blood by successive washing and centrifugation. RBC pellets (100 µL) were mixed with 100 µL DiI stock solution (0.5 mg DiI/mL ethanol) in 10 mL PBS and incubated in the dark at room temperature for 30 minutes. At the end of incubation, the cells were washed and centrifuged several times and then suspended in 1 mL PBS at a final concentration of 100 µL DiI-RBC/mL. The DiI-RBC were used within 2 days after the labeling.

Dorsal Skinfold Chamber and Tumor Models
Female athymic nude mice with a body weight of 20 to 26 g were purchased from the National Cancer Institute (Bethesda, MD) and anesthetized through an i.p. injection of a cocktail of 80 mg ketamine and 10 mg xylazine per kg body weight. Titanium window chambers were surgically implanted on the dorsal skin flap of nude mice (7, 14, 23, 24). After surgery, animals were allowed to recover for 48 hours before tumor cells were implanted into the chambers. All procedures were done under aseptic conditions. After surgery, animals were kept ad libitum in a warm room with food and water and a 12-hour light/dark cycle.

A human pharyngeal squamous carcinoma cell line (FaDu) was used to prepare two tumor models. One was the mouse dorsal skinfold chamber model in which 10 µL cell suspension (2 x 105) was implanted onto the center of the chambers. Angiogenesis in tumors could be observed on days 5 to 7. Since then, tumors started to grow progressively and reached ~3 mm in diameter at 10 to 12 days after tumor cell implantation. Tumors at this size were used in our experiments.

Another tumor model was prepared through s.c. inoculation of 106 tumor cells on the right hind limb of nude mice. The s.c. tumors were used in our experiments when they reached ~5 mm in diameter.

Microcirculation Experiments
The animal was anesthetized using the same method as that described above and placed on a temperature-controlled microscope stage to maintain the body temperature throughout the experiment. The dorsal skinfold chamber was fixed onto a hyperthermia stage designed specially for this kind of experiments (23–25). DiI-RBC (0.05 mL) were injected i.v. through the tail vein at 5 minutes before experiments.

In the experiments, the tumor was treated with one of the four protocols. (a) LTSL-DOX-HT: hyperthermia plus i.v. injection of LTSL-DOX (DOX dose: 5 mg/kg body weight; n = 6); (b) LTSL-HT: hyperthermia plus i.v. injection of empty LTSL (at the same lipid concentration as in the first group; n = 6); (c) LTSL-DOX-nHT: i.v. injection of LTSL-DOX without hyperthermia (DOX dose: 5 mg/kg body weight; n = 3); and (d) LTSL-nHT: i.v. injection of LTSL without hyperthermia (at the same lipid concentration as in the first group; n = 3). In the first two protocols, the tumors were first heated for ~2 minutes before the liposome injection in order for the tumor and surrounding tissues to reach a thermal equilibrium with the heating device maintained at 42°C. To evaluate the effects of LTSL-DOX and hyperthermia on normal microvessels in the skin, the dorsal skinfold chambers without tumors were treated with either the first (i.e., LTSL-DOX-HT) or the second (i.e., LTSL-HT) protocol as described above (n = 5 in each group).

At 0.5 hours before and 0, 6, and 24 hours after each treatment, tumor or normal microcirculation was examined under a fluorescence microscope with either transillumination or epi-illumination. For epi-illumination, a filter set for rhodamine was used. The microcirculation in five different areas of a tumor was recorded onto videotapes using a 20x objective and a SIT camera (C2400, Hamamatsu Photonics, Hamamatsu, Japan) connected to a S-VHS video recorder (BV-1000, Mitsubishi Electronics, Tokushima, Japan). The recorded videos were analyzed offline for determining RBC velocity and the density and the diameter of microvessels.

