Tumor Microenvironment Modulation by Cyclopamine Improved

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Tumor Microenvironment Modulation by Cyclopamine Improved Photothermal Therapy of Biomimetic Gold Nanorods for Pancreatic Ductal Adenocarcinomas Ting Jiang, Bo Zhang, Shun Shen, Yanyan Tuo, Zimiao Luo, Yu Hu, Zhiqing Pang, and Xinguo Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09458 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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ACS Applied Materials & Interfaces

Tumor Microenvironment Modulation by Cyclopamine Improved Photothermal Therapy of Biomimetic Gold Nanorods for Pancreatic Ductal Adenocarcinomas Ting Jianga,b,1, Bo Zhanga,1, Shun Shenb, Yanyan Tuob, Zimiao Luob, Yu Hua*, Zhiqing Pangb* and Xinguo Jiangb a

Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of

Science & Technology, Wuhan, Hubei, 430022, PR China; b

School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery, Ministry of

Education, 826 Zhangheng Road, Shanghai, 201203, PR China * Corresponding author: Yu Hu, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei, 430022, PR China Tel.: +86-27-85726335; fax: +86-27-85776343. E-mail address: [email protected] (Y. Hu). Zhiqing Pang, School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery, Ministry of Education, 826 Zhangheng Road, Shanghai, 201203, PR China Tel.: +86-21-51980069; fax: +86-21-51980069. E-mail address: [email protected] 1

Equal contribution to the work.

Abstract Due to the rich stroma content and poor blood perfusion, pancreatic ductal adenocarcinoma (PDA)

ACS Paragon Plus Environment


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is a tough cancer that can hardly be effectively treated by chemotherapeutic drugs. Tumor microenvironment modulation or advanced design of nanomedicine to achieve better therapeutic benefits for PDA treatment was widely advocated by many reviews. In the present study, a new photothermal therapy strategy of PDA was developed by combination of tumor microenvironment modulation and advanced design of biomimetic gold nanorods. On the one hand, biomimetic gold nanorods were developed by coating gold nanorods (GNRs) with erythrocyte membrane (MGNRs). It was shown that MGNRs exhibited significantly higher colloidal stability in vitro, stronger photothermal therapeutic efficacy in vitro, and longer circulation in vivo than GNRs. On the other hand, tumor microenvironment modulation by cyclopamine treatment successfully disrupted the extracellular matrix of PDA and improved tumor blood perfusion. Moreover, cyclopamine treatment significantly increased the accumulation of MGNRs in tumors by 1.8-fold and therefore produced higher photothermal efficiency in vivo than the control group. Finally, cyclopamine treatment combined with photothermal MGNRs achieved the most significant shrinkage of Capan-2 tumors xenografts among all the treatment groups. Therefore, with the integrated advantages of tumor microenvironment regulation and long-circulation biomimetic MGNRs, effective photothermal therapy of PDA was achieved. In general, this new strategy of combining tumor microenvironment modulation and advanced design of biomimetic nanoparticles might have great potential in PDA therapy.

Key words Tumor microenvironment modulation; cyclopamine; biomimetic gold nanorods; erythrocyte membrane; photothermal therapy; pancreatic ductal adenocarcinomas


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Introduction Photothermal therapy (PTT), in which photon energy is converted to thermal energy upon irradiation, is a minimally invasive hyperthermic treatment method for tumors, 1-3 and has aroused extensive attention worldwide. Different types of nanoparticles including those based on gold, carbon, copper and iron, et al have been used for PTT for a wide range of tumors and obtained therapeutic benefits to various extents. 1, 2, 4 Unlike conventional therapeutics, these nanoparticles can display a preferential tumor accumulation based on enhanced permeability and retention (EPR) effect and thus mediate a selective hyperthermia effect upon precise irradiation with near-infrared light. However, as far as we are concerned, researches about PTT for pancreatic ductal adenocarcinoma (PDA), a common and lethal malignancy resulting in tens of thousands of deaths every year, 5 were still limited. The main reasons behind this phenomenon might be due to the poor delivery of photothermal nanoparticles to PDA which was mainly hindered by the highly desmoplastic response of PDA and the short circulation of nanoparticles used for PTT. PDA is well known for the highly desmoplastic property and consequently the rich extracellular matrix (ECM). 5, 6 Besides, tumor vessels in PDA were also very scarce. The dense ECM not only isolated tumor cells into tumor nests, but also compressed tumor vessels to reduce tumor blood perfusion. The compromised tumor perfusion plus the blocking resistance from the dense ECM could reduce the transport of nanoparticles to arrive at tumor site and the following penetrating process to finally reach tumor cells. 7 The highly desmoplastic response of PDA was regulated by many signaling pathways, among which hedgehog (Hh) signaling pathway played a crucial role. 8-12

