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Journal of Controlled Release 210 (2015) 134–146
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Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Vascular-targeted TNFα improves tumor blood vessel function and
enhances antitumor immunity and chemotherapy in colorectal cancer
Lan Lu a,b, Zhi Jie Li b,⁎,1, Long Fei Li b, William Ka Kei Wu c, Jing Shen b, Lin Zhang b, Ruby Lok Yi Chan b, Le Yu d,
Ya Wei Liu e, Shun Xiang Ren b, Kam Ming Chan b, Chi Hin Cho b,⁎
a
Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu, PR China.
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, PR China
Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, PR China
d
School of Pharmacy, Southern Medical University, Guangzhou, PR China
e
Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, PR China
b
c
a r t i c l e
i n f o
Article history:
Received 7 November 2014
Received in revised form 20 March 2015
Accepted 20 May 2015
Available online 21 May 2015
Keywords:
Vascular targeting peptide
TNFα
Colorectal cancer
Drug delivery
Antitumor therapy
a b s t r a c t
Delivery and penetration of chemotherapeutic drugs into neoplasm through the tumor vasculature are essential
mechanisms to enhance the efficiency of chemotherapy. “Vascular targeting” strategy focuses on promoting the
infiltration of chemotherapeutic drugs into neoplastic tissues. In this study, we achieved a targeted therapy by
coupling tumor necrosis factor α (TNFα) with TCP-1, a novel vascular-targeting peptide, in an orthotopic
colorectal cancer model in mice. High dose of TCP-1-conjugated TNFα (TCP-1/TNFα: 5 μg/mouse) displayed
potent antitumor activity by inducing apoptosis and reducing microvessel number in tumors than unconjugated
TNFα, with no evidence of increased toxicity. In the combined therapy, the antitumor action of 5-fluorouracil
(5-FU) was potentiated when the mice were pretreated with a low dose of TNFα (1 ng/mouse) and to a greater
extent by the same concentration of TCP-1/TNFα. In this regard, TCP-1/TNFα combined with 5-FU synergistically
inhibited the tumor growth, induced apoptosis and reduced cell proliferation. More importantly, TCP-1/TNFα
normalized the tumor vasculature and facilitated the infiltration of immune cells to neoplasm as well as
attenuated the immunosuppressing effects of TNFα in bone marrow and spleen. At the same time, TCP-1/
TNFα significantly improved 5-FU absorption into the tumor mass. Taken together, these findings underscore
the therapeutic potential of TCP-1 as a drug carrier in cancer therapy. TCP-1 is a novel vascular-targeting peptide
and appears to be a promising agent for drug delivery. TCP-1 fused with TNFα holds great promise for colorectal
cancer therapy.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Colorectal cancer (CRC) remains one of the most frequently
diagnosed cancers and the leading cause of cancer deaths worldwide,
in spite of continuous advancements in its diagnosis and treatment.
For instance, EGFR antibody and VEGF antibody as targeted therapy
agents have been extensively applied in patients with advanced CRC
in combination with chemotherapeutic drugs. However, not all patients
with CRC are eligible for these two bioactive agents owing to intolerable
side effects, KRAS mutation and the development of drug resistance
[1–4]. Thus, new therapeutic options and bioactive agents are urgently
needed to further optimize the therapeutic strategy for a better outcome in CRC treatment. The development of drug delivery/imaging
⁎ Corresponding authors at: School of Biomedical Sciences, The Chinese University of
Hong Kong, Shatin, NT, Hong Kong, China.
E-mail addresses: [email protected] (Z.J. Li), [email protected] (C.H. Cho).
1
Present address: Harry Perkins Institute of Medical Research, University of Western
Australia, Crawley 6009, Western Australia, Australia.
http://dx.doi.org/10.1016/j.jconrel.2015.05.282
0168-3659/© 2015 Elsevier B.V. All rights reserved.
probe systems represents an ongoing effort to improve the specificity
and efficacy of antitumor therapy and diagnosis. Tumor-homing
peptides (THPs) are important drug delivery/imaging vectors that can
target predefined molecules, cells or tissues. In this regard, more attention has been paid to peptides homing to tumor vasculature since the
endothelial cells are the first accessible component after systemic drug
administration and drug resistance rarely occurs in endothelial cells
[5]. “Vascular targeting” strategy aims at enhancing the local efficacy
and improving chemotherapeutic drug delivery to increase the
therapeutic index of drugs.
Tumor necrosis factor α (TNFα), an inflammatory cytokine exerts its
potent antitumor activity through complex mechanisms, including induction of inflammatory and immune responses, tumor cell apoptosis/
necrosis, extensive thrombosis and destruction of tumor vasculature
[6,7]. However, early clinical trials have disappointingly shown that
systemic administration of TNFα to patients including CRC patients
was associated with prohibitive toxicity and the maximum tolerated
dose being 10–50 times lower than the expected effective doses for
cancer treatment [8,9]. To date, the clinical use of TNFα has been limited
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
to loco-regional administration in combination with chemotherapy
[10]. Nevertheless, an increasing body of evidence shows that some
functional properties of TNFα, such as alteration of endothelial barrier
function, reduction in tumor interstitial pressure as well as stabilization
of tumor vessel network, provides evidence to support exploration of its
therapeutic use [11,12].
Notably, effects of TNFα on vascular function present a rational basis
for exploiting the “vascular targeting” strategy to improve its therapeutic efficacy and application. To this end, several tumor vascular ligands
fused to TNFα have been devised, some of which have entered clinical
trials for cancer therapy [13–16].
Through biopanning, we previously identified a cyclic peptide
known as TCP-1 that specifically targets the vasculature of orthotopic
colorectal tumors [17]. We also showed that TCP-1 could be used for
targeted delivery of fluorescein and a pro-apoptotic peptide to tumor
vasculature [17]. The current study is to further investigate the targeting
ability of TCP-1 to deliver TNFα to tumor vessels in an orthotopic
colorectal cancer model established in immuno-competent mice. In
this study, we achieved the vascular-targeting delivery of small protein
agents, enhanced green fluorescent protein (EGFP) and TNFα, to a
tumor mass. We found that high-dose TCP-1-conjugated TNFα
(TCP-1/TNFα: 5 μg/mouse) per se potently induced apoptosis and
destructed neovasculature in tumors. Furthermore, low-dose TCP-1/
TNFα (1 ng/mouse) potentiated the antitumor effect of 5-fluorouracil
(5-FU). In addition, the low-dose TCP-1/TNFα strongly promoted
tumor blood vessel normalization and antitumor immunity. We also
provided evidence that TCP-1/TNFα could enhance the intratumoral
accumulation of 5-FU. In this study, we extended the therapeutic
application of the vascular-targeting peptide TCP-1 and investigated
extensively the related antitumor mechanisms of TCP-1/TNFα for
developing an effective drug carrier and a potential fusion protein for
CRC treatment.
2. Materials and methods
2.1. Cells and animals
The human colon cancer cell lines HT-29 (CCL-218), SW1116 (CCL233), the normal human colon fibroblast cell line CCD18Co (CRL-1459),
the human gastric epithelial mucosa cell line GES-1 and human gastric
carcinoma cancer cell line MKN45 were obtained from the American
Type Culture Collection (Manassas, VA, USA). The murine CRC cell
Colon 26 was obtained from the Health Science Research Resources
Bank (Osaka, Japan). Mouse fibroblast cell line L929 for TNFα activity determination was purchased from the Shanghai Cell Bank of the Chinese
Academy of Sciences (Shanghai, China). Cells were grown in recommended medium supplemented with 100 U/mL penicillin G, 100 μg/mL
streptomycin, and 10% fetal bovine serum (FBS) and maintained at
37 °C in a humidified atmosphere containing 5% CO2. Male BALB/c mice
aged 9 weeks were maintained at the Chinese University of Hong Kong
Animal Facility. Animal experiments in this project had been approved
by the Laboratory Animals Ethics Committee of the Chinese University
of Hong Kong.
2.2. Reagents and antibodies
The TCP-1 peptide CTPSPFSHC was synthesized commercially by GL
Biochem (Shanghai, China) to our specifications. The peptide was confirmed by high-performance liquid chromatography and mass spectrometry analyses and the purity was at least 95%. Ni-NTA resin and
biotin-labeled anti-His tag antibody were from Qiagen. Rat anti-mouse
CD31, rat anti-mouse CD4 and rat anti-mouse CD8 monoclonal antibodies were purchased from BD Pharmingen. FITC-labeled tomato lectin
was purchased from Vector. Alexa Fluor 488/568 goat anti-rat/mouse/
rabbit IgG (H + L) and Alexa Fluor 488-conjugated streptavidin were
purchased from Invitrogen. Anti-caspase-3, anti-cleaved caspase-3,
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anti-PARP, anti-cleaved PARP, anti-Desmin and anti-PDGFRβ antibodies
were purchased from Cell Signaling Technology. PE-Cy™ 7 anti-mouse
CD3, PE anti-mouse CD4 and FITC anti-mouse CD8a, PE anti-mouse
CD68, APC anti-mouse CD34, PE-Cy™7 anti-mouse CD45 antibodies,
and isotype IgG were purchased from Biolegend. Anti-Ki67 antibody
was purchased from Dako. In Situ Cell Death Detection Kit (Fluorescein)
was purchased from Roche.
