Synergistic inhibition of lung cancer cell invasion, tumor growth and angiogenesis using aptamer-siRNA chimeras

Biomaterials 35 (2014) 2905e2914

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Synergistic inhibition of lung cancer cell invasion, tumor growth and angiogenesis using aptamer-siRNA chimeras Wei-Yun Lai a, b, c, Wei-Ya Wang c, Yi-Chung Chang c, Cheng-Ju Chang c,1, Pan-Chyr Yang c, d, *,1, Konan Peck c,1, 2 a

Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 115, Taiwan Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan d National Taiwan University, Taipei 100, Taiwan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 October 2013 Accepted 19 December 2013 Available online 4 January 2014

Early metastasis is one of the major causes of mortality among patient with lung cancer. The process of tumor metastasis involves a cascade of events, including epithelial-mesenchymal transition, tumor cell migration and invasion, and angiogenesis. To specifically suppress tumor invasion and angiogenesis, two nucleolin aptamer-siRNA chimeras (aptNCL-SLUGsiR and aptNCL-NRP1siR) were used to block key signaling pathways involved in lung cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Through nucleolin-mediated endocytosis, the aptNCL-siRNA chimeras specifically and significantly knocked down the expressions of SLUG and NRP1 in nucleolin-expressing cancer cells. Furthermore, simultaneous suppression of SLUG and NRP1 expressions by the chimeras synergistically retarded cancer cell motility and invasive ability. The synergistic effect was also observed in a xenograft mouse model, wherein the combined treatment using two chimeras suppressed tumor growth, the invasiveness, circulating tumor cell amount, and angiogenesis in tumor tissue without affecting liver and kidney functions. This study demonstrates that combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR can synergistically suppress lung cancer cell invasion, tumor growth and angiogenesis by cancer-specific targeting combined with gene-specific silencing. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Aptamer Combination therapy Lung cancer Metastasis siRNA

1. Introduction Lung cancer is the leading cause of cancer-related death and non-small cell lung cancer (NSCLC) accounts for about 85% of cases [1,2]. Despite advances in conventional and targeted therapies for NSCLC, early metastasis is still the most recalcitrant factor in cancer treatment, which causes a high mortality rate in lung cancer patients [3,4]. The metastatic cascade involves a series of events that include epithelial-mesenchymal transition (EMT), cancer cell migration, invasion, intravasation into the systemic circulation, and subsequent adhesion to endothelial cells, followed by extravasation, colonization of distant organs, and induction of angiogenesis [5]. These consecutive events are characterized by the activations of

* Corresponding author. National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan. Tel.: þ886 2 33662000; fax: þ886 2 23621877. E-mail address: [email protected] (P.-C. Yang). 1 Drs. Cheng-Ju Chang, Pan-Chyr Yang, and Konan Peck contributed equally to this paper. 2 Dr. Konan Peck is deceased. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.12.054

different genes and signaling pathways at different stages of the cascade [6]. In previous studies, the over-expression of snail family zinc finger 2 (SLUG) and neuropilin 1 (NRP1) in NSCLC has been shown to promote malignant transformation and activate key signaling pathways during different stages of the metastasis cascade [7e10]. SLUG is a zinc-finger-containing transcriptional factor [11] that activates EMT and the migration, and invasion of lung cancer cells [12]. In addition, SLUG up-regulates both the expression and activity of matrix metalloproteinases-2 (MMP-2), which also promotes cancer cell invasion, tumor-induced angiogenesis, and colonization of metastatic foci in distant organs [7,13]. NRP1 is the co-receptor for vascular endothelial growth factor receptors (VEGFR) [14] and is involved in the VEGF-PI3k-Akt axis that enhances lung cancer cell migration and invasion, and induces angiogenesis in tumor tissues [8]. Although the invasion and metastasis capabilities of lung cancer cells may be inhibited by suppressing SLUG or NRP1 function, there is no available targeted drug against SLUG and only one small chemical inhibitor blocks the interaction between NRP1 and VEGF [15].

