35020982

Effect of Artificial Oxygen Carrier with

Chemotherapy on Tumor Hypoxia and

Neovascularization

Jianqun Han, Minghua Yu, Min Dai, Peilin Cui,

Hongwei Li, Jian Zhang, Qian Liu and Ruijuan Xiu

Peking Union Medical College and Chinese Academy of Medical Sciences,

Beijing, China

Abstract: In addition to transfusion alternatives, artificial oxygen carriers are a benefit

in ischemia disorders. This study aimed at evaluating the possible effects of PEG-

conjugated hemoglobin (PEG-Hb) plus cisplatin on tumor hypoxia and neovasculature.

Methods: HeLa cells were injected into submucosa of golden hamster cheek pouch to

build tumor model. Animals were randomly assigned to 4 groups (n�10) and treated

respectively: group 1, saline; group 2, cisplatin (5mg/kg); group 3, cisplatin (5mg/kg)

plus PEG-Hb (0.3g/kg); group 4, cisplatin (5mg/kg) plus PEG-Hb (0.6g/kg). Tumor

neovascularization morphological variation and tissue hypoxia were detected by

intravital microscopy and immunostaining, respectively. Results: Microvessel tortuos-

ity and area capillary density in peritumoral areas were notably depressed in group 4

compared with group 2 (pB0.05). Hypoxia markers pimonidazole and HIF-1aexpression were decreased significantly in group 4. Conclusion: PEG-Hb in high

concentration can notably improve tumor tissue oxygenation and normalize neovascu-

lature; it may be a potential adjuvant to chemotherapy in cancer.

Keywords: Oxygen carrier, tumor hypoxia, HIF-1a, angiogenesis, HeLa

We thank Prof. T.M.S. Chang, McGill University, Canada, for serious review of this

manuscript. This work was supported by Prof. Ruijuan Xiu’s UNESCO Award for

Women in Science 2000 and the grant of ‘‘Knowledge Innovation Project,’’ Academy

of Science, China (No.KJCX1-SW-07).

Address correspondence to Ruijuan Xiu, Institute of Microcirculation, Chinese

Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC), 5

Dong Dan San Tiao, Beijing, 100005, China. E-mail: [email protected]

Artificial Cells, Blood Substitutes, and Biotechnology, 36: 431�438, 2008

Copyright # Informa UK Ltd.

ISSN: 1073-1199 print / 1532-4184 online

DOI: 10.1080/10731190802369789

431

INTRODUCTION

It is now accepted that intratumoral hypoixa affects every major aspect of cancer

biology. Severe hypoxia is correlating with progression, angiogenesis, invation,

metastasis, and therapy resistance, etc. [12]. Thus, tumor hypoxia might

represent a novel potential target for therapeutic intervention in solid tumors [9].

A variety of experimental and clinical studies have demonstrated that the

structural and functional abnormalities of the vascular microenvironment such

as tortuosity, dilation, sacculation and permeability are the special character-

istics of cancer newly formed vessels [1,2,6]. The microvascular network

appears highly chaotic [22]; accordingly the irregular and less effective

mircovessels result in a small amount of anticancer drugs reaching the mass and

aggravation of tissue hypoxia. These changes make the situation more

complex. Microcirculation detection is a valuable parameter in the assessment

of the anticancer agent’s action on tumor vessels [16]. Previously we have

utilized a non-invasive computer-assisted intravital microscopy system to

observe and quantitatively analyze the vascular microenvironment repeatedly,

which enabled us to evaluate the influence of the drug on tumor microcircula-

tion [24,25]. So we utilize the model in the artificial oxygen carrier experiment

with emphasis on its possible chemo-sensitization effect in cancer treatment.

The development of an artificial oxygen carrier is associated with the

known and potential infective agents, ambulance service, and the shortage of

donor blood. To date, only nanotechnology based polyhemoglobin (PolyHb)

and conjugated Hb continue to show promise in clinical trials [5]. In addition

to transfusion alternatives, the artificial oxygen carrier is a benefit in ischemia

disorders [3,4,7]. The application of it in tumor tissue hypoxia was carried out

decades ago. Owing to the difference of tested samples and doses, some

controversial data exist, which indicate that there still will be a long way to go.

MATERIALS AND METHODS

Polyethyleneglycol-conjugated Hemoglobin (PEG-Hb)

The PEG-Hb used in this study was a Chinese domestic sample. Its

preparation processes and physiochemical properties belong to the manufac-

turer’s proprietary information and will not be discussed in this paper. The

sample was kept at 48C in the dark until use.

Tumor Cell Line

HeLa cells were obtained from the Cell Bank of the Chinese Academy of Medical

Sciences, Beijing, China, were grown in RPMI-1640 (Gibco, Life Technologies,

432 J. Han et al.

Vienna, Austria) supplemented with 10% heat-inactivated fetal bovine serum

(FBS), 100U/mL penicillin G, 0.1mg/ml streptomycin, termed the growth

medium, and maintained in a humidified atmosphere with 5% CO2 at 378C.