2.3. Orthotopic CRC model
The model was performed as previously described [17]. Briefly, all
mice were given tap water containing 3% dextran sulfate sodium
(DSS) for 7–9 days to induce colitis. Mice were fasted overnight after
colitis induction, and then anesthetized with sodium pentobarbital.
Colon 26 cells (4 × 106 cells/40 μL/mouse) were implanted intrarectally
with a micropipette inserted 2 cm into the anus of mice. The anus was
compressed with a noncrushing microclamp immediately after instillation of cancer cells for at least 30 min to prevent leakage. Successful
models were used for various in vivo analyses at 2 weeks after implantation of cancer cells.
2.4. Toxicitical analysis of TCP-1 peptide
Male BALB/c mice were randomly divided into two groups (n = 5).
The experimental group was intravenously injected with 100 μg/dose/
mouse of TCP-1 peptide and the control group received an equal volume
of phosphate-buffered saline (PBS) alone once every other day. Treatment was terminated 16 days after the first dose of peptide administration. Animals were euthanized 2 days after the last injection. Blood
samples were collected for hematological examination and biochemical
assays via the abdominal aorta. Hematological examinations were performed using an automatic blood cell counter (Sysmex KX-21, Japan)
for the red blood count (RBC), white blood cell count (WBC), hemoglobin (HGB) and platelet count (PLT). Plasma was harvested from blood
samples for biochemical determinations with a clinical automatic biochemical analyzer (HITACHI7020, Japan) for alanine aminotransferase
(ALT), lactic dehydrogenase (LDH), total protein (TP), albumin (ALB),
urea nitrogen (BUN) and creatinine (CREA) levels. Organs were collected
and fixed in 10% formalin solution for histological analysis.
2.5. Plasmid construction
All the plasmids were constructed by routine recombinant DNA
technology. TCP-1 gene was first introduced into our preserved
pET-14b/EGFP plasmid by PCR-mediated site-directed mutagenesis
with the primer pair 5′-ttttcgcattgcggaggtaccgtgagcaagggcgaggag3′ and 5′-aggactaggcgtacaagcgggccccatatggctgccgcgcgg-3′. The
cDNA of mature human TNFα was kindly provided by Prof. Sven
Pfeifer [18]. The TNFα fragment was amplified by PCR with primer pair
5′-catggtaccgtgcgtagcagcagccgtaccc-3′and 5′-catggatcccagcgcaataatgcc
aaaatacacc-3′ flanked by KpnI and BamHI (underlined) restriction enzyme sites. The PCR product was then cloned into a modified pET-14b
vector. Subsequently, the TCP-1 gene was introduced into constructed
pET-14b/TNFα plasmid by PCR as described above. All the constructs
were finally confirmed by DNA sequencing. The flow chart of plasmid
construction is shown in Fig S1.
2.6. Protein expression, purification and verification
The expression of EGFP, TCP-1/EGFP, TNFα and TCP-1/TNFα in the
transformed BL21(DE3) Escherichia coli cells was induced by 1 mM
Isopropyl β-D-Thiogalactoside (IPTG). Various soluble proteins were
purified from bacterial lysates by immobilized metal ion-affinity
chromatography with Ni-NTA resin following the manufacturer's
instructions. All solutions in the purification steps were prepared with
sterile and Milli-Q water. Purified proteins were finally confirmed by
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L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
SDS-PAGE and Western blot with anti-His tag antibody. The salts in the
purified protein solution were exchanged with PBS through Amicon
Ultra Centrifugal Filters (Millipore). A commercial Bradford protein
assay kit (BioRad) was used to quantify the purified protein. Proteins
with N 90% purity based on SDS-PAGE image were used for various examinations. The quantitative chromogenic Limulus amebocyte lysate
(LAL) test was used to quantitate Gram-negative bacterial endotoxin.
The endotoxin concentration in the purified proteins used in the study
is approximately 0.1 EU/μg.
2.7. TNFα activity assay
The activities of TNFα and TCP-1/TNFα were determined in L929 and
Colon 26 cells as previously described [18]. Briefly, 2 × 104 cells/well
were seeded in a 96-well plate with 100 μL growth medium. After 24 h,
cells were treated with 1 μg/mL actinomycin D for 30 min followed by
the treatment with different concentrations of TNFα or TCP-1/TNFα for
an additional 24 h. Subsequently, MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) method was used to detect the cell
viability.
2.8. Animal treatment
To investigate whether TCP-1 peptide is able to deliver large proteins
to tumor blood vessels, 50 nmol TCP-1/EGFP fusion protein or equal
molar EGFP was i.v. injected into mice bearing orthotopic CRC to examine the distribution of EGFP. The protein was allowed to circulate for 1 h.
Tumor and control tissues were collected and prepared for frozen
section. Blood vessels were stained with anti-CD31 antibody followed
by incubation with Alexa Fluor 568-conjugated secondary antibody.
EGFP signal was amplified by biotin-labeled anti-His tag antibody
followed by Alexa Fluor 488-conjugated streptavidin.
For antitumor treatment, mice bearing orthotopic CRC were randomized into three groups (n = 6). The therapeutic group was i.v.
injected with TCP-1/TNFα (5 μg/dose/mouse). The control groups
received an equal amount TNFα or PBS alone. Mice were euthanized
at 24 h after drug injection. Tumors and control organs were dissected
and prepared for frozen sections. Tumor microvessel density and
apoptosis were assessed.
For combination therapy of TCP-1/TNFα with 5-FU in orthotopic CRC
model, mice were treated in six groups (n = 5): PBS, 5-FU (20 mg/kg,
100 μL), TNFα + 5-FU, TCP-1/TNFα + 5-FU, TNFα and TCP-1/TNFα
(TNFα or TCP-1/TNFα: 1 ng/mouse, 100 μL). For TNFα + 5-FU and
TCP-1/TNFα + 5-FU groups, mice were i.v. injected with TNFα or
TCP-1/TNFα in a 0.9% NaCl solution. Two hours later, 5-FU diluted
with 0.9% NaCl was i.p. injected into mice. Mice were euthanized at
the 10th day after drug injection. Tumor volume was measured and
calculated by the formula length × width2 / 2. Tumor size and volume
were shown as mean ± SEM.
2.9. Measurements of intratumoral 5-FU and anti-tumor immunity
For the HPLC analysis of 5-FU, mice were i.v. injected with TNFα or
TCP-1/TNFα (1 ng/mouse, 100 μL) prepared in 0.9% NaCl solutions.
Two hours later, 5-FU (20 mg/kg, 100 μL) diluted with 0.9% NaCl was
i.p. injected into mice. After 30 min of circulation, tumors were dissected
and homogenized for the HPLC analysis of 5-FU. Various concentrations
of 5-FU were prepared for the standard curve construction. HPLC system
consisted of a HPLC pump, a UV Detector and Kromasil C18 (5 μm
200 mm × 4.6 mm) analytical column. The mobile phase consisted of
acetonitrile and H2O (1:99, v/v). The flow rate was 1.0 mL/min and
the detection wavelength was at 265 nm. Sample injection volume
was 10 μL and the column temperature was 35 °C.
For antitumor immunity assessment, mice bearing orthotopic CRC
were randomized into three groups (n = 5). The therapeutic group
was i.v. injected with TCP-1/TNFα (1 μg/dose/mouse). The control
groups received an equal amount TNFα or PBS alone. Mice were euthanized at 24 h after drug injection. Spleen, femur and whole blood were
collected for flow cytometry.
2.10. Histology
Frozen tissue sections (10 μm) were cut, air-dried on slides, and then
blocked with 10% normal goat serum in PBS. For immunofluorescent
staining, tissue sections were incubated overnight in 10% normal goat
serum containing various primary antibodies(anti-cleaved caspase-3,
anti-cleaved PARP, anti-Desmin, anti-PDGFRβ, anti-CD4 or anti-CD8 antibody). Slides were then rinsed three times with PBS for 5 min each and
incubated for 1 h with a 0.22 μm filtered secondary antibody solution
(10% normal goat serum) containing Alexa Fluor 488/568. For TUNEL
assay, In Situ Cell Death Detection Kit (Roche) was used for the detection of apoptotic cells in frozen tissues following the manufacturer's instructions. For lectin perfusion assay, mice were i.v. injected with 50 μg
FITC-labeled lectin. After 10 min, mice were heart-perfused with 2%
neutral-buffered formalin and tumors were obtained for frozen section.
Colocalization with endothelial cells (CD31-positive) was performed as
described above. Staining of nuclei was performed with DAPI and sections were mounted with nail polish. Fluorescent signals were detected
using a confocal fluorescence microscope (Nikon EZ-C1, Nikon, Tokyo,
Japan). For immunohistochemical staining, blood vessels and proliferating cells were stained by rat anti-CD31 and anti-Ki67 antibodies, respectively, followed by signal amplification using the anti-rat Ig HRP
Detection kit (BD Pharmingen™). Histological assessment was performed after H&E staining.