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Aptamers are single stranded DNA or RNA with high affinity and specificity to target molecules and have little or no toxicity or immunogenicity in standard assays [16]. For cancer treatment, aptamers deliver the drug or siRNA in a tumor cell-specific manner, thereby reducing unwanted side effects like cytotoxicity to normal cells [17]. An aptamer-siRNA chimera is an emerging class of targeted therapeutic agents that has been evaluated for HIV-1 treatment [18e20], drug hypersensitivity [21], and cancer therapy [22e27]. Several aptamers targeting cancer cell surface markers have been used for siRNA delivery, including prostate-specific membrane antigen (PSMA) and alpha V and integrin beta 3 (avb3) integrin for prostate cancer [23], human epidermal growth factor receptor 2 (HER2) for breast cancer [26], and B-cell-activating factor-receptor (BAFF-R) for Bcell non-Hodgkin lymphomas [27]. However, information on aptamer-siRNA chimera utilized in the treatment of lung cancer patients is limited [28]. Nucleolin (NCL) is highly expressed on the surface of lung cancer cells and is a shuttle protein across the nucleus, cytoplasm, and cell membrane [29]. The NCL-targeting aptamer (aptNCL) is a promising tumor cell-specific targeting carrier that specifically recognizes the NCL-expressing cell and is further internalized [29,30]. To apply the technology of aptamer-siRNA chimera to lung cancer and

treat lung cancer cell metastasis and tumor angiogenesis in multiple aspects, this study successfully conjugated aptNCL with siRNAs against two genes (SLUG and NRP1). The feasibility and efficacy of the combined treatment with two therapeutic chimeras were assessed. 2. Materials and methods 2.1. Cell culture and reagents CL1-5, a lung adenocarcinoma cell line, was cultivated in RPMI-1640 medium (Gibco BRL, Life Technologies, Grand Island, NY, USA). HUVEC, endothelial cells obtained from the endothelium of umbilical cord veins, were grown in Minimum Essential Medium (Gibco BRL). RAW-BlueÔ (InvivoGen, San Diego, CA, USA) cells were macrophages and were grown in Dulbecco’s modified Eagle medium (Gibco BRL). All of the media were supplemented with 10% FBS (Gibco BRL). HUVEC was provided by Dr. Danny Ling Wang of the Institute of Biomedical Sciences, Academia Sinica. 2.2. Nucleic acids reagents All of the aptamers were synthesized by Purigo Biotech (Taipei, Taiwan). The sequence of aptNCL was as follows: 50 -GGT GGT GGT GGT TGT GGT GGT GGT GG-30 [29]. A poly(dT) spacer and sulfhydryl group were introduced at the 50 -end. The target sequences of siRNAs (Dharmacon, Lafayette, CO, USA) targeting SLUG and NRP1 mRNAs were: 50 -CCG UAU CUC UAU GAG AGU UAC UCC A-30 and 50 -AAC ACC UAG UGG AGU GAU AAA-30 , respectively. All of the sense strands in siRNAs were modified with primary amine moiety at the 50 -end.

Fig. 1. AptNCL-siRNA chimeras are specifically internalized by NCL-expressing cells. (A) AptNCL with poly(dT) spacers at the 50 -end was conjugated to the 50 -end of the sense strand of siRNA through the sulfhydryl group (S) bridging of sulfo-SMPB linker. (B) NCL protein expression in CL1-5 and HUVEC were detected by Western blotting. (C) CL1-5 and HUVEC were incubated with Alexa FluorÒ 488-labeled aptNCL-siRNA for 30 min and then stained by Hoechst 33258. The fluorescence images were taken by a confocal system.

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Fig. 2. AptNCL-siRNA chimeras significantly silence target gene expression without immune stimulation. (A and B) CL1-5 cells were treated with 50 nM chimeras (n ¼ 3). The knockdown efficiency on (A) SLUG and (B) NRP1 expressions were analyzed by RT-qPCR (upper panel) and Western blotting (lower panel). ACTB mRNA and b-actin proteins were used as internal control for RT-qPCR and immunoblotting, respectively. (C) HUVEC cells were treated with 50 nM chimeras for 48 h. Cell viability was determined by MTT assay (n ¼ 3). (D) RAW-BlueÔ cells were treated with 50 nM chimeras and Poly(I:C) (1.56 mg/ml) were used as the positive control (n ¼ 3). The NF-kB signal was assessed using a QUANTIBlueÔ kit following the manufacturer’s protocol. All data was presented as mean  SD and analyzed by Student’s t-test.