Offline Analysis of RBC Velocity and Microvascular Density and Diameter
The videos of microcirculation were digitized using an image acquisition software (Osprey Multimedia Capture, version 5.0, Microsoft Corp., Redmond, WA). The DiI-RBC velocity in a microvessel was determined by the dual-window technique (26) based on a MATLAB program developed in-house. In our experiments, only the microvessels that satisfied the following criteria were selected for velocity measurement: (a) the microvessel segment was straight and without branches, (b) DiI-RBC could be reliably tracked in the microvessels, and (c) the cross-correlation coefficient was >0.70. The images from the digitized videos of tumor microcirculation in transilluminated regions were also used to measure the microvascular density and diameter. In each image, all microvessels were traced by a computer mouse using Image-Pro Plus (version 4.1, Media Cybernetics, Carlsbad, CA) and the density was defined as the total length of the microvessels per unit area of the image. The diameters of all tumor vessels in this image were also measured using a software tool provided in the Image-Pro Plus and the mean diameter in the image was calculated. The averages of the length densities and mean diameters from five different images were defined as the density and the diameter of microvessels in the tumor, respectively. Unless indicated specifically, all data in this article are reported as the means ± SE from different tumors.

Apoptosis of Endothelial and Tumor Cells
Tumors from the right hind limb in mice were treated with either the LTSL-DOX-HT or the LTSL-HT protocol as described above. During the hyperthermia treatment, the limb was wrapped with a plastic bag to prevent direct contact of the skin to the heated water. The wrapped limb was immersed into the water maintained at 42°C for 1 hour. The tumors were removed from animals at 1, 6, and 24 hours after treatment, fixed with 10% formalin solution, embedded in paraffin, and sectioned with a microtome. For labeling microvessels, tumor sections were deparaffinized, rehydrated, washed in PBS, pretreated with proteinase K (20 µg/mL, Sigma Chemical) in Tris buffer (10 mmol/L, pH 7.6) at 37°C for 15 minutes, and stained with a rabbit antibody against human von Willebrand factor (DAKO, Carpinteria, CA). Then, all sections were incubated with the biotinylated secondary antibody for 30 minutes according to the protocol of Vectastain ABC-AP kit (Vector Laboratories, Burlingame, CA). The secondary antibody in the sections was colored by the Vector Red (Alkaline Phosphatase Substrate Kit I, Vector Laboratories).

The terminal deoxynucleotidyl transferase–mediated UDP end labeling (TUNEL) assay was done using In situ Cell Death Detection Kit-Fluorescein (Roche Applied Science, Indianapolis, IN) on the tissue sections already stained with the antibody against human von Willebrand factor. In addition, a section of tumor tissues treated with DNase (IU/µL) at room temperature for 15 minutes was used as a positive control. All tissue sections including the positive control were incubated with TUNEL reaction mixture containing nucleotide mix and terminal deoxynucleotidyl transferase enzyme in the dark at 37°C for 60 minutes. The sections were mounted with Vetashield mounting medium (Vector Laboratories) and the apoptotic cells were examined under a fluorescence microscope equipped with a 40x objective. In each field under the microscope, two images were taken with a digital camera (Zeiss AxioCam, Carl Zeiss, Thornwood, NY). They were through the filter sets for fluorescein and rhodamine, respectively. The two images from the same field were overlaid with the Image-Pro Plus software, with red indicating microvessels and green indicating apoptotic cells. Apoptotic endothelial cells were shown as yellow dots.

