Carbon Nanotubes Conjugated to Tumor Lysate Protein Enhance the Efficacy of an Antitumor Immunotherapy

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Antitumor therapy

DOI: 10.1002/smll.200701059

Carbon Nanotubes Conjugated to Tumor Lysate ProteinEnhance the Efficacy of an Antitumor ImmunotherapyJie Meng, Jie Meng, Jinhong Duan, Hua Kong, Li Li, Chen Wang, Sishen Xie,Shuchang Chen, Ning Gu, Haiyan Xu,* and Xian-Da Yang*

Keywords:� biomedicine

� carbon nanotubes

� immunotherapy

� tumor lysate

The biomedical applications of carbon nanotubes (CNTs) have attracted

deep interest in recent years. Antitumor immunotherapy has the potential to

improve the prognosis of cancer treatment but the efficacy of current

immunotherapy generally needs further improvement. Multi-walled CNTs

conjugated to tumor lysate protein are investigated as to whether they would

enhance the efficacy of an immunotherapy employing a tumor-cell vaccine

in a mouse model bearing the H22 liver cancer. The tumor cure rate is found

to be markedly improved by CNTs conjugated to tumor lysate protein. The

cellular antitumor immune reaction is also enhanced. Moreover, the

observed antitumor immune response is relatively specific against the tumor

intended for treatment. These findings suggest that CNTs may have a

prospective role in the development of new antitumor immunotherapies.

1. Introduction

Carbon nanotubes (CNTs) have been shown to have

potential applications in multiple biomedical fields,[1–11]

particularly as transporters for delivery of various bioactive

molecules such as peptides,[12] proteins,[13–15] DNA,[16–18]

RNA,[19,20] or drugs.[4,21] One area of particular interest is

CNTs’ role in modulating immunological functions. Prior

study has revealed that viral peptides conjugated to CNTs can

elicit strong antipeptide antibody responses in mice with no

detectable cross reactivity to the CNTs.[22] It is also reported

[�] Prof. H. Xu, J. Meng, H. Kong

Department of Biomedical Engineering

Institute of Basic Medical Sciences

Chinese Academy of Medical Sciences and

Peking Union Medical College

5 Dong Dan San Tiao, Beijing 100005 (P.R. China)

E-mail: [email protected]

Prof. X.-D. Yang, J. Meng, J. Duan

Department of Pathophysiology

Institute of Basic Medical Sciences

Chinese Academy of Medical Sciences and

Peking Union Medical College

5 Dong Dan San Tiao, Beijing 100005 (P.R. China)

E-mail: [email protected]

Prof. C. Wang

National Center of Nanoscience and Technology

No.11 Bereitiao, Zhongguangcun, Beijing 100080 (P.R. China)

� 2008 Wiley-VCH Ver

that functionalized CNTs are noncytotoxic to immune cells.[23]

However, whether CNTs can be effectively applied in

antitumor immunotherapy has never been evaluated.

The significance of immunotherapy as an adjuvant anti-

caner treatment is well recognized.[24–26] While chemotherapy

faces the issues of accumulative toxicity and drug resistance,

antitumor immunotherapy usually has few adverse effects,

good patient tolerance, and the potential to significantly

improve the prognosis.[27] Some clinical trials of immuno-

therapy achieved promising results in treating malignancies

such as melanoma, malignant glioma, or renal cell carcinoma,

Prof. S. Xie

Institute of Physics

Chinese Academy of Sciences

P.O. Box 603, Beijing 100080 (P.R. China)

Dr. L. Li

Department of Thoracic Surgery

Peking Union Medical College Hospital

53 Dond Dan Bei Da Jie, Beijing 100005 (P.R. China)

Dr. S. Chen, X.-D. Yang

Department of Oncology

Peking Union Medical College Hospital

Beijing 100730 (P.R. China)

Prof. N. Gu

Department of Biological Science and Medical Engineering

Southeast University

2 Sipailou, Nanjing 210096 (P.R. China)

lag GmbH & Co. KGaA, Weinheim small 2008, 4, No. 9, 1364–1370

CNTs in Antitumor Immunotherapy

Figure 1. Characterization of MWCNTs functionalized with carboxyl

groups and tumor lysate protein. a) Schematic drawing of MWCNTs and

the functioinalizing process with tumor lysate protein. EDAC: coupling

agent N-ethyl-N0-(3-dimethylaminopropyl) carbodiimide. b) Scanning

electron microscopy image of functionalized CNTs. c) Image of

stable CNT solution with minimal aggregation. d) C1s spectrum

of CNTs, analyzed with X-ray photoelectron spectroscopy. The data

represents intensity versus binding energy. Peak 1 at a binding

energy of 284 eV was contributed by C–C and C–H bonds, while peak

2 at a binding energy of 289 eV was assigned to the carboxyl groups on

the surface of CNTs. e) Amounts of CNT-carried H22P with either

covalent binding or simple absorption.