2.3. Aptamer and siRNA conjugation

2.4. Confocal imaging

Amine-modified siRNA was incubated with 8 mM sulfo-SMPB (Thermo scientific, Hudson, NH, USA) and thiol-modified aptNCL was activated in 10 mM TCEP (SigmaeAldrich, St. Louis, MO, USA) in PBS (pH 7.2) at 37  C for 1 h. Both products were then purified using the Centri-Sep column (Princeton Separations, Inc., Adelphia, NJ) equilibrated with PBS (pH 7.2) containing 5 mM EDTA. Purified siRNA and aptNCL were then mixed together at 37  C for 2 h. The aptNCL-siRNA chimeras were purified by 10% non-denaturing polyacrylamide gel electrophoresis. The concentration of chimera was defined as the siRNA concentration in the chimera. The siRNA concentration was detected by QubitÔ RNA assay kit (Invitrogen, Carlsbad, CA, USA) using QubitÒ 2.0 Fluorometer (Invitrogen) following the manufacturer’s protocol.

CL1-5 and HUVEC were incubated with 50 nM Alexa FluorÒ 488-labeled aptNCLsiRNA for 30 min and stained with Hoechst 33258 (Invitrogen). Confocal fluorescence imaging was performed using an UltraVIEW RS confocal system (PerkinElmer Life Sciences Inc., MA, USA). 2.5. Reverse transcription-quantitative PCR (RT-qPCR) and Western immunoblotting CL1-5 cells were separately treated with 50 nM chimeras. At 48 h after treatment, total RNA purification was done by TRIzolÒ (Invitrogen) and RT-qPCR was performed on a LightCycler 480 system (Roche Applied Science, Mannheim, Germany). The relative amount of SLUG and NRP1 mRNA was normalized by b-actin (ACTB) mRNA.

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W.-Y. Lai et al. / Biomaterials 35 (2014) 2905e2914 The PCR primer sequences (Purigo Biotech) used were 50 -TGG TTG CTT CAA GGA CAC AT-30 (forward) and 50 -GCA AAT GCT CTG TTG CAG TG-30 (reverse) for SLUG; 50 -TGC AGA GCA GTG TCT CAG AAG-30 (forward) and 50 -AAG CTG TGA TCT GGT CAG AAT G30 (reverse) for NRP1; and 50 -ATT GGC AAT GAG CGG TTC-30 (forward) and 50 -CGT GGA TGC CAC AGG ACT-30 (reverse) for ACTB. At 72 h after treatment, total protein was extracted by RIPA buffer (Roche Applied Science). Tumor tissues from xenograft were also extracted by RIPA buffer. The primary antibodies were used at a 1:1000 dilution: NCL (Genetex, San Antonio, TX, USA), SLUG (Santa Cruz Biotechnology, Santa Cruz, CA), NRP1 (Genetex), Ecadherin (BD Biosciences, San Jose, CA, USA), MMP-2 (Cell Signaling Technology, Beverly, MA, USA), phospho-Akt (S473) (Cell Signaling Technology), Akt (Santa Cruz Biotechnology), and b-actin (Santa Cruz Biotechnology). The HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used at a 1:5000 dilution. 2.6. Cell viability assay and NF-kB reporter assay HUVEC and CL1-5 cells were separately treated with 50 nM chimeras for 48 h. Cell proliferation was measured by MTT assay. RAW-BlueÔ cells were treated with 50 nM chimeras. Cells transfected with polyinosinic-polycytidylic acid (Poly(I:C) 1.56 mg/ml, InvivoGen) by Lipofectamine 2000 (Invitrogen) was used as positive control. At 48 h after treatment, the cell supernatant was assayed using the QUANTI-BlueÔ (InvivoGen) kit following the manufacturer’s protocols. 2.7. Immunofluorescence imaging of filopodia formation CL1-5 cells were treated with 50 nM chimeras. At 48 h after treatment, the cells were stained with Texas RedÒ-X phalloidin (Molecular probes, Invitrogen). Fluorescence imaging was taken by Axiovert 200 Inverted Fluorescent Microscope (Carl Zeiss Inc., Thornwood, New York, USA). 2.8. Cell migration assay and cell invasion assay CL1-5 was seeded into wells of Culture-Inserts (Ibidi, Munich, Germany) leaving a defined cell-free gap. The cells were treated with 25 nM or 50 nM chimeras for 48 h. After treatment, Culture-Inserts were taken away and cell-free gap was photographed at 0 h and 12 h by microscopy. CL1-5 cells were treated with 25 nM or 50 nM chimeras. At 48 h after treatment, the cells were trypsinized and added to Millicell Cell Culture Inserts with Matrigel coating (Millipore). After 24 h, cell invasion assays were performed according to the manufacturer’s protocol. 2.9. Synergy CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA) was used to calculate the combination index (CI) values in cell migration and invasion assays. The CI values <1 indicated synergistic effects between the two chimeras (aptNCL-SLUGsiR and aptNCL-NRP1siR) [31]. 2.10. In vivo tumorigenesis assay All of the animal studies were performed under the guidance of the Laboratory Animal Center, Academia Sinica. NOD-SCID mice 8-weeks old (BioLASCO, Taipei, Taiwan) were subcutaneously inoculated with 2  106 CL1-5 cells. Seven days after inoculation, the mice were randomly divided into five groups (n ¼ 6 per group): (1) PBS buffer control, (2) aptNCL-ControlsiR, (3) aptNCL-SLUGsiR, (4) aptNCL-NRP1siR, and (5) combined treatment. For single chimera injection, 2 mM chimera in 50 ml PBS buffer (aptNCL-ControlsiR, aptNCL-SLUGsiR, or aptNCL-NRP1siR) were injected. For combined chimera injection, 1 mM of each chimera (aptNCL-SLUGsiR and aptNCLNRP1siR) in total 50 ml PBS buffer were injected. The reagents were injected intratumor three times per week until completion of the experiments. After the mice were sacrificed, the tumor tissues were analyzed by Western blotting and immunohistochemical staining. 2.11. In vivo cell invasiveness assay Peripheral blood samples were taken from mice in MicrotainerÒ tubes with dipotassium EDTA (BD Biosciences) at 10 and 40 days after xenografting. Genomic DNA was extracted from blood samples using a QIAamp DNA mini kit (Qiagen, Hilden, Germany). The circulating tumor cell (CTC) levels were measured by qPCR for human Alu sequence. The primer sequences (Purigo Biotech) were 50 -GTC AGG AGA TCG AGA CCA TCC C-30 (forward) and 50 -TCC TGC CTC AGC CTC CCA AG-30 (reverse) [32]. The relative amount of CTCs was normalized by the tumor size.