Statistics
Man-Whitney U test was used to determine the difference between different groups. The difference was considered to be statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RBC Velocities in Tumor and Normal Tissues
Tumors in dorsal skinfold chambers were treated with one of the four protocols as described in Materials and Methods. They are (a) LTSL-DOX-HT: hyperthermia plus i.v. injection of LTSL-DOX; (b) LTSL-HT: hyperthermia plus i.v. injection of empty LTSL; (c) LTSL-DOX-nHT: i.v. injection of LTSL-DOX without hyperthermia; and (d) LTSL-nHT: i.v. injection of LTSL without hyperthermia. Before any treatment, there was no significant difference in the distribution of RBC velocities among different tumors. The RBC velocity (mm/s) was decreased from 0.428 ± 0.037 at 0.5 hour before treatment to 0.043 ± 0.026, 0.012 ± 0.012, and 0.003 ± 0.003 at 0, 6, and 24 hours, respectively, after the LTSL-DOX-HT treatment (Fig. 1). The medians of those data were 0.431, 0.019, 0, and 0, respectively. In LTSL-HT-treated tumors, the RBC velocity was decreased transiently after the treatment, but the amount of decrease was much less than that in LTSL-DOX-HT-treated tumors (see Fig. 1). At 24 hours after LTSL-HT treatment, the RBC velocity recovered to 0.385 ± 0.049 mm/s, which was insignificantly different from the pretreatment level (Fig. 1). LTSL-DOX-nHT or LTSL-nHT treatment had minimal effects on the RBC velocity in tumors (Fig. 1).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. RBC velocity in tumors treated with either LTSL-DOX-HT (n = 6), LTSL-HT (n = 6), LTSL-DOX-nHT (n = 3), or LTSL-nHT (n = 3). RBC velocity was measured at either 0.5 hour before or 0, 6, and 24 hours after the treatment. A, distribution of RBC velocity in LTSL-DOX-HT- and LTSL-HT-treated tumors. Symbols, RBC velocity in a specific microvessel. B, average RBC velocities in tumors treated with one of the four protocols. Points, mean; bars, SE. *, P < 0.01, LTSL-DOX-HT group is statistically different from LTSL-HT group.

 
In contrast to blood flow in tumors, the RBC velocity in normal tissues was much less sensitive to LTSL-DOX-HT treatment (Fig. 2). The RBC velocities (mm/s) were reduced only to 0.208 ± 0.007 and 0.174 ± 0.018 at 6 and 24 hours, respectively, after the treatment. There was no significant change in the RBC velocity after LTSL-HT treatment (Fig. 2).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. RBC velocity in normal s.c. tissues treated with either LTSL-DOX-HT (n = 5) or LTSL-HT (n = 5). Labels as in Fig. 1. A, distribution of RBC velocity in LTSL-DOX-HT- and LTSL-HT-treated tissues. Symbols, RBC velocity in a specific microvessel. B, average of the RBC velocities as in A. Points, mean; bars, SE. *, P < 0.01, LTSL-DOX-HT group is statistically different from LTSL-HT group.

 
We also noticed that the baseline velocity before any treatment in normal tissues was ~60% of that in tumors (Figs. 1 and 2). If normalized by the baseline values, the RBC velocities at 24 hours after LTSL-DOX-HT treatment were 0.677 and 0.007 in normal and tumor tissues, respectively.

Microvascular Density and Vessel Diameter in Tumors
The diameter of tumor microvessels was increased after LTSL-DOX-HT treatment (Fig. 3A). The increase was statistically significant at 6 and 24 hours after the treatment. The microvascular density (mm/mm2) was 3.93 ± 0.44 before the treatment (Fig. 3B) and decreased to 0.86 ± 0.27 at 24 hours after treatment (Fig. 3B). The medians of those data were 3.60 and 0.64, respectively. The amount of decrease was statistically significant. In addition, we observed blood flow stasis and severe hemorrhage immediately after treatment and no microvessels with blood flow in five of six tumors at 6 and 24 hours after the treatment.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Average microvessel diameter (A) and microvascular density (B) in tumors treated with either LTSL-DOX-HT (n = 6), LTSL-HT (n = 6), LTSL-DOX-nHT (n = 3), or LTSL-nHT (n = 3). Labels as in Fig. 1. Points, mean; bars, SE. *, P < 0.05, **, P < 0.01, LTSL-DOX-HT group is statistically different from LTSL-HT group.