2.11. Western blots
Protein concentration was determined by the BCA protein assay
(Pierce, Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE
and were transferred to PVDF membranes. The membranes were
probed with primary antibodies: anti-cleaved caspase-3 and cleaved
PARP antibody (1:1000) and anti-GAPDH antibody (1:5000), overnight
at 4 °C and followed by 1 hour incubation with horseradish peroxidase
(HRP)-conjugated secondary antibody. The membrane signals were
developed with LumiGLO reagent and detected and quantified by the
ChemiDoc XRS gel documentation system (Bio-Rad Laboratories,
Hercules, USA).
2.12. Flow cytometry
Twenty-four hours after drug administration, mice were anesthetized
by i.p. injection of a mixture of ketamine (75 mg/kg) and xylazine
(10 mg/kg). For the preparation of plasma samples, whole blood was collected in a 1.5 mL centrifuge tube pre-coated with 20 μL of 1250 U/mL
heparin by intra-cardiac puncture. Hematopoietic cells were collected
from femurs by bone marrow aspiration and washed with PBS supplemented with 2% FBS. After treatment with erythrocyte lysis buffer
(BD, Biosciences), cells were resuspended in 100 μL PBS supplemented
with 2% FBS. The mouse spleens were immediately removed after cervical dislocation, and single-cell suspension of splenocytes was produced
using gentle homogenizing by a plunger of a 5-mL syringe (BD, USA)
and passing through a 200-mesh sieve [19]. Splenocytes were freshly
used for the following experiments after treatment with erythrocyte
lysis buffer. The cells were resuspended at 2 × 106 cells/mL in RPMI1640 medium supplemented with 10% FBS. For determination of
lymphocytes/macrophages, samples were stained with anti-mouse
antibodies: PE-Cy™ 7 anti-mouse CD3, PE anti-mouse CD4 and FITC
anti-mouse CD8a, PE anti-mouse CD68 or IgG isotype for 30 min at
4 °C in the dark. For measurement of hematopoietic cells, cells were
stained with APC anti-mouse CD34, PE-Cy™ 7 anti-mouse CD45 or IgG
isotype for 30 min at 4 °C in the dark. Cells were washed thrice with
PBS and analyzed by flow cytometry. The CD34bright CD45dim cells
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
were selected for comparison and data from 10,000 cells were collected
and analyzed.
2.13. Statistical analysis
The results are expressed as mean ± SEM. Toxicity assessments of
TCP-1 were analyzed by two-tailed t-test between two groups. For the
in vivo treatment experiment with TCP-1 fusion protein, comparisons
among all groups were analyzed by one-way ANOVA followed by the
Tukey's test. P value below 0.05 was considered statistically significant.
3. Results
3.1. Toxicity assessment of TCP-1 peptide
The toxicity of TCP-1 peptide was examined in vitro and in vivo
(Fig. 1). In the in vitro testing, we investigated the effect of TCP-1 peptide on cell viability by MTT assay in a human normal colon fibroblast
cell line, a murine CRC cell line and a panel of human colon cancer cell
lines (Fig. 1A). TCP-1, even at a high concentration (100 nM), exhibited
no significant effect on cell viability in these cell lines. We also examined
the possible toxicity of TCP-1 in mice. Results indicated that 100 μg TCP1 which was more than a thousand times higher than the therapeutic
dose we used, did not induce any toxicity in mice after 8 successive injections based on the measurements of various parameters, including
body weight (Fig. 1B), the hematological profiles (Fig. 1D), renal and hepatic functions (Fig. 1C and E). Meanwhile, there was no detectable
pathological change in various organs in the two groups, suggesting
that TCP-1 did not produce any pathological tissue damage after repeated administrations (Fig. S2). We also confirmed that TCP-1 peptide did
not induce obvious immunogenic reaction when compared with the
phage particles after repeated administration of these compounds in
mice (Fig. S3). All these findings imply the safety of using TCP-1 as a
drug carrier in vivo.
3.2. Delivery of EGFP by TCP-1 to tumor vasculature
To confirm the homing ability of TCP-1 peptide, we constructed the
plasmid integrated TCP-1 gene with EGFP gene into the pET-14b vector
(Figs. 2A and S1). The clones efficiently expressing EGFP and TCP-1/
EGFP were rapidly and successfully screened through IPTG induction
(Fig. 2B). Two recombinant proteins were purified by NI-NTA resin
which could bind to His tag of the proteins and showed bright green
color in appearance (Fig. 2C and D). His tag antibody confirmed that
the fusion proteins were attained (Fig. 2E). Previously we have clarified
that TCP-1 was capable of carrying fluorescein and a short peptide to a
tumor mass [17]. However, its capacity to carry large proteins remains
to be tested. Herein, we assessed the delivering ability of TCP-1 through
TCP-1/EGFP fusion protein. We injected 50 nmol EGFP or TCP-1/EGFP
into mice bearing orthotopic CRC through tail veins. Data showed that
TCP-1/EGFP could colocalize with tumor vasculature, but not with
blood vessels in normal organs including heart, brain, liver and normal
colon tissues (Fig. 2F). Meanwhile, EGFP was not detectable in the
tumor vasculature (Fig. 2F). EGFP didn't show homing ability on
tumor blood vessels without TCP-1delivery. These results indicated
that the large protein did not negatively affect the binding ability of
TCP-1 to tumor vasculature. We therefore hypothesized that TCP-1
peptide could deliver large therapeutic proteins (e.g. TNFα) to tumor
vasculature, although TNFα of equal molar to EGFP could not be injected
into mice for the targeting assessment due to its lethal toxicity.
3.3. Targeted delivery of TNFα by TCP-1 in the orthotopic CRC model
To investigate the effect of TCP-1 coupling with TNFα, TNFα and
TCP-1/TNFα fusion proteins were prepared. TNFα and TCP-1/TNFα
proteins were expressed and purified in E. coli by similar procedures
137
(Fig. 3 A–C). Consistent with the fact that TNFα could form a
homotrimer, reducing SDS-PAGE of TNFα and TCP-1/TNFα mainly
showed a single band of around 20 kD (TNFα, 20 kD; TCP-1/TNFα,
22 kD after fusion with His tag), which was expected for monomeric
TNFα (Fig. 3C), whereas nonreducing SDS-PAGE of these two proteins
showed three forms of around 20, 40, 60 kD, corresponding to
monomers, dimers, and trimers, respectively (data not shown). The
toxic activities of TNFα and TCP-1/TNFα were determined in a murine
fibrosarcoma cell line (L929) and a colonic adenocarcinoma cell line
(Colon 26). TCP-1/TNFα had no obvious difference from TNFα as determined by the standard cytotoxicity assay (Fig. 3D). Fusion of TNFα with
TCP-1 did not affect the cytotoxicity of TNFα on these cell lines. These
findings indicated that TCP-1 peptide did not change TNFα folding,
oligomerization, activity and binding to TNFα receptors, thereby producing equipotent cytotoxicity on cells. It has been shown that highdose TNFα in vivo could inhibit tumor vascularization by damaging vascular endothelial cells, thereby decreasing the blood flow and oxygen
required for the progression of tumor growth. Nevertheless, high-dose
TNFα was accompanied by severe toxicity in normal tissues [6,7,20].
We briefly investigated whether TCP-1 peptide could improve the therapeutic effect of TNFα on tumor vasculature. Results showed that 5 μg
TNFα produced a slight vasculature injury when compared with the
PBS control. In contrast, 5 μg TCP-1/TNFα resulted in a more obvious
tumor vascular damage and significant reduction in microvessel density
in tumor tissues at 24 h after treatment when compared with 5 μg TNFα
or PBS (Fig. 3E–F). TUNEL assay also showed that TCP-1/TNFα induced
more apoptosis in tumors and the associated blood vessels when compared with PBS or unconjugated TNFα (Fig. 3G). Consistently, the
level of cleaved caspase-3 was higher in the TCP-1/TNFα group than
the TNFα or PBS alone group (Fig. 3H). Loss of body weight has been
shown to be a systemic toxicity of TNFα treatment [21]. Nevertheless,
short-term administration of TNFα or TCP-1/TNFα groups did not
show a marked loss of body weight (b 5% of body weight) (Fig. S4).
Our results indicate that TCP-1 could be used to deliver TNFα as an
anti-angiogenesis drug, which exerted antitumor activity in an
orthotopic CRC model.