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2.12. Immunohistochemical staining and microvessel number calculation After the mice were sacrificed, the tumor tissues were fixed and embedded in paraffin, stained with antibody against mouse CD31 (BD Biosciences) at 1:100 dilution, and counterstained with hematoxylin (Merck, Millipore). The micro-vessel morphology of tumor tissues was evaluated by Axiovert 200 Inverted Microscope (Carl Zeiss Inc.). Brown immuno-stained endothelial cell clusters that separated from one another and had a size >15 mm were considered single microvessel [33]. The microvessels in the tumor area were counted in 200  fields and the number of microvessels was calculated by MetaMorph software (Molecular Devices). 2.13. Blood chemistry analysis Before the mice were sacrificed, whole blood was collected. Levels of glutamate oxaloacetate transaminase (GOP), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), and ammonia (NH3) were detected by Fuji Dri-Chem 4000i (Fujifilm Co., Tokyo, Japan). 2.14. Statistical analysis The data were presented as mean  SD. All statistical tests were two-sided and statistical significance was set at p < 0.05.

3. Results 3.1. Cell specificity of aptNCL-siRNA chimeras The aptNCL-siRNA chimeras contained two entities, aptNCL and siRNA, linked together by a hetero-bifunctional crosslinker, sulfoSMPB (Fig. 1A). In the aptNCL portion, a poly(dT) spacer was introduced at the 50 -end to minimize steric interference between aptNCL and siRNA. The incorporation of oligo (dT) spacer to the aptamer terminal was easy for manipulation and did not interfere with the folding of the aptamer structure [34]. To validate the ability of aptNCL-siRNA chimera in cell-specific targeting, two cell lines with different NCL expression levels were selected. The blotting results revealed that CL1-5 cells had higher NCL expression while the HUVEC cells had lower expression (Fig. 1B). Cell imaging showed that the aptNCL-siRNA chimeras were internalized into higher NCL-expressing CL1-5 cells within 30 min at 37  C, while no significant signals were detected from HUVEC cells with lower NCL expression (Fig. 1C). Moreover, scrambled oligonucleotides with random sequences were not internalized into the CL1-5 cells in the same time span (data not shown). The results indicated that the aptNCL-siRNA chimeras were specifically internalized into NCL-expressing cells. 3.2. Gene silencing ability of aptNCL-siRNA chimeras To investigate whether chimeras (aptNCL-SLUGsiR and aptNCLNRP1siR) could efficiently suppress target gene expression, the extents of SLUG and NRP1 silencing were assessed by RT-qPCR and Western blotting. Compared to treatment with aptNCL-ControlsiR, the aptNCL-SLUGsiR and aptNCL-NRP1siR significantly silenced the relative gene expression in both mRNA and protein levels (Fig. 2A and B). There were no differences in SLUG and NRP1 expressions between untreated group and aptNCL-ControlsiR groups. The expression of SLUG was not affected by aptNCL-NRP1siR chimera treatment and vice-versa. Furthermore, to determine whether the chimeras affected normal cell viability, MTT assay was done on HUVEC cells. The cell viability in three chimera treatment groups (aptNCL-ControlsiR, aptNCL-SLUGsiR, and aptNCL-NRP1siR) did not differ from that of