Animal Preparation and Drug Administration

Forty male Syrian hamsters (Mesocricetus auratus), 8 weeks old with an initial

average body weight of 85g, were used throughout the following experiments

performed in compliance with the guidelines of authorities. The tumor model

was built in the cheek pouch of the hamster as we described previously

[24,26]. Briefly, after acclimation, 40 animals were anesthetized with

pentobarbital sodium (30mg/kg b.w. i.p., sigma), and 5�106 HeLa cell

suspension (100mL) was injected into the submucosa cheek pouch of

hamsters. Two days after tumor-cell inoculation, animals all had an observable

tumor xenograft in inoculated point. The animals were then randomly assigned

to 4 groups (n�10) and treated respectively: group 1 was given saline via a

jugular catheter; group 2, cisplatin (5mg/kg in 0.5mL of saline, Mayne Pharma

Pty Ltd, Mulgrave, Australia); group 3, cisplatin (5mg/kg) combined with

PEG-Hb (0.3g/kg); group 4, cisplatin (5mg/kg) combined with PEG-Hb (0.6g/

kg). Five days after tumor-cell inoculation, hamsters in each group received

repeated treatment, respectively.

Intravital Microscopy

The development of the newly formed vessels was observed on days 0, 3, 5,

and 7 after tumor implantation with an intravital microscope (ACM, Zeiss,

Germany). Simultaneously, tumor volume was measured by sliding caliper.

During preparation and observation, the hamster’s cheek pouch was

continuously superfused with warmed physiological solution composed of

(in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 18 NaHCO3, PH

7.4 at 378C, and equilibrated with oxygen-containing gas. Then the recorded

microscopic images were analyzed off-line by a professional staff in a blinded

fashion. A computer-assisted digital image processing system (Institute of

Microcirculation, PUMC&CAMS, China) was used to quantitatively analyze

the changes of the microvessel tortuosity and area functional capillary density.

Detection of Tumor Hypoxia

Animals were sacrificed after a week of respective treatment, intraperitoneally

injected hamsters with pimonidazol hydrochloride (60mg/kg) (Hypoxyprobe-1,

Oxygen Carrier and Tumor Neovasculature 433

Chemicon, USA) 1hr before being sacrificed. A tumor mass of appropriate

size was fixed in neutral-buffered formalin (10%) following paraffin

imbedding. Immunostaining for pimonidazole and HIF-1a was carried out

on contiguous sections using procedures described previously [11,15]. The

primary antibodies were polyclonal anti-HIF-1a (1:25 dilution) from Santa

Cruz Biotechnology (Santa Cruz, CA, USA) and Hypoxyprobe-1Mab1 (1:50

dilution) from Chemicon (California, USA). The resultant immunocomplexes

were detected with horseradish peroxidase-conjugated antirabbit IgG (Santa

Cruz Biotechnology). Eosin (HIF-1a sections) or hematoxlin (pimonidazole

sections) were used as counterstains.

Statistical Analysis

All data were presented as mean9S.E.M. Statistical analysis was performed

using the statistical program SPSS 10.0 for Windows (SPSS Inc., Chicago, IL,

USA) and differences were analyzed by one-way ANOVA. The level of

significance was taken as PB0.05.

RESULTS

1. Tumor Growth and Development of Neovasculature

On the 2nd day after implantation, neoplasma were visible in all hamsters;

after respective drugs administration, tumor volume was decreased sharply in

groups 2, 3 and 4 compared with group 1 (PB0.01). There was no significant

difference in tumor volume when cisplatin combined with or without artificial

oxygen carrier in a short time period (Figure 1).

Compared with contralateral normal cheek pouch mucosa microvessels,

chaotic and heterogeneous microvasculature was presented in tumor side

characterized by dilation, tortuosity, sacculation and permeability profiles.

Quantitatively evaluation neoplasma neovascular network revealed that

increasing microvessel tortuosity and area capillary density were obvious in

peritumoral areas after tumor cell inoculation in group 1. However, these

changes were notably depressed after cisplatin combined with or without the

oxygen carrier. The interesting thing is that group 4, combined with a higher

dose of oxygen carrier, had a further inhibiting effect on these changes

compared with group 2 (cisplatin alone) on the fifth day (tortuosity) and fifth

and seventh day (capillary density), respectively (Figure 2 and 3). But in

group 3, combined with a low dose of oxygen carrier, this effect was not

significant.