3.4. Antitumor activity of TCP-1/TNFα in combination with 5-FU in the
orthotopic colorectal cancer model
Several lines of evidence suggested that targeted delivery of
subnanogram TNFα improved the drug accumulation and absoption
into tumor tissues and obviously enhanced the efficacy of various chemotherapeutic drugs [6,7,22–26]. We next examined if a low dose of
TCP-1-conjugated TNFα could be used for CRC treatment in combination with 5-FU, a commonly used chemotherapeutic agent, and further
enhance the anti-tumor activity of 5-FU. For in vivo anticancer study, the
dose of 5-FU normally ranges from 10 to 50 mg/kg [27–30]. We chose
the moderate dose (i.e. 20 mg/kg) that could partly inhibit tumor
growth and avoid masking the action of TCP-1/TNFα. The antitumor activity of low-dose TCP-1/TNFα in combination with 5-FU in the
orthotopic CRC model was studied in six groups: Control, 5-FU,
TNFα + 5-FU, TCP-1/TNFα + 5-FU, TNFα alone and TCP-1/TNFα
alone (5-FU: 20 mg/kg, 100 μL; TNFα or TCP-1/TNFα: 1 ng/mouse,
100 μL). The dose of TNFα, 1 ng/mouse, was regarded as an extra low
dose [31]. Compared with the control, 5-FU slightly but not significantly
decreased tumor growth. However, when combined with TNFα or TCP1/TNFα, the single administration of 5-FU (20 mg/kg) significantly decreased tumor growth to a greater extent. TCP-1/TNFα showed more
prominent antitumor effect than TNFα in the combined treatment
(P b 0.01, Fig. 4B). Parameters of systemic toxicity were also determined,
including loss of body weight and blood cell count. These parameters
are known to be the vital signs of systemic toxicity following 5-FU treatment during anti-cancer therapy [32]. The current data indicated that
there was no significant systemic toxicity (Fig. S5), which was likely
due to administration of a single, modest dose of 5-FU and a low dose
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Fig. 1. Systemic toxicity assessment of TCP-1 peptide in vitro and in vivo. (A) Effects of TCP-1 peptide on cell viability. Cells were incubated with increasing concentrations (0.5, 1, 5, 10, 50
and 100 nM) of TCP-1 peptide for 48 h. Cell viability was determined by MTT assay. (B) Body weights of mice following 8-successive i.v. injections every other day. (C) Renal functions,
(D) hematological parameters and (E) hepatic functions were analyzed on the next day after the final TCP-1 administration. The experimental groups were i.v. injected with TCP-1 peptide
(100 μg/dose/mouse) and the control group received an equal volume of PBS alone throughout 8-successive injections every other day (n = 5). Hematological examinations were
performed for the red blood count, white blood cell count, hemoglobin and platelet count. Biochemical analysis included measuring the plasma levels of alanine aminotransferase, lactic
dehydrogenase, total protein, albumin, urea nitrogen and creatinine. Data were presented as mean ± SEM.
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
139
Fig. 2. Characterization of TCP-1/EGFP fusion protein and biodistribution of TCP-1/EGFP in tumor-bearing mice. (A) Schematic representation of TCP-1/EGFP and EGFP fusion proteins.
TCP-1 was fused to the N-terminal of EGFP. (B) Induction of EGFP and TCP-1/EGFP in E. coli by IPTG. IPTG addition could obviously induce the expression of both fusion proteins.
(C) Purification of EGFP and TCP-1/EGFP by Ni-NTA resin. SDS-PAGE result indicated the attainment of high-purity proteins with approximately 30 kD molecular weight. (D) Bright
green color appearance of EGFP and TCP-1/EGFP. (E) Verification of both fusion proteins by the Western blots with anti-His tag antibody. The specific bands shown in the membrane
corresponded to 30 kD as expected. (F) EGFP or TCP-1/EGFP was i.v. injected into tumor-bearing mice. Mice were sacrificed 1 h later and the EGFP localization was detected in various
tissues by anti-His tag antibody (green). TCP-1/EGFP was colocalized with CD31 (red) in the tumor blood vessels but not in the control organs including the heart, brain, liver and normal
colon tissues. EGFP alone did not bind to the blood vessels of tumor tissues. Original magnification: 400×. Scale bar: 50 μm.
of TNFα. 5-FU, principally used as a thymidylate synthase inhibitor in
anticancer therapy, could induce apoptosis [33]. To further elucidate
the antitumor action of TCP-1/TNFα + 5-FU, the level of apoptosis was
examined. As expected, the group treated with TCP-1/TNFα + 5-FU
had the highest number of apoptotic cells (P b 0.05, Fig. 4C and D),
disclosing that TCP-1/TNFα produced synergism with 5-FU in the
induction of apoptosis. Concordantly, western blot and immunofluorescence staining for cleaved caspase-3 and cleaved PARP, two crucial markers involved in the apoptotic signaling, revealed that TCP1/TNFα + 5-FU markedly induced more apoptosis as compared
with other groups (Fig. 4E and F). As 5-FU has been reported to inhibit cell proliferation by inducing G1/S phase arrest, we examined the
inhibitory action on cancer cell proliferation by detecting the expression of Ki67 (Fig. 4G). TCP-1/TNFα markedly potentiated the inhibitory effect of 5-FU on cell proliferation when compared with TNFα
(P b 0.05, Fig. 4H).
3.5. TCP-1/TNFα significantly improved 5-FU absorption in tumor mass
We next determined if TCP-1/TNFα led to increased absorption of
5-FU in tumors by HPLC (Fig. 5). The blank sample and standard are
shown in Fig. 5A and B, respectively. Retention time was found to be
about 5.5 min with resolution value N2. Various concentrations of
5-FU were prepared for the standard curve construction and
measurement. Correlative coefficient of regression under our experimental conditions was over 0.99. Results showed that 2-hour pretreatment with TCP-1/TNFα (1 ng/mouse) significantly increased 5-FU
concentration in the tumor tissue (Fig. 5C and D), which may explain
the synergistic effect of TCP-1/TNFα and 5-FU in our orthotopic CRC
model.
3.6. Effect of TCP-1/TNFα on tumor blood vessel normalization
Morphologically, the tumor vasculature is very distinct from the
normal blood vessels. Many studies disclosed that low-dose targeted
TNFα could stabilize, remodel and normalize tumor blood vessels, so
as to enhance active immunotherapy or drug absorption [22–26]. In
our study, vascular alteration and morphology were observed by immunohistochemical staining (Fig. 6A). The microvessel density showed no
significant difference in all groups (Fig. 6C), indicating that unlike the
high dose, TNFα or TCP-1/TNFα at low dose did not affect angiogenesis
in the tumor. However, in the groups treated with TCP-1/TNFα (including TCP-1/TNFα and TCP-1/TNFα + 5-FU), the number of blood vessels
with open blood vessel lumen were remarkably increased (Fig. 6B,
P b 0.05) while the tumor tissue became looser (Fig. 6A). Accordingly,
the cell density in tumors was also markedly reduced in the groups
treated with TCP-1/TNFα (Fig. 6D). Pericyte coverage is an important indicator for vessel maturation and functionality. In the untreated tumors,
immature PDGFRβ+ pericytes were found to closely attach to tumor
blood vessels while mature desmin+ cells located near the blood vessels
were mostly detached. In contrast, low-dose TNFα or TCP-1/TNFα with
or without 5-FU markedly increased the coverage of mature desmin+
pericytes and reduced the coverage of immature PDGFRβ+ pericytes,
suggesting normalization of tumor blood vessels (Fig. 6E, G and H). Furthermore, such normalization resulted in increased vascular perfusion,
which was proven by delivery of FITC-conjugated lectin to tumor
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Fig. 3. TCP-1/TNFα significantly increased tumor vasculature destruction. (A) Schematic representation of TCP-1/TNFα and TNFα fusion proteins. The TCP-1 peptide was fused to the Nterminal of TNFα. (B) Induction of TNFα and TCP-1/TNFα in E. coli by IPTG. IPTG addition slightly increased the expression of both fusion proteins. (C) Purification of TNFα and TCP-1/TNFα
by Ni-NTA resin. A large quantity of proteins was obtained in soluble form. (D) Activity analysis of TCP-1/TNFα and TNFα on L929 and Colon 26. Cell viability was determined by MTT assay.
TCP-1/TNFα had no significant difference as compared with TNFα alone. (E) TCP-1/TNFα significantly increased tumor vasculature destruction. Immunohistochemical staining (DAB development) of endothelial cells of blood vessel and hematoxylin staining for locations of total cells. Localization for endothelial cells was performed using anti-CD31 antibody. Original
magnification: 200×. Scale bar: 50 μm. (F) Microvessel density (MVD) quantitation. TCP-1/TNFα significantly increased tumor vasculature destruction compared with non-targeted
TNFα (P b 0.05) or PBS alone (P b 0.001) 24 h after a single injection. (G) Tumor vasculature and tumor cell apoptosis. Apoptotic cells in tumor mass (green) were detected by TUNEL
assay and colocalization with endothelial cells of blood vessels (red) was marked using anti-CD31 antibody. Staining of nuclei was performed with DAPI. TCP-1/TNFα could obviously
induce more apoptosis of endothelial cells and tumor cells when compared with the non-targeted TNFα. Images were captured by confocal immunofluorescence microscopy. Original
magnification: 400×. Scale bar: 50 μm. (H) Western blot for detecting the protein levels of cleaved caspase-3 (pooled samples in each group). *P b 0.05. Data were presented as
mean ± SEM.
vessels after treatment (Fig. 6F and I). Such effects were more prominent in TCP-1/TNFα + 5-FU as compared with other groups. In addition,
such vascular alterations were very similar in the groups treated with
TCP-1/TNFα (including TCP-1/TNFα and TCP-1/TNFα + 5-FU), indicating that these actions derive from TCP-1/TNFα but not 5-FU.