Fig. 3. Combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR synergistically inhibits cell migration and invasion ability. (A and B) CL1-5 cells were treated with 50 nM chimeras. (A) The SLUG and NRP1 expression levels were analyzed by Western blotting. (B) The F-actin of treated cells was stained with Texas RedÒ-X phalloidin. (C) CL1-5 were seeded into wells of Culture-Insert and treated with 25 or 50 nM chimeras for 48 h (n ¼ 3). After the Culture-Insert was removed, the cell-free gap was photographed at 0 h and 12 h and the migrated cell number was counted. (D) CL1-5 cells were treated with 50 nM chimeras for 48 h and re-seeded to Millicell Cell Culture Insert coated with Matrigel (n ¼ 3). The invaded cell number was counted 24 h after cell seeding. The data was presented as mean  SD and analyzed by Student’s t-test.

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500

PBS buffer control aptNCL-ControlsiR aptNCL-SLUGsiR aptNCL-NRP1siR Combined treatment

400 300

aptNCL-SLUGsiR

100 50 0

CTC amount ormalized to tumor volume) (No

7

14

21

28

35

42

10 d

40 d

1.8

1.2

P < 0.03*

* 0.6

*

*

* *

0 aptNCL-ControlsiR

-

+

-

-

-

-

+

-

-

-

aptNCL-SLUGsiR

-

-

+

-

+

-

-

+

-

+

aptNCL-NRP1siR

-

-

-

+

+

-

-

-

+

+

C

PBS buffer control

Combined treatment

H&E

CD31

No. of vessel per field

D

400 P < 0.03*

300

* *

200

*

100

* *

0 aptNCL-ControlsiR

-

+

-

-

-

aptNCL-SLUGsiR

-

-

+

-

+

aptNCL-NRP1siR

-

-

-

+

+

aptNCL-ControlsiR

aptNCL-NRP1siR

P < 0.001*

200

0

B

PBS buffer control

n.s.

* **

Tumor volume (mm3)

A

Combined treatment

PBS buffer control

E

Combined treatment

W.-Y. Lai et al. / Biomaterials 35 (2014) 2905e2914

2911

F RNA

SLUG mRNA

NRP1 mRNA aptNCL-siRNA chimera

Protein

NRP1

SLUG Transcriptional regulation

SLUG

Phosphorylation

E-cadherin Signaling pathway

E-cadherin

MMP-2

Akt

MMP-2 NRP1

Phenotype

EMT

Migration

Invasion

Angiogenesis

pAkt Akt β-actin Fig. 4. (continued).