 
In LTSL-HT-treated tumors, there was no significant change in the vessel diameter and microvascular density (Fig. 3). Only minor hemorrhage was observed in these tumors. LTSL-DOX-nHT and LTSL-nHT treatments had minimal effects on microvascular density (Fig. 3B). In the normal s.c. tissues, no hemorrhage was observed and the microvascular networks was apparently intact after the LTSL-DOX-HT treatment.

Apoptotic Cells in Tumors
Apoptotic cells and tumor microvessels were labeled with TUNEL and an antibody against von Willebrand factor, respectively. We observed very few spots to be both TUNEL and von Willebrand factor positive at 1, 6, and 24 hours, respectively, after either LTSL-DOX-HT or LTSL-HT treatment (Fig. 4), indicating that few apoptotic cells in tumors were endothelial cells.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Double staining of tumor microvessels and apoptotic cells with anti–von Willebrand factor antibody and TUNEL, respectively, at 1, 6, and 24 hours after treatment with either LTSL-DOX-HT or LTSL-HT. Green dots, apoptotic cells; red dots, tumor microvessels. As a result, apoptotic endothelial cells were yellow. The width of all images was ~250 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We quantified the RBC velocity, microvascular density, and microvessel diameter in normal and tumor tissues after they were treated with different protocols. We observed that LTSL-DOX-HT treatment could shutdown blood flow, result in a significant decrease in the microvascular density, and cause a vessel dilation in tumor tissues. However, the same treatment had only minor effects on the microcirculation in normal s.c. tissues.

Mechanisms of the blood flow shutdown in tumors remain to be determined. Previous studies have shown that the rapid release of DOX from LTSL during hyperthermia can largely increase the concentration of free DOX in perivascular regions and lead to a cure of FaDu tumors in mice (16, 19). When DOX released slowly from other temperature sensitive liposomes, it cannot stop the growth of the same tumor in mice (19), indicating that tumor microcirculation is not severely impaired after the treatment. We observed that LTSL, LTSL-DOX, or LTSL-HT alone caused at most a minor and transient changes in the RBC velocity in tumors. Taken together, these experimental observations suggest that the shutdown of tumor blood flow after LTSL-DOX-HT treatment is caused by free DOX at a very high concentration in the vicinity of vascular endothelial cells.

DOX can damage both endothelial and tumor cells (27, 28). The death of endothelial cells may directly cause intravascular blood coagulation (29, 30), which in turn will reduce the blood flow. The death of tumor cells, on the other hand, may indirectly cause intravascular blood coagulation because it reduces the production of vascular endothelial growth factor, an endothelial cell survival factor (31, 32). Although we did not investigate, in the present study, whether it was the death of tumor or endothelial cells that caused the shutdown of blood flow, previous studies have shown that the killing of tumor cells alone does not result in a significant decrease in the RBC velocity within 24 hours (32) and that the neutralization of vascular endothelial growth factor in tumors has minimal effects on the microvascular density (33). Based on these data, we might conclude that the shutdown of tumor blood flow observed in this study was caused by the direct effects of DOX on endothelial cells. Further studies will be done to investigate molecular mechanisms of DOX/endothelial cell interactions.

The RBC velocity in LTSL-HT-treated tumors was decreased by ~25% at 0 and 6 hours after treatment (see Fig. 1). This decrease was caused by hyperthermia treatment (34, 35), because the RBC velocity was not changed in LTSL- and LTSL-DOX-treated tumors. At 24 hours after LTSL-HT treatment, the RBC velocity recovered to the pretreatment level (see Fig. 1). The same phenomenon has been observed in a previous study in which the tumor blood flow was reduced by ~50% after a mild hyperthermia treatment (40–41°C for 40 min) and returned to the pretreatment value within 72 hours (36). However, the reduction in the blood flow can be irreversible if the temperature is higher than 43°C (36). In this case, tumor microvessels are severely damaged, which causes the shutdown of blood flow (36).