3.7. TCP-1/TNFα enhanced antitumor immunity
Targeted low-dose TNFα into solid tumors with resultant vessel
stabilization can be exploited to “precondition” the tumor microenvironment for active immunotherapy. The highly stabilized vascular network facilitates immune cell extravasation and infiltration, thereby
enhancing active antitumor immunity. Importantly, the composition
of tumor-targeted T cell infiltration is a major prognostic factor in CRC
outcome [34]. Immunofluorescence staining was thus used to quantify
the number of intratumoral CD3+CD4+ and CD3+CD8+ lymphocytes.
Our data suggested that CD3+CD8+ lymphocytes were obviously increased in the groups treated with low-dose TCP-1/TNFα (including
TCP-1/TNFα and TCP-1/TNFα + 5-FU, Fig. 7A and B), indicating that
the effect of lymphocyte accumulation was derived from TCP-1/TNFα
but not 5-FU.
Next, we asked whether targeted TNFα by TCP-1 could alleviate
immunosuppression and elicit mobilization of immune cells from
normal tissues such as bone marrow, spleen and peripheral blood. To
this end, a higher dose of TCP-1/TNFα (1 μg/mouse) was utilized to obtain a clear demonstration for immune cell analysis. This dose of TNFα
can't result in acute vessel destruction like 5 μg. The analysis of bone
marrow hematopoietic cells suggested that TNFα (1 μg/mouse) can
slightly inhibit the CD34bright CD45dim hematopoietic cells, whereas
TCP-1/TNFα (1 μg/mouse) can restore the loss of CD34bright CD45dim hematopoietic cells (P b 0.01, Fig. 7C), indicating that targeted TNFα is instrumental in alleviating immunosuppression in cancer therapy.
Similarly, the peripheral blood cell analysis further disclosed that
TNFα decreased the number of CD3+, CD3+CD4+ and CD3+CD8+
cells, but TCP-1/TNFα can restore them (Fig. 7D). On the other hand,
in spleens, the percentages of CD3+, CD3+CD4+ and CD68+ cells
were found decreased and CD3+CD8+ lymphocytes were as well significantly reduced in the mice treated with TCP-1/TNFα when compared
with TNFα treatment (P b 0.05, Fig. 7E). Taken together, these data
strongly imply that targeted TNFα by TCP-1 could obviously ameliorate
the immunosuppression which is an adverse effect associated with
TNFα monotherapy, and consequently cause CD3+CD8+ cells to extravasate from the spleen and then infiltrate into CRC tissues. Meanwhile,
the normalized tumor blood vessels induced by targeted TNFα enable
these immune cells to effectively penetrate into the tumor mass,
which resultantly enhances the antitumor immunity.
4. Discussion
To improve the therapeutic index of TNFα in cancer therapy,
targeted delivery of this cytokine to tumor vasculature has been
developed for its systemic administration with promising potential in
preclinical settings [35–37]. TNFα can be fused with peptide ligands
or antibody fragments specifically homing to tumor blood vessels
which are distinguished from normal tissue vasculature structurally
and functionally and exclusively express their own specific molecular
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
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Fig. 4. Antitumor activity of TCP-1/TNFα in combination with 5-FU in the orthotopic CRC model. (A) Schematic representation of treatment regimen in the orthotopic CRC model.
(B) Tumor size and weight in the six groups: control, 5-FU, TNFα + 5-FU, TCP-1/TNFα + 5-FU, TNFα and TCP-1/TNFα (n ≥ 4 per group). Compared with the control group, 5-FU slightly
decreased tumor weight and size but markedly reduced the tumor growth when combined with TNFα (P b 0.01) or TCP-1/TNFα (P b 0.001). TCP-1/TNFα showed more prominent
inhibitory effect than TNFα in this combined drug treatment (P b 0.01). (C) Apoptosis in tumor mass detected by TUNEL assay. Apoptotic cells in tumor mass (green) were detected by
TUNEL assay and endothelial cells (red) were visualized using anti-CD31 antibody. Staining of nuclei was performed with DAPI (blue). Original magnification: 200×. Scale bar: 50 μm.
(D) Quantitation of apoptotic cell number. TCP-1/TNFα combined with 5-FU significantly increased number of apoptotic cells in tumors. The effect was more prominent as compared
with other groups. (E) Western blot for cleaved caspase-3 and cleaved PARP (pooled samples in each group). (F) Immunofluorescence staining for cleaved caspase-3 and cleaved
PARP. Expression for cleaved caspase-3 and cleaved PARP in tumor tissues (green) were detected by anti-cleaved caspase-3 antibody and blood vessels were labeled by anti-CD31 antibody
(red). Staining of nuclei was performed with DAPI (blue). Original magnification: 400×. Scale bar: 50 μm. (G) Histological analysis of proliferating cells. Immunohistochemical staining
(DAB development) and hematoxylin staining were used for visualizing Ki67-positive cells and total cells, respectively. (H) Quantitation of proliferating cells. The number of proliferating
cells in the TCP-1/TNFα + 5-FU group was significantly decreased when compared with the TNFα + 5-FU group and other groups. Original magnification: 400×. Scale bar: 50 μm. Data
were presented as mean ± SEM. *P b 0.05. **P b 0.01. ***P b 0.001.
biomarkers. The fusion modifications with targeted agents could enable
TNFα to specifically accumulate in the tumor mass and exert its antitumor activity to the greatest extent. Further investigations also demonstrated that targeted TNFα can greatly enhance the efficacy of
chemotherapeutic drugs when they were administered in combination
with other anti-cancer drugs [11,22,24]. Based on these findings, several
clinical trials at different phases are underway in the treatment of solid
tumors [13,15,38,39]. A number of peptides against tumor blood vessels
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Fig. 5. TCP-1/TNFα significantly improved 5-FU accumulation in tumors. HPLC chromatograms of (A) blank sample, (B) standard and (C) tumor samples in groups treated with 5-FU,
TNFα + 5-FU or TCP-1/TNFα + 5-FU. Retention time and peak area are shown. Arrows indicate the location of 5-FU peaks in the chromatographs. (D) 5-FU concentration in
samples from 5-FU, TNFα + 5-FU and TCP-1/TNFα + 5-FU groups. Results show that TCP-1/TNFα significantly increased 5-FU concentration in tumor mass. Data are presented as
mean ± SEM. *P b 0.05.
or tumor cells have been identified by phage display biopanning and
applied for targeted diagnosis and therapy [40–44]. Unlike the NGR
and RGD vascular-targeting peptide which can generally target
neovasculature in various tumors, TCP-1 has a unique homing ability
to the vasculature only in the CRC, indicating its specificity and accuracy
as a drug carrier in CRC therapy [17].
In general, the mechanisms related to antitumor functions of TNFα
emphasize the direct cytotoxic effects on tumor cells and indirect
destruction of tumor-associated blood vessels [10,45,46]. However,
different doses of TNFα induce different effects on cancer therapy. It has
been shown that a high dose of TNFα could inhibit tumor angiogenesis
through damaging vascular endothelial cells in the vicinity of the tumor
mass, thereby decreasing the blood flow and oxygen required for the progression of tumor growth. In this regard, our study demonstrated that a
high dose of TCP-1/TNFα exerted more potent antitumor activity than
the unconjugated TNFα in the induction of apoptosis and reduction of
microvessel density in the orthotopic CRC model, which is compatible
with human CRC, with no obvious systemic toxicity. However, a low
dose of TNFα, acts through distinct mechanisms from a high dose. For
example, it could not only alter tumor vessel permeability and further
improve the drug penetration into neoplastic tissues but also normalize
the tumor blood vessels. This in turn could promote drug delivery into
the tumor mass [6,11,12,47]. Drug-penetration barriers and interstitial
fluid pressure are believed to be the two major barriers preventing drug
penetration into a tumor mass [48–56]. Low-dose of targeted TNFα
could increase endothelial permeability and decrease interstitial
fluid pressure, thereby accelerating drug penetration into neoplasm. A
growing body of evidence also suggested that targeted delivery of
subnanogram TNFα could obviously enhance the efficacy of various
chemotherapeutic drugs through different mechanisms to increase the
therapeutic potential in antitumor therapy [22–26]. Targeted delivery of
a low-dose of TNFα by NGR, a vascular-targeted peptide selectively recognizing an aminopeptidase N (CD13) isoform expressed on endothelial
cells in tumor vessels, increases the antitumor efficacy of several chemotherapeutic drugs [24,57]. In our study, we investigated the synergistic
antitumor activity of 5-FU, a first-line anti-cancer agent for CRC treatment, together with a low dose of TCP-1/TNFα in an orthotopic CRC animal model. Following this treatment schedule, we found that TCP-1/
TNFα pretreatment could significantly potentiate the anti-cancer action
of 5-FU through reduction in tumor size and weight, induction of apoptosis and inhibition of cell proliferation when compared with the
unconjugated TNFα. Further investigation disclosed that TCP-1/TNFα
promotes the accumulation of 5-FU in the tumor mass. Because the
2-hour pretreatment paradigm was employed in our treatment schedule
[24], we speculate that the increased 5-FU in the tumor mass is more
likely a result of the acute alteration of vessel permeability rather than
of the tumor vasculature normalization which should be elicited in the
later stage of TCP-1/TNFα treatment. Nevertheless, all these alterations
in blood vessels could enhance the anti-cancer action of 5-FU in CRC.