which tended to respond poorly to chemotherapies.[28–31]

Cancer cells often develop immune tolerance and immune

escape mechanisms.[32,33] In order to mount an anticancer

immune reaction, current immunotherapies employ tumor-

cell vaccines (TCV) made of inactivated cancer cells, dendritic

cells (DC) that have been exposed to tumor antigens or

cytokines that modulate the immune function.[34–36] Although

these techniques have achieved various degrees of success, the

efficacy of immunotherapy generally needs additional

improvement.[37,38] A key issue of the antitumor immunother-

apy field, therefore, is to develop new technologies that can

further improve the treatment outcome. Given the excellent

features of CNTs as transporters for bioactive molecules,[39] it

will be of great interest to evaluate if CNTs can be applied to

improve antitumor immunotherapy.

In this work, we investigated whether multi-walled (MW)

CNTs conjugated to tumor lysate protein would enhance the

efficacy of an antitumor immunotherapy employing TCV in a

mouse model bearing the H22 liver cancer. Tumor lysate

protein contains a mixture of various tumor proteins. Unlike

tumor-specific antigens that are rare and difficult to obtain,[40]

tumor lysate proteins are readily obtainable and have been

investigated in antitumor immunotherapy studies with

promising results.[21,41–45]

2. Results

In order to conjugate tumor lysate proteins to CNTs, an

oxidation/sonication procedure was used to introduce suffi-

cient carboxyl groups to the CNTs for solubilization and

subsequent protein conjugation. The characterization of the

functionalized CNTs was carried out with standard meth-

odologies[39,46] (Figure 1a, c, and d). The tubelike structure

of the CNTs was maintained, with an average length of

500–800 nm and an average diameter of 20–30 nm (Figure 1b).

The stable CNT solution had a concentration of 0.2mgmL�1

(Figure 1c). H22 liver-cancer cells were lysed and the tumor

lysate protein (H22P) extracted per a standard protocol. H22P

was then covalently conjugated to the oxidized CNTs

(Figure 1a). TCV was prepared from H22 cancer cells with

a protocol involving mitomycin.

CNTs may carry tumor proteins either via absorption or

covalent binding. To compare the amount of H22P carried with

these two mechanisms, a certain amount of protein was first

added to the reaction medium for either absorption or covalent

binding byCNTs. TheCNT-carried proteinwas then calculated

through assaying the protein amount in the post-reaction

medium, after high-speed centrifugation removed the CNTs

from the supernatant. It was found that the covalent-binding

procedure consistently resulted in more CNT-carried proteins

than the absorption procedure (Figure 1e). Since the main

purpose of this study was to evaluate CNTs’ role as carrier of

tumor antigens in the induction of antitumor immune response,

the covalent-binding procedure, rather than the absorption

procedure, was adopted in this immunotherapy study.

The murine hepatoma H22 was employed in this study

because it was a mature tumor model that had been frequently

used in immunotherapy research in the past.[47] The experi-

small 2008, 4, No. 9, 1364–1370 � 2008 Wiley-VCH Verlag

ments of antitumor immunotherapy were carried out in five

groups of mice, with one control group and four treatment

groups, and 24 mice in each group. The four treatment groups

included the group that received TCV only (TCV), the group

that received TCV plus H22P (TCVþH22p), the group that

received TCV plus CNTs (TCVþCNT), and the group that

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full papers H. Xu, X.-D. Yang, et al.

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received TCV plus CNTs conjugated to H22P (TCVþCNT-

H22P). All animals received subcutaneous inoculation of H22

cancer cells on day 1 at the beginning of the experiments.

Except for the control group, all treatment groups received

two subcutaneous doses of TCVon days 7 and 14, to trigger the

antitumor immune reaction. On day 2, the following three

groups also received additional subcutaneous treatments: the

TCVþH22P group received a dose of H22P, the TCVþCNT

group a dose of CNTs, and the TCVþCNT-H22P group a dose

of CNTs conjugated to H22P. Tumor dimensions were

monitored using calipers every 48–72 h. Survival situations

were recorded for up to 90 days.