Figure 3 shows that there is a progressive reduction in the microvascular density in tumors treated with LTSL-DOX-HT. It was likely to be caused by the shutdown of blood flow. Without blood supply, all cells within microvessels and in perivascular tissues will die. The RBC in dead tissues would eventually lose their membrane integrity and the hemoglobin content. In our experiments, the measurement of microvascular density was based on the difference in green light absorption between hemoglobin and other molecules. The microvessels without intact RBC were invisible under the light microscope.

In addition to the blood flow and the microvascular density, we observed that microvessels were dilated in tumors treated with LTSL-DOX-HT but were unaffected in tumors treated with LTSL-HT (Fig. 3). Although previous studies had shown that hyperthermia treatment might result in vessel dilation in some tumor models under specific conditions (34–39), the results shown in Fig. 3 indicated that the vessel dilation in our experiment was caused mainly by free DOX released from LTSL. The mechanisms of vessel dilation are not completely understood. It is possible that the vessel dilation is a passive consequence in response to the damage of endothelial cells, pericytes, and tumor cells in perivascular regions. The damaged cells cannot provide sufficient mechanical support to balance the pressure difference across the microvessel wall.

Normal microvessels were less sensitive to LTSL-HT and LTSL-DOX-HT treatments compared with tumor microvessels (see Figs. 1 and 2). This was consistent with the results in previous studies in which vascular endothelial cells and blood flow in tumors were more thermosensitive than those in normal tissues (40). This difference between normal and tumor tissues might be caused by three factors. First, tumor microvessels are more permeable to liposomes than normal microvessels (6, 7, 14). Hyperthermia can further increase the microvascular permeability in tumors while having minor effects on liposome extravasation in normal tissues (25). As a result, the concentrations of LTSL-DOX and free DOX in tumors are much higher than those in normal tissues. Second, tumor microvessels lack basement membrane and other supporting structures (41). They are immature and remodel constantly during tumor growth (42). Third, tumor microvessels contain more proliferating endothelial cells than normal microvessels due to active angiogenesis and remodeling processes in tumors. Proliferating endothelial cells are more sensitive to thermal injury (40) and DOX treatment (43) than resting cells. The factors described above may act alone or in concert, making tumor microvessels prone to damage during LTSL-HT or LTSL-DOX-HT treatment. The difference in thermosensitivity between normal and tumor tissues provides an opportunity for specific reduction in tumor blood flow without causing significant, adverse effects in normal tissues.


   Footnotes
 
Grant support: NIH grant CA87630.

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.

3 Wright AM, Needham D. Evaluation of LTSL-doxorubicin loading and release performance characteristics, manuscript in preparation. Back