In addition to the improvement of drug accumulation in the tumor
mass, a low dose of targeted TNFα is capable of increasing vascular functionality and thus enhancing antitumor immunity. Abnormal tumor
vasculature can serve as a barrier for T lymphocyte adhesion and migration into neoplastic tissues, limiting the therapeutic effectiveness of immunotherapies. A low dose of targeted TNFα could stabilize the vascular
network, promote vasculature remodeling and improve blood vessel
perfusion. All these actions could facilitate the access of effector cells
into tumor tissues, resulting in more effective antitumor immune
responses [58]. In our study, targeted delivery of TNFα by TCP-1 effectively causes tumor blood vessel normalization and remodeling. To a
large extent, this could effectively reverse the “angiogenic switch”
in tumor tissue and transform tumor blood vessels from abnormal to
normalized vasculature. Normalized tumor blood vessels could potentially prime the tumor microenvironment and further facilitate
intratumoral immune cell infiltration [59]. As expected, TCP-1/TNFα
markedly enhanced intratumoral CD8+ T lymphocyte infiltration and
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
143
Fig. 6. Effects of TCP-1/TNFα on tumor blood vessel normalization. (A) Histological analysis and quantitation of tumor blood vessels. Immunohistochemical staining of blood vessel was
performed using endothelial marker CD31. Blue arrows indicate the blood vessels with collapsed lumens in TNFα and TNFα + 5-FU groups and red arrows indicate the blood vessels with
open lumens in TCP-1/TNFα and TCP-1/TNFα + 5-FU groups. Original magnification: 200×. Scale bar: 50 μm. (B) Percentage of blood vessels with open lumens in all groups. TCP-1/TNFα
and TCP-1/TNFα + 5-FU groups show the highest percentage of blood vessels with open lumens (P b 0.05). (C) The numbers of blood vessels in all groups showed no significant difference,
suggesting that low-dose TNFα or TCP-1/TNFα did not affect angiogenesis. (D) Cell number per field in tumors in each group. The cell number per field in the TCP-1/TNFα and TCP-1/
TNFα + 5-FU group were reduced significantly (P b 0.05). (E) TCP-1/TNFα enhanced blood vessel normalization as revealed by increased coverage of mature pericytes and reduced
coverage of immature pericytes. Mature pericytes (green; upper) and immature pericytes in tumor (green; lower) was detected by anti-desmin and anti-PDGFRβ antibodies, respectively.
Endothelial cells of blood vessels (red) are visualized using anti-CD31 antibody. Staining of nuclei was performed with DAPI (blue). Original magnification: 400×. Scale bar: 50 μm.
(F) Tumor blood vessels (left) in relation to FITC-lectin (right) after injection. FITC-lectin (i.v. injected) in tumor mass (green) was amplified by anti-fluorescein/Oregon Green, Alexa
Fluor 488-conjugated antibody and blood vessels (red) are visualized using anti-CD31 antibody. TCP-1/TNFα markedly increased lectin accumulation in the tumor blood vessels indicating
higher blood perfusion efficiency when compared with the non-targeted TNFα. Dashed line separates perfused and nonperfused tumor areas. (G) Percentage of CD31-positive endothelial
cells covered by desmin-positive pericytes. (H) Ratios of PDGFRβ-positive pericytes to CD31-positive endothelial cells. (I) Ratios of lectin-positive vessels to CD31-positive blood vessels.
Original magnification: 400×. Scale bar: 50 μm. Data are presented as mean ± SEM. *P b 0.05. **P b 0.01.
simultaneously alleviated immunosuppression in bone marrow
through restoring the loss of hematopoietic stem cells. CD8+ lymphocytes as crucial effectors benefit the antitumor immunity. Besides the effects on the vascular compartment, other activities of TNFα may also
include intratumoral upregulation of endothelial leukocyte adhesion
molecules, release of proinflammatory cytokines and effects on macrophage polarization [12,26].
For the receptor of TCP-1 peptide, a series of pull-down assays have
been performed in our laboratory to isolate the receptor of TCP-1 in CRC
tissue which should be a biomarker exclusively expressed or highly
expressed in the tumor blood vessels. But no confirmed candidate
has been identified at present. Although the receptors of TNFα are
expressed in both tumor and normal tissues, more TNFα will relatively
be accumulated into tumor tissue after TCP-1/TNFα injection. Locally
concentrated TNFα maximizes its various anti-tumor activities
described above and minimizes its toxic side effects.
TNFα as an inflammatory cytokine with potent anti-tumor activity
has failed in clinical trials against diverse types of cancers including
colorectal cancer owing to prohibitive toxicity after systemic administration. TNFα fused with various targeting agents such as peptides and
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Fig. 7. TCP-1/TNFα enhanced active immunotherapy. (A) Immunofluorescence staining of CD3+CD4+ and CD3+CD8+ cells (red) in the normal colon and tumor tissues. Mice bearing
orthotopic CRC were treated with PBS, 5-FU, TNFα + 5-FU, TCP-1/TNFα + 5-FU, TCP-1/TNFα (5-FU: 20 mg/kg, 100 μL; TNFα or TCP-1/TNFα: 1 ng/mouse, 100 μL). For TNFα + 5-FU
and TCP-1/TNFα + 5-FU groups, mice were pretreated with the therapeutic proteins 2 h before 5-FU injection. Tissue sections were incubated with anti-CD4/CD8 antibody. Staining of
nuclei was performed with DAPI. Original magnification: 200×. Scale bar: 50 μm. (B) Quantitation of CD3+CD4+/CD3+CD8+ cells in the tumor tissues. TCP-1/TNFα + 5-FU and TCP-1/
TNFα significantly increased CD8+, but not CD4+ cells, in the tumor when compared with the TNFα + 5-FU group (P b 0.05). (C) CD34bright CD45dim hematopoietic cells in bone marrow.
Mice bearing orthotopic colorectal cancer were i.v. injected with TCP-1/TNFα, TNFα, or PBS alone (TCP-1/TNFα or TNFα: 1 μg TNFα /mouse), allowing circulation for 24 h. TNFα induced
myelosuppression in bone marrow, while TCP-1 could restore the loss of hematopoietic cells (P b 0.01). (D) Lymphocytes in peripheral blood. TNFα induced immunosuppression in peripheral blood, while TCP-1 could partially restore the loss of lymphocytes in the peripheral blood circulation. (E) Percentage of CD3+, CD3+CD4+, CD3+CD8+ and CD68 cells in spleens.
TCP-1/TNFα decreased the percentages of CD3+, CD3+CD4+ and CD68 (P b 0.05) and significantly reduced CD3+CD8+ lymphocytes in spleen (P b 0.01). Data are presented as mean ±
SEM. *P b 0.05.
antibody fragments have been widely investigated in different studies,
and some of them have been tested in clinical [8,25,36,37,60]. The research evidence has revealed that targeted delivery of TNFα could dramatically minimize the side effects of this cytokine, further significantly
improving its local concentration and increasing its anti-tumor activity,
which paves the way to redeveloping the TNFα as a potent anti-cancer
agent. In our study, we introduced a novel peptide TCP-1 and developed
the fusion protein of TCP-1/TNFα which was extensively investigated in
the colorectal cancer model. Our study provides proof that targeted delivery of TNFα could be used to treat colorectal cancer, and the conjugate holds great promise to be translated into the clinical setting.
5. Conclusion
Vascular-targeted delivery of TNFα by TCP-1 peptide is a new strategy to increase the therapeutic index of this cytokine in CRC-related
clinical settings. In this regard, the combination of the conjugated
TNFα together with 5-FU might be a promising option for CRC therapy.
Targeted delivery of TNFα by TCP-1 peptide would extend therapeutic
strategy regarding CRC therapy through synergistic administration of
targeted TNFα and chemotherapeutic agents. Moreover, it would be
highly possible to exploit TCP-1 to enhance the effectiveness of other
therapeutic proteins and imaging agents in CRC therapy and diagnosis.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2015.05.282.
Acknowledgments
The authors would like to thank the following granting agents: the
Innovation and Technology Fund from the Innovation and Technology
Commission (Grant No.: ITS/212/12) and the General Research Fund
from the Hong Kong Research Grant Council (Grant No.: CUHK
463613), for their generous support of this project. The financial
support from the National Natural Science Foundation of China (Grant
No.:81473269) is also appreciated.
References
[1] C. Rolfo, A. Russo, D. Santini, G. Bronte, M. Peeters, Dilemma in metastatic colorectal
cancer: VEGF versus EGRF targeting, Expert Opin. Ther. Targets 17 (2013) 869–871.
[2] H. Prenen, L. Vecchione, E. Van Cutsem, Role of targeted agents in metastatic
colorectal cancer, Target. Oncol. 8 (2013) 83–96.
[3] M.W. Saif, M. Cohenuram, Role of panitumumab in the management of metastatic
colorectal cancer, Clin. Colorectal Cancer 6 (2006) 118–124.