the untreated control group (Fig. 2C), indicating that these chimeras were not toxic to the HUVEC cells. Similar results were detected in CL1-5 cells (data not shown) which indicated that cell viability was not affected by silencing SLUG and NRP1 expression in in vitro assay. To verify whether the chimeras induced NF-kB signaling in immune cells, NF-kB reporter assay was performed on chimeratreated RAW-BlueÔ cells. RAW-BlueÔ cells expressed many pattern-recognition receptors (PRRs), including toll-like receptors (TLRs) and NOD-like receptors (NLRs). Treatment of agonists such as Poly(I:C) triggered PRR downstream signaling pathways and activated NF-kB signaling [35]. Compared to the untreated group, Poly(I:C) induced strong NF-kB signal in RAW-BlueÔ cells while NF-kB signaling was slightly activated by three aptNCL-siRNA chimeras (1% by aptNCL-ControlsiR, 4.8% by aptNCL-SLUGsiR, and 2.6% by aptNCL-NRP1siR) (Fig. 2D). These suggested that the chimeras did not induce the PRR signaling pathways and downstream NF-kB signaling. 3.3. Synergistic effect between aptNCL-SLUGsiR and aptNCLNRP1siR chimeras Combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR significantly and simultaneously silenced the expressions of the two target genes (Fig. 3A). Silencing of SLUG or NRP1 expression resulted in decreased cell migration and invasion ability [7,8,10]. However, the suppressive effect on cell migration and invasion when co-inhibiting SLUG and NRP1 expressions remained unclear. Filopodia are actin-rich protrusions along cell membrane for microenvironment sensing [36]. The formation of filopodia functions as guidance for cell migration. To examine whether combined

treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR chimeras could suppress cell motility, the inhibitory effect on filopodia formation was evaluated. While aptNCL-ControlsiR-treated cells revealed highly visible filopodia along the cell membrane (Fig. 3B, left panel), the combined treatment group had few filopodia (Fig. 3B, right panel). To examine whether combined treatment could suppress cell motility in a synergistic manner, the inhibitory effects on wound healing and matrigel invasion assay were evaluated. In the wound healing assay, treatment with aptNCL-SLUGsiR or aptNCLNRP1siR significantly suppressed cell migration in a dosedependent manner. AptNCL-SLUGsiR or aptNCL-NRP1siR (50 nM) decreased the number of migrated cells to 59.6% or 39.9%, respectively, compared to the number in aptNCL-ControlsiR-treated controls (Fig. 3C). At half-dose administration (25 nM of each chimera), the combined treatment group had significantly decreased migrated cell number to 20.6%. In the cell invasion assay, a similar inhibitory effect was observed (Fig. 3D). Single treatment of aptNCL-SLUGsiR or aptNCLNRP1siR suppressed cell invasion to 69.4% or 47.2%, respectively. At half-dose administration of each chimera, the combined treatment further inhibited cell invasion to 34.6%. The combined effect of aptNCL-SLUGsiR and aptNCL-NRP1siR was determined by CI value calculation. The combined treatment inhibited cell migration (CI ¼ 0.35) and invasion (CI ¼ 0.33) in a synergistic manner. 3.4. Suppression effect of combination treatment on xenograft tumor model Compared to the PBS buffer control group, the tumor growth rate was 3-fold to 4-fold slower in the three chimera-treated groups (aptNCL-SLUGsiR, aptNCL-NRP1siR, and combined treatment)

Fig. 4. Combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR synergistically inhibits tumor growth, circulating tumor cell amount, and angiogenesis in tumor tissue. (AeE) CL1-5 cells were subcutaneously inoculated to NOD-SCID mice (n ¼ 6, per group). (A) Tumor volume was measured every 7 days. (B) The circulating tumor cells were detected by qPCR for human Alu sequence 10 and 40 days after inoculation. (C) Xenograft tumor tissues were processed for immunostaining. Scale bars represent 100 mm in H&E images and CD31 images. (D) The microvessels in the tumor area were detected by immunohistochemical staining using anti-CD31 antibody. The number of microvessels per field was counted. (E) The SLUG and NRP1 expression levels and downstream signaling were determined by Western blotting. (F) The proposed mechanism of aptNCL-siRNA chimeras.