Received 3/ 8/04; revised 7/29/04; accepted 8/20/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Papahadjopoulos D, Allen TM, Gabizon A, et al. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc Natl Acad Sci U S A 1991;88:11460–4.[Abstract/Free Full Text]
  2. Lasic DD, Papahadjopoulos D. Liposomes revisited. Science 1995;267:1275–6.[Free Full Text]
  3. Gregoriadis G. Liposome technology. Vol. I, II, III. Boca Raton: CRC Press; 1993.
  4. Ranson M, Howell A, Cheeseman S, et al. Liposomal drug delivery. Cancer Treat Rev 1996;22:365–79.[CrossRef][Medline]
  5. Tardi PG, Boman NL, Cullis PR. Liposomal doxorubicin. J Drug Target 1996;4:129–40.[Medline]
  6. Jain RK. Delivery of molecular and cellular medicine to solid tumors. Microcirculation 1997;4:1–23.[Medline]
  7. Yuan F, Leunig M, Huang SK, et al. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft. Cancer Res 1994;54:3352–6.[Abstract/Free Full Text]
  8. Yatvin MB, Weinstein JN, Dennis WH, et al. Design of liposomes for enhanced local release of drugs by hyperthermia. Science 1978;202:1290–3.[Abstract/Free Full Text]
  9. Needham D, Dewhirst MW. The development and testing of a new temperature-sensitive drug delivery system for the treatment of solid tumors. Adv Drug Deliv Rev 2001;53:285–305.[CrossRef][Medline]
  10. Hong MS, Lim SJ, Oh YK, et al. pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system. J Pharm Pharmacol 2002;54:51–8.[CrossRef][Medline]
  11. Batist G, Ramakrishnan G, Rao CS, et al. Reduced cardiotoxicity and preserved antitumor efficacy of liposome-encapsulated doxorubicin and cyclophosphamide compared with conventional doxorubicin and cyclophosphamide in a randomized, multicenter trial of metastatic breast cancer. J Clin Oncol 2001;19:1444–54.[Abstract/Free Full Text]
  12. Harris L, Batist G, Belt R, et al. Liposome-encapsulated doxorubicin compared with conventional doxorubicin in a randomized multicenter trial as first-line therapy of metastatic breast carcinoma. Cancer 2002;94:25–36.[CrossRef][Medline]
  13. Harrington KJ, Lewanski CR, Northcote AD, et al. Phase I-II study of pegylated liposomal cisplatin (SPI-077) in patients with inoperable head and neck cancer. Ann Oncol 2001;12:493–6.[Abstract/Free Full Text]
  14. Wu NZ, Da D, Rudoll TL, et al. Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor tissue. Cancer Res 1993;53:3765–70.[Abstract/Free Full Text]
  15. Hobbs SK, Monsky W, Yuan F, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Nat Acad Sci U S A 1998;95:4607–12.[Abstract/Free Full Text]
  16. Kong G, Anyarambhatla G, Petros WP, et al. Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release. Cancer Res 2000;60:6950–7.[Abstract/Free Full Text]
  17. Greene R, Collins J, Jenkins J, et al. Plasma pharmacokinetics of Adriamycin and Adriamycinol: implications for the design of in vitro experiments and treatment protocols. Cancer Res 1983;47:3417–21.
  18. Anyarambhatla GR, Needham D. Enhancement of the phase transition permeability of DPPC liposomes by incorporation of MPPC: a new temperature-sensitive liposome for use with mild hyperthermia. J Liposome Res 1999;9:491–506.
  19. Needham D, Anyarambhatla G, Kong G, et al. A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model. Cancer Res 2000;60:1197–201.[Abstract/Free Full Text]
  20. Mayer LD, Bally MB, Cullis, PR. Uptake of Adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta 1986;857:123–6.[Medline]
  21. Wu NZ, Braun RD, Gaber MH, et al. Simultaneous measurement of liposome extravasation and content release in tumors. Microcirculation 1997;4:83–101.[Medline]
  22. Unthank JL, Lash JM, Nixon JC, et al. Evaluation of carbocyanine-labeled erythrocytes for microvascular measurements. Microvasc Res 1993;45:193–210.[CrossRef][Medline]
  23. Kong G, Braun RD, Dewhirst MW. Characterization of the effect of hyperthermia on nanoparticle extravasation from tumor vasculature. Cancer Res 2001;61:3027–32.[Abstract/Free Full Text]
  24. Yuan F, Leunig M, Berk DA, et al. Microvascular permeability of albumin, vascular surface area, and vascular volume measured in human adenocarcinoma LS174T using dorsal chamber in SCID mice. Microvasc Res 1993;45:269–89.[CrossRef][Medline]