[4] A.S. Chung, X. Wu, G. Zhuang, H. Ngu, I. Kasman, J. Zhang, J.M. Vernes, Z. Jiang, Y.G.
Meng, F.V. Peale, W. Ouyang, N. Ferrara, An interleukin-17-mediated paracrine
network promotes tumor resistance to anti-angiogenic therapy, Nat. Med. 19
(2013) 1114–1123.
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
[5] T.L. ten Hagen, A.L. Seynhaeve, A.M. Eggermont, Tumor necrosis factor-mediated
interactions between inflammatory response and tumor vascular bed, Immunol.
Rev. 222 (2008) 299–315.
[6] F.J. Lejeune, C. Ruegg, Recombinant human tumor necrosis factor: an efficient agent
for cancer treatment, Bull. Cancer 93 (2006) E90–E100.
[7] L. Mortara, E. Balza, F. Sassi, P. Castellani, B. Carnemolla, A. De Lerma Barbaro, S.
Fossati, G. Tosi, R.S. Accolla, L. Borsi, Therapy-induced antitumor vaccination
by targeting tumor necrosis factor alpha to tumor vessels in combination with
melphalan, Eur. J. Immunol. 37 (2007) 3381–3392.
[8] F. Curnis, A. Gasparri, A. Sacchi, R. Longhi, A. Corti, Coupling tumor necrosis
factor-alpha with alphaV integrin ligands improves its antineoplastic activity,
Cancer Res. 64 (2004) 565–571.
[9] J.H. Schiller, B.E. Storer, P.L. Witt, D. Alberti, M.B. Tombes, R. Arzoomanian, R.A.
Proctor, D. McCarthy, R.R. Brown, S.D. Voss, et al., Biological and clinical effects of
intravenous tumor necrosis factor-alpha administered three times weekly, Cancer
Res. 51 (1991) 1651–1658.
[10] F.J. Lejeune, D. Lienard, M. Matter, C. Ruegg, Efficiency of recombinant human TNF in
human cancer therapy, Cancer Immun. 6 (2006) 6.
[11] A.L. Seynhaeve, S. Hoving, D. Schipper, C.E. Vermeulen, G. de Wiel-Ambagtsheer, S.T.
van Tiel, A.M. Eggermont, T.L. Ten Hagen, Tumor necrosis factor alpha mediates
homogeneous distribution of liposomes in murine melanoma that contributes to a
better tumor response, Cancer Res. 67 (2007) 9455–9462.
[12] A. Johansson, J. Hamzah, C.J. Payne, R. Ganss, Tumor-targeted TNFalpha stabilizes
tumor vessels and enhances active immunotherapy, Proc. Natl. Acad. Sci. U. S. A.
109 (2012) 7841–7846.
[13] V. Gregorc, F.G. De Braud, T.M. De Pas, R. Scalamogna, G. Citterio, A. Milani, S. Boselli,
C. Catania, G. Donadoni, G. Rossoni, D. Ghio, G. Spitaleri, C. Ammannati, S. Colombi, F.
Caligaris-Cappio, A. Lambiase, C. Bordignon, Phase I study of NGR-hTNF, a selective
vascular targeting agent, in combination with cisplatin in refractory solid tumors,
Clin. Cancer Res. 17 (2011) 1964–1972.
[14] A. Santoro, L. Rimassa, A.F. Sobrero, G. Citterio, F. Sclafani, C. Carnaghi, A. Pessino, F.
Caprioni, V. Andretta, M.C. Tronconi, G. Finocchiaro, G. Rossoni, A. Zanoni, C.
Miggiano, G.P. Rizzardi, C. Traversari, F. Caligaris-Cappio, A. Lambiase, C.
Bordignon, Phase II study of NGR-hTNF, a selective vascular targeting agent, in
patients with metastatic colorectal cancer after failure of standard therapy, Eur. J.
Cancer 46 (2010) 2746–2752.
[15] V. Gregorc, A. Santoro, E. Bennicelli, C.J. Punt, G. Citterio, J.N. Timmer-Bonte, F.
Caligaris Cappio, A. Lambiase, C. Bordignon, C.M. van Herpen, Phase Ib study of
NGR-hTNF, a selective vascular targeting agent, administered at low doses in
combination with doxorubicin to patients with advanced solid tumours, Br. J.
Cancer 101 (2009) 219–224.
[16] V. Gregorc, P.A. Zucali, A. Santoro, G.L. Ceresoli, G. Citterio, T.M. De Pas, N. Zilembo, F.
De Vincenzo, M. Simonelli, G. Rossoni, A. Spreafico, M. Grazia Vigano, F. Fontana, F.G.
De Braud, E. Bajetta, F. Caligaris-Cappio, P. Bruzzi, A. Lambiase, C. Bordignon, Phase II
study of asparagine-glycine-arginine-human tumor necrosis factor alpha, a selective
vascular targeting agent, in previously treated patients with malignant pleural
mesothelioma, J. Clin. Oncol. 28 (2010) 2604–2611.
[17] Z.J. Li, W.K. Wu, S.S. Ng, L. Yu, H.T. Li, C.C. Wong, Y.C. Wu, L. Zhang, S.X. Ren, X.G. Sun,
K.M. Chan, C.H. Cho, A novel peptide specifically targeting the vasculature of
orthotopic colorectal cancer for imaging detection and drug delivery, J. Control.
Release 148 (2010) 292–302.
[18] A. Hoffmann, M.Q. Muller, M. Gloser, A. Sinz, R. Rudolph, S. Pfeifer, Recombinant
production of bioactive human TNF-alpha by SUMO-fusion system—high yields
from shake-flask culture, Protein Expr. Purif. 72 (2010) 238–243.
[19] H. Yuan, J. Song, X. Li, N. Li, J. Dai, Immunomodulation and antitumor activity of
kappa-carrageenan oligosaccharides, Cancer Lett. 243 (2006) 228–234.
[20] I. Petrache, A. Birukova, S.I. Ramirez, J.G. Garcia, A.D. Verin, The role of the
microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability,
Am. J. Respir. Cell Mol. Biol. 28 (2003) 574–581.
[21] J.E. Morley, D.R. Thomas, M.M. Wilson, Cachexia: pathophysiology and clinical
relevance, Am. J. Clin. Nutr. 83 (2006) 735–743.
[22] M.T. Bertilaccio, M. Grioni, B.W. Sutherland, E. Degl'Innocenti, M. Freschi, E. Jachetti,
N.M. Greenberg, A. Corti, M. Bellone, Vasculature-targeted tumor necrosis
factor-alpha increases the therapeutic index of doxorubicin against prostate cancer,
Prostate 68 (2008) 1105–1115.
[23] E. Balza, L. Mortara, F. Sassi, S. Monteghirfo, B. Carnemolla, P. Castellani, D. Neri, R.S.
Accolla, L. Zardi, L. Borsi, Targeted delivery of tumor necrosis factor-alpha to tumor
vessels induces a therapeutic T cell-mediated immune response that protects the
host against syngeneic tumors of different histologic origin, Clin. Cancer Res. 12
(2006) 2575–2582.
[24] A. Sacchi, A. Gasparri, C. Gallo-Stampino, S. Toma, F. Curnis, A. Corti, Synergistic
antitumor activity of cisplatin, paclitaxel, and gemcitabine with tumor
vasculature-targeted tumor necrosis factor-alpha, Clin. Cancer Res. 12 (2006)
175–182.
[25] T. Hemmerle, P. Probst, L. Giovannoni, A.J. Green, T. Meyer, D. Neri, The antibodybased targeted delivery of TNF in combination with doxorubicin eradicates
sarcomas in mice and confers protective immunity, Br. J. Cancer 109 (2013)
1206–1213.
[26] A. Calcinotto, M. Grioni, E. Jachetti, F. Curnis, A. Mondino, G. Parmiani, A. Corti, M.
Bellone, Targeting TNF-alpha to neoangiogenic vessels enhances lymphocyte
infiltration in tumors and increases the therapeutic potential of immunotherapy, J.
Immunol. 188 (2012) 2687–2694.
[27] A. Ashkenazi, R.C. Pai, S. Fong, S. Leung, D.A. Lawrence, S.A. Marsters, C. Blackie, L.
Chang, A.E. McMurtrey, A. Hebert, L. DeForge, I.L. Koumenis, D. Lewis, L. Harris, J.
Bussiere, H. Koeppen, Z. Shahrokh, R.H. Schwall, Safety and antitumor activity of
recombinant soluble Apo2 ligand, J. Clin. Invest. 104 (1999) 155–162.
145
[28] F. Bunz, P.M. Hwang, C. Torrance, T. Waldman, Y. Zhang, L. Dillehay, J. Williams, C.
Lengauer, K.W. Kinzler, B. Vogelstein, Disruption of p53 in human cancer cells alters
the responses to therapeutic agents, J. Clin. Invest. 104 (1999) 263–269.
[29] C.L. Farrell, J.V. Bready, K.L. Rex, J.N. Chen, C.R. DiPalma, K.L. Whitcomb, S. Yin, D.C.
Hill, B. Wiemann, C.O. Starnes, A.M. Havill, Z.N. Lu, S.L. Aukerman, G.F. Pierce, A.