434 J. Han et al.

2. Expression of Exogenous and Endogenous Hypoxia Marker in Tumor

Tissue

To evaluate the degree of tumor hypoxia environment and the changes of

oxygenation after respective drugs administration, we carried out immuno-

histochemical analysis to detect pimonidazole and HIF-1a expression in tumor

sections (Table 1). In group 1 the tissue was severe hypoxia with

pimonidazole (27.694.7) and HIF-1a (29.295.5). After cisplatin combined

with oxygen carrier treatment (groups 3 and 4), the two hypoxia markers both

were significantly decreased compared with the control group (pB0.05). A

different concentration of oxygen carrier agent demonstrated capability in

improving the tumor hypoxia microenvironment. A high dose of oxygen

carrier (0.6g/kg) could markedly increase tumor oxygenation.

Figure 1. Tumor volume was decreased sharply in G2,G3 and G4 after chemotherapy

alone or/and combined with artificial oxygen carrier. *pB0.01 vs.saline.

Figure 2. Microvessel tortuosity in peritumoral areas on day0,day3,day5 and day7

after innoculation. in (# pB0.05 vs. cisplatin alone. * pB0.05 vs.saline).


DISCUSSION

More attention has recently focused on tumor hypoxia in tumor treatment.

Severe hypoxia is correlating with progression, angiogenesis, metastasis and

therapy resistance. Previous studies documented that in hypoxia condition

tumor cells were susceptible to chemoresistance, with the possible reasons

including poor drug distribution, quiencent tumor cell existence, acquired drug

resistance feature, and loss of apoptotic potential [23,27]. Many attempts have

been performed based on these experimental and preclinical observations

targeting tumor hypoxia.

Artificial oxygen carrier is a perfect mediator in oxygen transmission and

offloading. One type of oxygen carrier perfluorocarbon (PFC) emulsions plus

carbogen breathing have definite adjuvant effects in radiotherapy as well as

chemotherapy in tumor treatment [13,20,21]. Teicher further explored

hemoglobin preparation on the response of tumor models to radiotherapy

and chemotherapy [17�19]. Contrary data have also been reported [10,14].

Figure 3. Microvessel area capillary density in peritumoral areas on day0,day3,day5

and day7 after respective treatment. (# pB0.05 vs. cisplatin alone. * pB0.05

vs.saline).

Table 1. Pimonidazole and HIF-1a expression in neoplasma (mean9S.E.M.)

Group Number Pimonidazole% HIF-1a%

group 1 10 27.694.7 29.295.5

group 2 10 22.593.8 24.196.4

group 3 10 20.594.1* 22.594.7*

group 4 10 14.893.2* # 17.895.3* #

% percentage of tumor area staining positive for HIF-1aimmunoreactivity or pimoni-

dazole binding. * pB0.05 vs.saline # pB0.05 vs. cisplatin alone. group 1 saline; group

2 cisplatin alone; group 3 cisplatin with PEG-Hb (0.3g/kg); group4 cisplatin with PEG-

Hb(0.6/kg).

436 J. Han et al.

The reported results indicate the oxygen carrier’s adjuvant effect in tumor

therapy may depend on the tested sample type and dose. In this experiment,

we have designed two different concentration samples to test the possible

effect of PEG-Hb on chemo-sensitization. Although the low concentration of

PEG-Hb had some influence, it was not a statistical significance. Herein, PEG-

Hb had notable influence on tumor neovasculature and tissue oxygenation

induced by PEG-Hb in high concentration; this effect was based on lable

tumor hypoxia and microvessels morphological observations.

As we mentioned above, tumor neovascularization has its special

charicteristics. In our established tumor model chaotic and heterogeneous

microvasculature was presented. A similar phenomenon existed in Gim-

brone’s tumor model [8]. Tissue hypoxia induce angiogenesis; conversely, the

irregular and less effective microvessels result in a small amount of anticancer

drugs reaching the mass and aggravation of tissue hypoxia. Therefore, these

changes make the situation more complex. Microcirculation detection is a

valuable parameter in assessing the anticancer agent’s action on tumor vessels

[16]. Previously, we have utilized a non-invasive computer-assisted intravital

microscopy system to observe and quantitatively analyze the vascular

microenvironment repeatedly, which enables us to evaluate the influence of

the drug on tumor microcirculation [24,25]. Herein, we also measured the

tumor volume. PEG-Hb had no marked effect on it compared with cisplatin

alone. It might be due to the relatively short period of drug administration. We

have tested PEG-Hb in another tumor model on BALB/C nude mice with

conventional therapy time and a better effect on tumor volume was observed

(in press), whereas the microvessels structure normalization was obvious. We

are the first to show a direct relationship of oxygen carrier to tumor

neovascularization, although this study has some shortcomings.

In summary, tumor angiogenesis can be directly observed in our tumor

model, which is a non-invasive ideal platform to quantitatively analyze the

microvasculature repeatedly and assess the drugs’ action on it. PEG-Hb in

high concentration can notably improve tumor tissue oxygenation and

normalize neovasculature. It is indicated that the evaluated artificial oxygen

carrier (PEG-Hb) may be a potential adjuvant to chemotherapy in cancer.

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