The efficacy of the immunotherapy was measured using

the tumor cure rate. An animal was considered cured when its

tumor disappeared and remained nondetectable for the entire

study duration. After inoculation with H22 cells, most animals

gradually developed detectable subcutaneous tumor mass in

about 10 days. For the control group, the tumor size gradually

increased, eventually killing the animals when the tumor

reached a diameter of 4–5 cm. For the treatment groups,

however, the tumor masses in some animals stopped growing

after 2–4 weeks, then gradually shrank in size, and eventually

disappeared after about 6 weeks. These animals achieved long-

term functional survival and were considered cured. The

treatment outcomes are shown in Figure 2: the TCVþCNT-

H22P group had a cure rate of 54.2%, significantly higher than

Figure 2. Tumor-cure rates of the various treatment groups. a,b)

Examples of the tumor curing process. The arrow in (a) points to the spot

where a tumor used to grow but shrank to a minimal residue in an

animal of the TCVþCNT-H22P group, while the tumor in the control

group (arrow in b) enlarged significantly. c) The highest tumor-cure rate

was observed in the TCVþCNT-H22P group.

www.small-journal.com � 2008 Wiley-VCH Verlag Gm

the TCV group’s 37.5% (p< 0.01, x2 test). The cure rates of

the TCVþH22P and the TCVþCNT groups were 37.5 and

45.6%, respectively.

It is important to investigate whether the higher cure rates

in the TCVþCNT and TCVþCNT-H22P groups were due to a

toxic effect of CNTs on the tumor cells. To address this issue,

the H22 cells was first incubated for 8 h with either normal

culture medium or medium containing CNTs with a

concentration of 0.2mgmL�1; the viability of the H22 cells

was then evaluated with MTS assay 1 and 3 days later. The

results showed no significant difference in the number of live

cells in the two groups (Figure 3a), suggesting that CNTs’

cytotoxicity was not the major mechanism for the higher cure

rates of the CNT-containing immunotherapies. In addition,

CNTs alone were given as the only treatment agent to a

separate group of tumor-bearing animals (n¼ 8) per protocol

and failed to produce any obvious antitumor effect in terms of

cure rate (data not shown). The result again suggested that

CNTs’ toxicity was not the main mechanism for the cure of the

tumor. It should also be noted that in the immunotherapy

study, the CNTs were injected onto the right hind leg of the

animal, tominimize the direct effect of the CNTs on the tumor,

which was inoculated onto the left hind leg of the animal.

To further explore the mechanism of the improved

therapeutic outcome, cellular immunological experiments

were conducted. Specifically, the antitumor cytotoxicity of

the splenic lymphocytes of the mice was evaluated and

compared to the treatment groups. Surviving animals

randomly picked from various groups were terminated on

day 90 and the splenic lymphocytes were extracted with a

Ficoll centrifugation protocol. The lymphocytes were then co-

incubated with the H22 tumor cells. The cytotoxic effects of

the lymphocytes against the H22 cells were measured using a

lactate dehydrogenase assay kit following the manufacturer’s

protocol. As shown in Figure 3b, the TCVþCNT-H22P group

had a significantly higher cytotoxicity against the H22 cancer

cells by the lymphocytes, compared to the other treatment

groups (p< 0.05). The results suggested that an enhanced

antitumor immune reaction contributed to the higher cure rate

in the TCVþCNT-H22P group.

Histological studies of the tumor tissue also suggested that

immune mechanism played a major role in the cure of H22

cancer. As shown in Figure 3c, in the control group, the tumor

tissue was well maintained without much lymphocyte infiltra-

tion. In the TCVþCNT-H22P group (Figure 3d), however, the

tumor tissue was heavily infiltrated with small lymphocytes,

with obvious tumor necrosis, suggesting that antitumor

immune reaction was actively in process.

An important question here was whether the enhanced

antitumor immunity was specific and mainly against the H22

cancer, the tumor intended to be treated, or nonspecific and

against other types of cancer as well. To address this issue, we

performed tumor-challenge experiments with two types of

cancer cell, H22 and EMT, with the latter being a mouse

breast-cancer cell line. Six animals cured of H22 cancer from

the TCVþCNT-H22P group were used for the tumor

challenge tests on day 91. These mice first received a

subcutaneous injection of live H22 cells again. All six animals

successfully rejected the newly inoculated tumor, presumably

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Figure 3. Cellular immunological studies and histological slides. a) Assay of viable H22 cells by the MTS-absorbance method, with or without CNTs