As known to all, Hh ligands derived from tumor cells could act on Patched1 receptor in

tumor-associated fibroblasts to alleviate the inhibition of the 12-transmembrane protein



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Smoothened (SMO), resulting in activation of the transcription factor glioma-associated oncogene family zinc finger-1 (GLI-1) and expression of ECM-related proteins including collagens and fibronectins. 8 Consequently, cyclopamine, a specific inhibitor of Hh signaling pathway, which was able to significantly inhibit the desmoplastic response, might be utilized to modulate the tumor microenvironment of PDA to help enhance nanoparticle delivery to PDA (Figure 1). 13, 14 It was well documented that the circulation time of nanoparticles was a critical factor in dominating tumor nanoparticle accumulation, especially for tumors like PDA with scarce tumor vessels. Therefore, the circulation time of those nanomaterials for PTT was also a crucial issue deserving further exploration to improve PTT efficacy. Red blood cell (RBC) membrane-coated nanomedicines were reported to harbor the properties of RBC such as minimal uptake by phagocytes and long-circulation lifetime and have attracted increasing attention recently. 15-18 It was now well known that RBC membrane modification on nanoparticles could significantly increase the circulation time of nanoparticles compared with PEGylation, a classical method used for avoiding phagocyte uptake and increasing circulation time of nanoparticles. Furthermore, RBC membrane modification would not significantly impact the photothermal conversion efficiency of photothermal nanoparticles. 19, 20 Therefore, in this study, RBC membrane might be utilized to modify photothermal nanoparticles to elongate their circulation time, and help increase their delivery to PDA (Figure 1). To test our hypothesis mentioned above, modulation of ECM of PDA and optimization of the circulation time of photothermal nanoparticles simultaneously were performed to obtain more PTT benefits of PDA (Figure 1). Human pancreatic cancer cell line Capan-2 xenografts with rich ECM were used as the tumor models. Cyclopamine was used as the modulator of ECM of PDA and gold


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nanorods (GNRs), which have aroused extensive attention for PTT because of advantages including small size, high absorption coefficient and narrow spectral band width, were selected as the model photothermal nanoparticles. As far as we were concerned, it was the first time that tumor microenvironment modulation and advanced design of photothermal nanoparticles were combined to achieve better PTT benefits of PDA. In this study, GNRs were synthesized by a seed-mediated growth method and were further coated with RBC membrane (MGNRs). 21 In addition, the photothermal efficiency and long circulation of MGNRs were assessed by PTT tests in vitro and pharmacokinetics and biodistribution studies in vivo, and compared with those of GNRs. Furthermore, the effect of cyclopamine treatment on the tumor microenvironment of PDA including ECM morphology and tumor perfusion was investigated. Finally, therapeutic benefit of MGNRs plus cyclopamine treatment was evaluated.

Materials and methods Materials Chloroauric acid (HAuCl4·3H2O), cetyltrimethyl ammonium bromide (CTAB), silver nitrate (AgNO3), sodium borohydride (NaBH4), L-ascorbic acid (AA), Ethylenediaminetetraacetic acid EDTANa2

were

purchased

from

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazdium

Aladdin bromide

(Shanghai, (MTT)

assay

China). and

11-mercaptoundecaonic acid (MUDA) were purchased from Sigma-Aldrich (Saint Louis, USA). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin solution, and Trpsin-EDTA solution were purchased from Gibico (USA). Cylopamine were obtained from Struchem Co., Ltd (Wujiang, China). Cremophor® EL



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(polyoxyethlene castor oil derivatives) was purchased from BASF (Ludwigshafen, Germany). Propidium iodide (PI) and 3',6'-Di(O-acetyl)-4',5'-bis[N,N-bis(carboxymethyl)aminomethyl] fluorescein, tetraacetoxymethyl ester (Calcein-AM) were purchased from KeyGEN BioTECH (China). DyLight®488-labeled tomato lectin (Lycopersicon esculentum) was from Vector (USA). CD31 goat polyclonal primary antibody was ordered from R&D (USA). Hoechst 33342 was purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). The microbubble contrast agent was provided by VisualSonics (Vevo MicroMarkerTM, Toronto, Canada). Human-derived pancreatic cancer Capan-2 cell lines were from American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 medium supplemented with 10 % FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 ºC in a humidified atmosphere containing 5 % carbon dioxide (CO2). Male Balb/c nude mice and Balb/c mice aged 6-8 weeks were bought from the Shanghai Slac Lab Animal Ltd. (Shanghai, China). All animal experiments were carried out in accordance with the protocol evaluated and approved by the Experimental Animal Ethics Committee of School of Pharmacy, Fudan University (2014-03-YJ-PZQ-01). Synthesis of gold nanorods GNRs were synthesized by a seed-mediated growth method developed by Jana et al and