Thomason, C.S. Potten, T.R. Ulich, D.L. Lacey, Keratinocyte growth factor protects
mice from chemotherapy and radiation-induced gastrointestinal injury and mortality, Cancer Res. 58 (1998) 933–939.
[30] D.M. Pritchard, A.J. Watson, C.S. Potten, A.L. Jackman, J.A. Hickman, Inhibition by
uridine but not thymidine of p53-dependent intestinal apoptosis initiated by
5-fluorouracil: evidence for the involvement of RNA perturbation, Proc. Natl. Acad.
Sci. U. S. A. 94 (1997) 1795–1799.
[31] A. Corti, F. Curnis, G. Rossoni, F. Marcucci, V. Gregorc, Peptide-mediated targeting of
cytokines to tumor vasculature: the NGR-hTNF example, BioDrugs 27 (2013)
591–603.
[32] M.F. Sorrentino, J. Kim, A.E. Foderaro, A.G. Truesdell, 5-Fluorouracil induced
cardiotoxicity: review of the literature, Cardiol. J. 19 (2012) 453–458.
[33] H. Yin, F. Xie, J. Zhang, Y. Yang, B. Deng, J. Sun, Q. Wang, X. Qu, H. Mao, Combination
of interferon-alpha and 5-fluorouracil induces apoptosis through mitochondrial
pathway in hepatocellular carcinoma in vitro, Cancer Lett. 306 (2011) 34–42.
[34] C. Reissfelder, S. Stamova, C. Gossmann, M. Braun, A. Bonertz, U. Walliczek, M.
Grimm, N.N. Rahbari, M. Koch, M. Saadati, A. Benner, M.W. Buchler, D. Jager, N.
Halama, K. Khazaie, J. Weitz, P. Beckhove, Tumor-specific cytotoxic T lymphocyte
activity determines colorectal cancer patient prognosis, J. Clin. Invest. 125 (2015)
739–751.
[35] A. Tandle, E. Hanna, D. Lorang, A. Hajitou, C.A. Moya, R. Pasqualini, W. Arap, A. Adem,
E. Starker, S. Hewitt, S.K. Libutti, Tumor vasculature-targeted delivery of tumor
necrosis factor-alpha, Cancer 115 (2009) 128–139.
[36] F. Curnis, A. Sacchi, L. Borgna, F. Magni, A. Gasparri, A. Corti, Enhancement of tumor
necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery
to aminopeptidase N (CD13), Nat. Biotechnol. 18 (2000) 1185–1190.
[37] L. Borsi, E. Balza, B. Carnemolla, F. Sassi, P. Castellani, A. Berndt, H. Kosmehl, A. Biro,
A. Siri, P. Orecchia, J. Grassi, D. Neri, L. Zardi, Selective targeted delivery of TNFalpha
to tumor blood vessels, Blood 102 (2003) 4384–4392.
[38] G. Spitaleri, R. Berardi, C. Pierantoni, T. De Pas, C. Noberasco, C. Libbra, R. GonzalezIglesias, L. Giovannoni, A. Tasciotti, D. Neri, H.D. Menssen, F. de Braud, Phase I/II
study of the tumour-targeting human monoclonal antibody-cytokine fusion protein
L19-TNF in patients with advanced solid tumours, J. Cancer Res. Clin. Oncol. 139
(2013) 447–455.
[39] P.A. Zucali, M. Simonelli, F. De Vincenzo, E. Lorenzi, M. Perrino, M. Bertossi, R.
Finotto, S. Naimo, L. Balzarini, C. Bonifacio, I. Timofeeva, G. Rossoni, G. Mazzola, A.
Lambiase, C. Bordignon, A. Santoro, Phase I and pharmacodynamic study of
high-dose NGR-hTNF in patients with refractory solid tumours, Br. J. Cancer 108
(2013) 58–63.
[40] J. Zhang, H. Spring, M. Schwab, Neuroblastoma tumor cell-binding peptides
identified through random peptide phage display, Cancer Lett. 171 (2001) 153–164.
[41] J. Kang, G. Zhao, T. Lin, S. Tang, G. Xu, S. Hu, Q. Bi, C. Guo, L. Sun, S. Han, Q. Xu, Y. Nie,
B. Wang, S. Liang, J. Ding, K. Wu, A peptide derived from phage display library exhibits anti-tumor activity by targeting GRP78 in gastric cancer multidrug resistance
cells, Cancer Lett. 339 (2013) 247–259.
[42] L. Chen, Y. Wang, X. Liu, S. Dou, G. Liu, D.J. Hnatowich, M. Rusckowski, A new TAG-72
cancer marker peptide identified by phage display, Cancer Lett. 272 (2008)
122–132.
[43] Z.J. Li, C.H. Cho, Development of peptides as potential drugs for cancer therapy, Curr.
Pharm. Des. 16 (2010) 1180–1189.
[44] Z.J. Li, C.H. Cho, Peptides as targeting probes against tumor vasculature for diagnosis
and drug delivery, J. Transl. Med. 10 (Suppl. 1) (2012) S1.
[45] W.M. Chu, Tumor necrosis factor, Cancer Lett. 328 (2013) 222–225.
[46] C. Menon, A. Ghartey, R. Canter, M. Feldman, D.L. Fraker, Tumor necrosis factoralpha damages tumor blood vessel integrity by targeting VE-cadherin, Ann. Surg.
244 (2006) 781–791.
[47] R.K. Jain, Normalization of tumor vasculature: an emerging concept in antiangiogenic
therapy, Science 307 (2005) 58–62.
[48] A.M. Eggermont, H. Schraffordt Koops, D. Lienard, B.B. Kroon, A.N. van Geel, H.J.
Hoekstra, F.J. Lejeune, Isolated limb perfusion with high-dose tumor necrosis
factor-alpha in combination with interferon-gamma and melphalan for
nonresectable extremity soft tissue sarcomas: a multicenter trial, J. Clin. Oncol. 14
(1996) 2653–2665.
[49] J. Brett, H. Gerlach, P. Nawroth, S. Steinberg, G. Godman, D. Stern, Tumor necrosis
factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins, J. Exp. Med. 169 (1989) 1977–1991.
[50] S.E. Goldblum, W.L. Sun, Tumor necrosis factor-alpha augments pulmonary arterial
transendothelial albumin flux in vitro, Am. J. Physiol. 258 (1990) L57–L67.
[51] A.H. van der Veen, J.H. de Wilt, A.M. Eggermont, S.T. van Tiel, A.L. Seynhaeve, T.L. ten
Hagen, TNF-alpha augments intratumoural concentrations of doxorubicin in
TNF-alpha-based isolated limb perfusion in rat sarcoma models and enhances
anti-tumour effects, Br. J. Cancer 82 (2000) 973–980.
[52] F.J. Lejeune, High dose recombinant tumour necrosis factor (rTNF alpha) administered by isolation perfusion for advanced tumours of the limbs: a model for
biochemotherapy of cancer, Eur. J. Cancer 31A (1995) 1009–1016.
[53] C.A. Kristensen, M. Nozue, Y. Boucher, R.K. Jain, Reduction of interstitial fluid
pressure after TNF-alpha treatment of three human melanoma xenografts, Br. J.
Cancer 74 (1996) 533–536.
[54] S. Suzuki, S. Ohta, K. Takashio, H. Nitanai, Y. Hashimoto, Augmentation for intratumoral
accumulation and anti-tumor activity of liposome-encapsulated adriamycin by tumor
necrosis factor-alpha in mice, Int. J. Cancer 46 (1990) 1095–1100.
146
L. Lu et al. / Journal of Controlled Release 210 (2015) 134–146
[55] J.H. de Wilt, T.L. ten Hagen, G. de Boeck, S.T. van Tiel, E.A. de Bruijn, A.M. Eggermont,
Tumour necrosis factor alpha increases melphalan concentration in tumour tissue
after isolated limb perfusion, Br. J. Cancer 82 (2000) 1000–1003.
[56] R.K. Jain, Barriers to drug delivery in solid tumors, Sci. Am. 271 (1994) 58–65.
[57] F. Curnis, A. Sacchi, A. Corti, Improving chemotherapeutic drug penetration in
tumors by vascular targeting and barrier alteration, J. Clin. Invest. 110 (2002)
475–482.
[58] J.E. Talmadge, H.R. Tribble, R.W. Pennington, H. Phillips, R.H. Wiltrout, Immunomodulatory and immunotherapeutic properties of recombinant gamma-interferon and
recombinant tumor necrosis factor in mice, Cancer Res. 47 (1987) 2563–2570.
[59] I.A. Khawar, J.H. Kim, H.J. Kuh, Improving drug delivery to solid tumors: priming the
tumor microenvironment, J. Control. Release 201C (2015) 78–89.
[60] H.W. van Laarhoven, G. Gambarota, A. Heerschap, J. Lok, I. Verhagen, A. Corti, S.
Toma, C. Gallo Stampino, A. van der Kogel, C.J. Punt, Effects of the tumor vasculature
targeting agent NGR-TNF on the tumor microenvironment in murine lymphomas,
Invest. New Drugs 24 (2006) 27–36.