in the culture medium (n¼5). b) Cytotoxicities against the H22 tumor cells by splenic lymphocytes in all treatment groups. The columns represent

the number of killed H22 cells per well of the six mice in each group, in the form of mean�SE. The TCVþCNT-H22P group had the highest

cytotoxicity against the H22 tumor cells (p< 0.05, ANOVA). c,d) Representative histological slides of the control group (c) and the TCVþCNT-H22P

group (d) during the tumor-forming phase, with the red arrows pointing to H22 tumor cells. The tumor cells in the control group (c) had minimal

infiltration by the inflammatory cells, whereas the treatment group (d) had heavy infiltration of inflammatory cells (most of them small lymphocytes,

green arrow), and obvious tumor necrosis (black arrow).

due to their prior-developed immunity against the H22 cancer,

whereas the animals in the control group developed H22

tumormass (Figure 4a). The same six animals previously cured

of H22 cancer were then injected with another kind of cancer

cell (EMT) at a different body site 2 weeks later. This time the

EMT cancer grew and formed tumor mass (Figure 4b). In

other words, although these animals successfully rejected the

H22 cells due to their prior-developed immunity, they did not

have sufficient immunity against the EMT cancer. The results

suggested that the prior-developed antitumor immunity was

relatively tumor specific against the H22 cancer cells.

To further evaluate the specificity of the antitumor

immune reaction, the anti-H22 and anti-EMT cytotoxicities

by the lymphocytes in the TCVþCNT-H22 group were

assayed and compared. As shown in Figure 4c, the immune

reaction against theH22 cells was significantly higher than that

against the EMT cells (p¼ 0.001). The results again suggest

that the observed antitumor immune response was relatively

tumor specific against the H22 cancer.

3. Discussion

The aim of this study was to investigate whetherMWCNTs

could be applied to improve the outcome of an antitumor


immunotherapy that employed vaccines of inactivated tumor

cells. We found that CNTs conjugated to tumor lysate protein

improved the cure rate of H22 liver cancer in mice (Figure 2).

Moreover, elevated antitumor immune reaction was probably

the major mechanism that contributed to the higher cure rate

of the TCVþCNT-H22P group, since the antitumor cytotoxi-

city by the lymphocytes was significantly enhanced (Figure 3).

The presence of the antitumor immunity was also clearly

demonstrated by the result of the tumor-challenge study, as all

the cured animals successfully rejected the second injection of

the H22 tumor cells, whereas the control animals did not

(Figure 4a). Interestingly, the enhanced antitumor immune

reaction in the TCVþCNT-H22P group remained relatively

tumor specific against the cancer intended to be treated

(Figure 4), suggesting that CNTs had features ideal for

immunotherapy applications.

Pantarotto et al.[22] have found that viral peptides

conjugated to CNTs can improve the antipeptide immune

response. In agreement with their findings, our data shows that

tumor lysate protein conjugated to CNTs can be used to

enhance antitumor immune reactions. There aremultiple ways

to induce antitumor immune response. For example, the TCV

could be either unmodified or modified biologically or

chemically or various kinds of adjuvant such as Bacille

Calmette-Guerin (BCG) or aluminum could be used.[24,27] In



Figure 4. Tumor-challenge and specificity studies with H22 and EMT

cells. a,b) Typical in vivo responses of the animals to tumor challenges

by H22 (a) and EMT (b) cancer cells. The downward arrows indicate the

time of tumor inoculation. a) H22 cells produced tumor mass in a

control animal but not in an animal that had been cured of H22 cancer

by the TCVþCNT-H22P treatment. b) EMT cells resulted in a growing

tumor mass in both the control and the animal that had been cured of

H22 cancer previously. c) The anti-H22 cytotoxicity by lymphocytes is

significantly higher than the anti-EMT cytotoxicity in animals cured of

H22 cancer (p< 0.05, ANOVA). The columns represent number of killed

target cells per well from data of six mice, in the form of mean�SE.

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this study, we investigated CNTs’ role in an immunotherapy

that uses unmodified TCV in a murine tumor model. To

investigate the role of CNTs in more complicated immu-

notherapy regimens, further research is necessary. CNTs

might enhance the antitumor immune reaction through

several mechanisms. CNTs have been reported to be good

carriers of proteins,[13–15] presumably due to the hydrophobic

nature of CNTs and their high affinity to cell membranes. As


an excellent protein carrier, CNTs conjugated to tumor lysate

proteins might bring tumor antigens into antigen presenting

cells of the immune system more efficiently. Other mechan-

isms, such as adjuvant effects, are also possible. Much research

is warranted to explore these hypothetical mechanisms in

future work.