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(Fig. 4A). Even after the treatment dosage of individual chimera was reduced by half, the combined treatment group still revealed a similar inhibitory effect on xenograft tumor growth. To evaluate cell invasiveness, the CTCs were monitored by qPCR using human Alu sequence as the detection marker and normalized with the tumor size. The results revealed significant suppression of the CTC number by the chimera treatment groups (aptNCL-SLUGsiR, aptNCL-NRP1siR, and combined treatment) comparing to the PBS group (Fig. 4B). More importantly, combined treatment with aptNCL-SLUGsiR and aptNCL-NRP1siR synergistically downregulated cell invasiveness even at half-dose administration, consistent with results from in vitro cell migration and invasion assays. After the mice were sacrificed, immunohistochemical analysis of tumor tissues was performed to evaluate tissue morphology and microvessel formation. There was severe necrosis in tumor tissues with decreased microvessel number and length in sections from the combined treatment group but not in tissues from the PBS group (Fig. 4C). At half the treatment dosage of individual chimera, the combined treatment group showed the highest potency of all in suppressing microvessel formation (Fig. 4D). The decreased angiogenesis in tumor tissue might lead to smaller tumor size and fewer CTCs in blood observed in the combined treated groups. In addition, the liver and kidney functions of chimera-treated mice were examined by blood chemistry. There were no significant differences between the PBS and combined treatment groups in terms of level of GOP, GPT, BUN, and NH3 (Table 1), suggesting that the liver and kidney functions were not affected by continued chimera administration. 3.5. Effect of combination treatment on SLUG and NRP1 signaling AptNCL is used as an anti-cancer molecule and IC50 in cell culture system is at micro-molar range [30]. In our studies, there is no difference between PBS group and aptNCL-ControlsiR group on the tumor growth rate (Fig. 4A), the number of CTC in blood stream (Fig. 4B), and microvessel number in xenograft tumor tissue (Fig. 4D). The observation indicated that the suppression effects on cell invasion and angiogenesis were derived from SLUGsiR and NRP1siR not aptNCL itself under the dosage we used. As mentioned before, SLUG regulates EMT, invasion, and angiogenesis through E-cadherin and MMP-2 while NRP1 modulates invasion and angiogenesis via VEGF-PI3k-Akt axis [7,8]. To assess the effects of the chimera on the target gene expression and downstream signaling, xenograft tumors were processed for Western blotting. The results showed that combined treatment significantly down-regulated SLUG and NRP1 expression in xenograft tumor tissue (Fig. 4E). Decreased SLUG expression was accompanied by a concomitant increase in E-cadherin expression and decrease in MMP-2 expression. Down-regulated NRP1 expression suppressed the phosphorylation level on Akt. The results indicated that combined treatment significantly reduced SLUG and NRP1 expression simultaneously. Co-inhibition of SLUG and NRP1 signaling synergistically suppressed tumor growth, lung cancer cell invasive ability, circulating tumor cell amount, and angiogenesis in tumor tissue (Fig. 4F).

Table 1 The results of blood chemistry analysis. Group

GOP (U/l)

GPT (U/l)

BUN (mg/dl)

NH3 (mg/dl)

PBS buffer control Combined treatment

89  19.5 90  27.2

23  5.0 27  6.7

23  2.2 25  1.8

310  19.4 301  32.0

4. Discussion Aptamers are an emerging class of molecules that rival antibodies in molecular recognition, but with lower toxicity and immunogenicity [37]. Although there is an aptamer database available in public domain, few aptamers target cell-surface receptors are used in cell-specific siRNA delivery [28]. Surface NCL serves as a promising target for tumor-specific drug delivery due to the increased expression levels of surface nucleolin on various types of cancer cells and the rapid endocytosis after aptNCL binding [30]. The aptNCL itself possesses anti-proliferative effects on almost 60 cancer cell lines in the National Cancer Institute (NCI) with IC50 at 6.3 mM. Aside from the anti-proliferation ability of aptNCL, the aptNCL-siRNA chimeras provide an alternative application of aptNCL at a lower treatment dosage (nM rather than mM). Besides, the broad application of these chimeras on the treatment of different types of cancers can be anticipated. Until now, there has been no ideal cancer-specific surface marker in lung cancer that can be used to distinguish cancer cells from normal ones and the off-target effect remains a problem. To minimize side effects to normal cells, an important issue is target gene selection for diminishing cancer cells but not normal cells. Metastatic cancer cells have certain characteristics that are not usually found in normal well-differentiated cells. As such, instead of silencing cell survival-related genes, targeted treatment to lung metastasis and minimal side effects to normal cells can be ensured by cell-specific delivery and knocking down only the essential genes involved in metastatic signaling pathways. SLUG and NRP1 are two important genes that regulate lung cancer cell metastasis and angiogenesis in tumor tissue. This study demonstrates that co-inhibition of SLUG and NRP1 expressions by aptNCL-siRNA chimeras synergistically suppress the number of circulating tumor cells in blood and reduce the length and density of vessel in xenograft tumor tissue. Nonetheless, the detailed mechanism behind this synergistic effect warrants further investigation. A recent report demonstrated that NRP1 promotes the nuclear translocation of SNAI1, a family member of SLUG, and induces EMT in prostate cancer cells [38]. It can be speculated that similar phenomenon can be seen in lung cancer. In present study, combined treatment of chimeras not only silences SLUG expression but also has the potential of inhibiting the nuclear translocation of SLUG through NRP1. Studies have shown that anti-angiogenesis treatment can significantly suppresses primary tumor growth but will also contribute to increased local invasion and metastasis, resulting in high recurrence rates [39]. The combination of aptNCLSLUGsiR and aptNCL-NRP1siR chimeras, an anti-invasion agent with an anti-angiogenesis treatment, may therefore improve overall anti-tumor efficacy in the clinical setting. Apart from the role of SLUG and NRP1 in regulating tumor cell metastasis, both are involved in crucial pathways that confer chemo- or radio-resistance [40,41]. Previously, increased SLUG expression in non-small cell lung cancer patients with acquired resistance to EGFR tyrosine kinase inhibitor, gefitinib, has been demonstrated [42]. By silencing SLUG expression, resistant cells restore the sensitivity to gefitinib. The restoration of sensitivity to various clinical drugs through SLUG knockdown also occurs in other type of cancers, including malignant mesothelioma [43], cholangiocarcinoma [44], chronic myelocytic leukemia [45], and neuroblastoma [46]. These results suggest that the aptNCL-SLUGsiR chimera can be used to overcome SLUG-mediated chemo- and radio-resistance. Moreover, nearly 20% of epithelial tumor cells harbor activating mutations on K-Ras and there is no drug that directly targets mutant K-Ras [47]. SLUG has proven to be a synthetic lethal pair gene to mutant K-Ras in colon cancer [48]. Based on this finding, it