  25. Kong G, Braun RD, Dewhirst MW. Hyperthermia enables tumor-specific nanoparticle delivery: effect of particle size. Cancer Res 2000;60:4440–5.[Abstract/Free Full Text]
  26. Lee TQ, Schmid-Schonbein GW, Zweifach BW. The application of an improved dual-slit photometric analyzer for volumetric flow rate measurements in microvessels. Microvasc Res 1983;26:351–61.[CrossRef][Medline]
  27. Wang S, Kotamraju S, Konorev E, et al. Activation of nuclear factor-{kappa}B during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide. Biochem J 2002;367:729–40.[CrossRef][Medline]
  28. Zhang L, Yu D, Hicklin DJ, et al. Combined anti-fetal liver kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits angiogenesis and growth of human soft tissue sarcoma xenografts by induction of endothelial cell apoptosis. Cancer Res 2002;62:2034–42.[Abstract/Free Full Text]
  29. Caine GJ, Stonelake PS, Lip GY, et al. The hypercoagulable state of malignancy: pathogenesis and current debate. Neoplasia 2002;4:465–73.[CrossRef][Medline]
  30. Haller H. Endothelial function. General considerations. Drugs 1997;53 Suppl 1:1–10.[Medline]
  31. Alon T, Hemo I, Itin A, et al. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1995;1:1024–8.[CrossRef][Medline]
  32. Jain RK, Safabakhsh N, Sckell A, et al. Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor. Proc Natl Acad Sci U S A 1998;95:10820–5.[Abstract/Free Full Text]
  33. Yuan F, Chen Y, Dellian M, et al. Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Nat Acad Sci U S A 1996;93:14765–70.[Abstract/Free Full Text]
  34. Dudar TE, Jain RK. Differential response of normal and tumor microcirculation to hyperthermia. Cancer Res 1984;44:605–12.[Abstract/Free Full Text]
  35. Song CW. Effect of hyperthermia on vascular functions of normal tissues and experimental tumors; brief communication. J Natl Cancer Inst 1978;60:711–3.
  36. Emami B, Nussbaum GH, TenHaken RK, et al. Physiological effects of hyperthermia: response of capillary blood flow and structure to local tumor heating. Radiology 1980;137:805–9.[Abstract/Free Full Text]
  37. Eddy HA, Chmielewski G. Effect of hyperthermia, radiation and Adriamycin combinations on tumor vascular function. Int J Radiat Oncol Biol Phys 1982;8:1167–75.[Medline]
  38. Onishi T, Machida T, Iizuka N, et al. Influence of differences in tumor vascularity upon the effects of hyperthermia. Urol Res 1990;18:313–8.[CrossRef][Medline]
  39. Song CW. Effect of local hyperthermia on blood flow and microenvironment: a review. Cancer Res 1984;44:S4721–30.
  40. Fajardo LF, Prionas SD. Endothelial cells and hyperthermia. Int J Hyperthermia 1994;10:347–53.[Medline]
  41. Hashizume H, Baluk P, Morikawa S, et al. Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 2000;156:1363–80.[Abstract/Free Full Text]
  42. Risau W. Mechanisms of angiogenesis. Nature 1997;386:671–4.[CrossRef][Medline]
  43. Abou El Hassan MAI, Verheul HMW, Jorna AS, et al. The new cardioprotector monohydroxyethylrutoside protects against doxorubicin-induced inflammatory effects in vitro. Br J Cancer 2003;89:357–62.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J BiochemHome page
N. Yoshida, T. Mita, and M. Onda
Susceptibilities of Phospholipid Membranes Containing Cholesterol or Ergosterol to Gramicidin and its Derivative Incorporated in Lysophospholipid Micelles
J. Biochem., August 1, 2008; 144(2): 167 - 176.
[Abstract] [Full Text] [PDF]

Home page
 RadioGraphicsHome page
J. R. MacFall and B. J. Soher
From the RSNA Refresher Courses: MR Imaging in Hyperthermia
RadioGraphics, November 1, 2007; 27(6): 1809 - 1818.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
A. M. Ponce, B. L. Viglianti, D. Yu, P. S. Yarmolenko, C. R. Michelich, J. Woo, M. B. Bally, and M. W. Dewhirst
Magnetic Resonance Imaging of Temperature-Sensitive Liposome Release: Drug Dose Painting and Antitumor Effects
J Natl Cancer Inst, January 3, 2007; 99(1): 53 - 63.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Q.
Right arrow Articles by Yuan, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Q.
Right arrow Articles by Yuan, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online