Tumor lysate protein is a mixture of various tumor

proteins. The advantage of tumor lysates is that they can be

applied to a large number of tumors and patients, irrespective

of the genetic makeup of the tumors. However, the lack of

defined tumor markers, such as target antigens, renders the

antibody responses difficult to assess. Because the nature of

the immunogens was not known, the efficacy of therapy

involving the tumor lysate proteins was generally assessed by

tumor rejection, tumor growth retardation, or prolonged

survival of the immunized mice but not by antibody

production.[40] Nevertheless, human clinical studies based

on such vaccines have shown promising results in some of the

trials.[29,41–46] Fifis et al.[48] have reported that carboxylated

polystyrene nanospheres conjugated to ovalbumin (OVA) can

significantly enhance the immune reaction against an OVA-

expressing tumor. The results of this study showed that CNTs

conjugated to tumor lysate protein enhanced the antitumor

immune response initiated by TCV. Since tumor lysate protein

is readily obtainable frommost solid tumors, our finding points

to a practical way of potentially using CNTs to improve the

anticancer immune response against multiple tumors.

4. Conclusions

In summary, this study showed that CNTs conjugated to

tumor lysate protein enhanced the specific antitumor immune

response and the cancer cure rate of a TCV immunotherapy in

mice. The results suggest that CNTs may play a role in

development of new antitumor immunotherapies.

5. Experimental Section

Preparation and characterization of functionalized CNTs:

MWCNTs were purchased from Chengdu Organic Chemicals Co.

Ltd., with a purity of >95%, diameter of 20–30 nm, length of

50mm, amorphous carbon <3%, ash (catalyst residue) <1.5%,

special surface area >233 m2 g�1, and thermal conductivity of

about 2000 W m�1 k�1. Stable aqueous suspensions of purified

and shortened CNTs were prepared by oxidation and sonication of

the purchased commercial product. In brief, CNTs were suspended

in a 3:1 mixture of concentrated H2SO4/HNO3 and sonicated at

540 W for 45 s. The resulting mixture was then filtered through a

polycarbonate filter membrane with 2-mm pores (Millipore) and

rinsed thoroughly till neutralized. The obtained CNTs were dried

completely and suspended in pure water at a concentration of

0.3 mg mL�1 by sonication. Centrifugation (5000 rpm, 30 min)

then removed unreacted components from the solution to afford a

stable suspension of CNTs.

Conjugation of tumor protein to CNTs: H22 tumor lysate

protein (H22P) was extracted from H22 cancer cells per a routine

bH & Co. KGaA, Weinheim small 2008, 4, No. 9, 1364–1370


protocol. Briefly, H22 cells from mouse ascites were washed thrice

with aseptic normal saline. Following a 10 min centrifugation at

1000 rpm, the spun-down cells were mixed well with a cell lysis

buffer containing 20 mM of tris–HCl of pH 7.5, 150 mM NaCl, 1 mM

ethylenediaminetetraacetic acid (EDTA), 0.5% triton, 0.1% sodium

dodecyl sulfate, 1 mM dithiothreitol, and 0.5 mM of freshly made

phenylmethylsulfonyl fluoride. After sitting on ice for 90 min, the

mixture was centrifuged at 12 000 rpm for 30 min at 4 8C. The

supernatant was quantified for protein concentration with a

standard protein assay kit (BioRad) and kept at �80 8C till further

usage. The surface carboxyl groups were utilized to conjugate

H22P to the CNTs. Two mg of H22P was mixed with 0.5 mL CNTs

solution at the concentration of 0.2 mg mL�1. N-Ethyl-N0-(3-

dimethylaminopropyl) carbodiimide (EDC, Sigma) was added into

the solution at a concentration of 4 mg mL�1. The mixture was

stirred for 30 min at room temperature and then transferred to a

membrane tubing that had a cutoff molecular weight of 12 000 for

dialysis against phosphate buffered solution (PBS, pH¼7.4) to

remove the free EDC molecules. Following dialysis for at least

12 h, the mixture was carefully dripped out, yielding a dark-

colored homogeneous solution ready for further application.