W.-Y. Lai et al. / Biomaterials 35 (2014) 2905e2914

is feasible for the aptNCL-SLUGsiR chimera to kill cancer cells with mutant K-Ras in a synthetic lethal manner. On the other hand, NRP1 plays an important role in chemo-resistance partially through integrin-dependent survival pathway [41]. Knockdown of NRP1 expression has been shown to re-sensitize lung cancer cells to 5-FU and paclitaxel treatment, and increase the chemo-sensitivity of pancreatic cancer cells to gemcitabine [41,49]. Taken together, these results suggest that the aptNCL-NRP1siR chimera may be a potent adjuvant treatment in combination with other chemotherapeutic drugs for increasing chemo-sensitivity. A tumor is a heterogeneous tissue that contains several cell types, including cancer cells, cancer-associated fibroblasts, and immune and inflammatory cells [50]. All types of cells contribute to different hallmark traits, such as sustained proliferative signaling, induced angiogenesis, activated invasion and metastasis, and induced tumor-promoting inflammation. Since a repertoire of siRNA is readily available to silence human genes, using cell-specific aptamer targeting different cell types and blocking key signaling pathways is a very promising direction for developing new therapeutic modalities. 5. Conclusion The present study provides evidence that the combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR silences the expression and downstream signaling of SLUG and NRP1 in lung cancer cells. In a mouse model, the combined treatment synergistically suppresses tumor growth, lung cancer cell invasiveness, and angiogenesis in tumor tissue without affecting normal physiological functions. The results suggest that simultaneously silence SLUG and NRP1 signaling with aptNCL-siRNA chimeras may be a new and feasible targeted therapy for lung cancer patients. Grant support This work was supported by grants from the National Science Council of the Republic of China (NSC100-2325-B001-016, NSC1012325-B001-038, NSC102-2911-I-002-303). Conflict of interest The authors have declared that no conflicts of interest exist. Acknowledgments The authors would like to thank Dr. Konan Peck, who unfortunately passed away during the period of this research. The rational designed aptamer-siRNA chimera cocktail therapy originated from Dr. Peck. This paper was completed to honor of his devotion to the development of lung cancer treatment. The authors also thank the Taiwan Mouse Clinic, which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the National Science Council (NSC) of Taiwan for the technical support in blood chemistry analysis. References [1] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics. CA Cancer J Clin 2009;59:225e49. [2] Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008;359: 1367e80. [3] Pao W, Chmielecki J. Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer. Nat Rev Cancer 2010;10:760e74. [4] Temel JS, Greer JA, Muzikansky A, Gallagher ER, Admane S, Jackson VA, et al. Early palliative care for patients with metastatic non-small-cell lung cancer. N Engl J Med 2010;363:733e42.

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