To measure the amount of protein carried by CNTs with either

simple absorption or covalent binding, two groups of experiments

were performed. In one group, a suspension of the oxidized CNTs

of 0.2 mg mL�1 was mixed with various concentrations of H22P of

0.25, 0.5, 1, 2, and 4 mg mL�1 for 15 min at room temperature,

allowing the protein to be absorbed onto CNTs. In another group,

EDC (Sigma, 4 mg mL�1) was also added into the protein solutions

to facilitate the covalent linking of H22P to CNTs. The mixtures

from both groups were then stirred for 30 min at room temperature

and then centrifuged at 17 000g for 30 min at 4 8C. The

supernatants, free of CNTs after the centrifugation, were quanti-

fied for protein concentration with a protein assay kit (BioRad).

The amount of CNT-carried protein was calculated via deducting

the protein in the supernatant from the initial protein amount in

the reaction medium.

Immunotherapy studies: TCV was made by incubating 106 H22

cancer cells in PBS with mitomycin C of 80 mg L�1 for 60 min,

followed by thorough wash with PBS five times. Immunotherapy

experiments were performed using female adult BALB/c mice, 4–8

weeks old, with body weight ranging from 18–24 g. The animal

studies were conducted in accordance with the approved

protocols of the Chinese Academy of Medical Science. One

hundred and twenty mice were divided into five treatment groups:

control, TCV, TCV with H22 lysate protein (TCVþH22P), TCV with

CNTs (TCVþCNT), and TCV with protein conjugated to CNTs

(TCVþCNT-H22P). On day 1, every mouse was inoculated with

2�106 live H22 cells subcutaneously in the right hind leg. No

further treatment was offered to the control group. On day 2, the

TCVþH22P group received subcutaneously in the left hind leg an

injection of 2 mg of H22P, the TCVþCNT group an injection of

0.1 mg of CNTs, and the TCVþCNT-H22P group an injection of 2 mg

of H22P conjugated to CNTs (CNT-H22P). On days 7 and 14, every

mouse in all four treatment groups also received TCV at a dose of

106 subcutaneously in the left front leg. Tumor dimensions were

monitored using calipers at right angles every 48–72 h.

Cell viability assays: H22 cells harvested from ascites were

washed twice with aseptic normal saline, followed by a 10 min


centrifugation at 1000 rpm. The spun-down cells were resus-

pended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco)

supplemented with 10% fetal bovine serum (FBS). CNTs (0.1 mg)

were added to 500mL DMEM and thoroughly mixed, then 1�106

H22 cells were added in and incubated at 37 8C with 5% CO2 for

8 h. After co-incubation the cells were washed and resuspended in

culture medium, then plated in a 96-well plate at a density of

4�103 cells per well. At either 24 or 72 h, Celltiter96 reagent

(MTS cell viability assay kit, Promega) was added to the wells and

allowed to incubate for 90 min at 37 8C with 5% CO2. The reaction

was stopped after 90 min with 20mL of 10% Sodium dodecyl

sulfate (SDS). The absorbance at 490 nm was then measured and

the proportion of viable cells calculated after deducting the

background absorbance. The above MTS viability data was double

checked and confirmed with another assay for viability, the Trypan

Blue method, in which the H22 cells were stained with trypan blue

dye at either 24 or 72 h into the incubation, and then counted

under the microscope. The number of viable cells was calculated

through deducting the number of stained cells from the total cell

count.

Cytotoxic immunological studies: 10 000 tumor cells (target

cells) were seeded on round-bottomed 96-well tissue culture

plates (Falcon) at a density of 2�108 L�1; effecter cells (1�105

lymphocyte in 50mL) were then added, keeping the lymphocyte-

to-tumor cell ratio at 10:1. The final combined volume was 100mL.

Tissue culture plate was centrifuged at 250 rpm for 5 min at 4 8C to

ensure cell–cell contact, then incubated at 37 8C with 5% CO2 for

4 h. After the reagents were added per manufacture’s (Promega)

instruction, the optical density of the supernatant was then

measured by using an ELISA plate reader with a 490-nm filter. The

values of effecter cells’ spontaneous Lactate dehydrogenase (LDH)

release, target cells’ spontaneous LDH release, target cells’

maximum LDH release, and culture-medium background were

also measured and subtracted per manufacture’s protocol. In

each experiment, quadruplicate wells were analyzed to ensure

reliable readings. Results were compared by analysis of variance

(ANOVA), using the SPSS10.0 software. p-Value of <0.05 was

considered significant.!

Acknowledgements

J. M. and J. M. contributed equally to this work. This work was

supported by the Natural Science Foundation of China

(90306004, 90406024), National Center of Nanoscience and

Technology of China, National Key Scientific Projects of China

(2006CB933204), and Natural Science Foundation of Beijing

(Z0005190043511).

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