Nikoobakht et al. 21 Briefly, to make the seed solution, 72 µL of 1 % HAuCl4 and 20 mL of 0.1 M CTAB were mixed and stirred vigorously. After 60 µL ice-cold 0.1 M NaBH4 was added, the mixed solution was stirred for another 2 min. The brown solution was kept at 25 ºC for at least 3 h before further use. To make the growth solution, 110 µL of 0.1 M AA was dropwise added to the yellow mixture of 20 mL of 0.1M CTAB , 200 µL of 0.01 M AgNO3, 286 µL of 1 % HAucl4 and 40 µL of 1M HCl under gentle stirring. After the growth solution turned colorless, 24 µL of seed


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solution was added and the solution was kept at 28 ºC with a water bath for 4 h to obtain GNRs. RBC ghosts derivation RBC ghosts were prepared as previously reported by Zhang et al. 15 Briefly, the whole blood was collected from the Balb/c mice by cheek porch puncture with heparin-treated sterile centrifuge tube and was centrifuged at 800 × g for 5 min at 4 °C to remove the serum and the buffy coat. The resulting packed RBCs were then washed with ice cold PBS containing 1 mM EDTANa2 for 3 times and spun down at 800 × g at 4 ºC. For hypotonic medium treatment, the washed RBCs were suspended in ice cold 0.25× PBS for 10 min and were centrifuged at 20000 × g for 4 min at 4 ºC. The released hemoglobin was removed, and the resulting RBC ghosts were collected. Preparation of MGNRs The obtained GNRs were washed with deionized water twice to remove the excess CTAB by centrifugation at 8000 g for 8 min and resuspended in 5 mL of deionized water. The solution was then mixed with 50 µL of 20 mM MUDA-ethanol solution and kept at room temperature overnight for MUDA activation. After centrifugation at 6000 g for 8 min, the GNRs were collected and resuspended in 1.2 mL of RBC ghosts. Then the mixture was sonicated in a capped glass vial at a frequency of 53 kHz and a power of 100 W for 2 min. The resulting MGNRs were kept at 4 ºC in dark place for later use. Characterization of MGNRs and GNRs The concentration of the atomic gold in the MGNRs and GNRs solution was determined by inductively coupled plasma mass (ICP-MS, Nexlon 300X, USA). Briefly, the sample was added into 1 mL of aqua regia, mixed at room temperature overnight, and kept at 60 ºC for 12 h to remove acids. The sample was subsequently resuspended in deionized water at a final volume of 5



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mL, filtered through a 0.22-µm membrane filter unit (Millipore), and subjected to ICP-MS for Au content quantification. The morphologies of MGNRs and GNRs were directly observed under a transmission electron microscope (TEM, JEM-1400plus, JEOL, Japan) operating at 100 kV. The zeta potentials of MGNRs and GNRs were measured using a Malvern Nano ZS (Malvern Instruments, UK) at 25 ºC, respectively. The UV-VIS absorption spectra of MGNRs and GNRs were recorded by a UV-VIS recording spectrophotometer (UV-2401, Japan), respectively. To investigate the stability of MGNRs, MGNRs and GNRs were dispersed in phosphate buffer saline (PBS, 0.01 M, pH=7.4) or 10 % FBS in water and stored at room temperature. The plasmon resonance peak shifts, absorbance at 808 nm, and zeta potentials of MGNRs and GNRs were monitored at preset time points. In vitro photothermal conversion effect The in vitro photothermal conversion effect of MGNRs and GNRs were measured by monitoring the temperature changes of MGNRs and GNRs solution when radiated with an 808-nm laser (Changchun New Industries Optoelectronics Technology, Changchun, China) at varying power densities. Briefly, 100 µl of MGNRs and GNRs solution with Au content of 42 µg/mL in 96-well plates were radiated with an 808-nm laser at different power densities including 0.75 W/cm2, 1 W/cm2, and 2 W/cm2 for 5 min and cooled down naturally to room temperature for 5 min. The solution temperature was recorded every 10 s during the whole experiment. To assess the effect of Au concentration on the photothermal conversion effect, 100 µl of MGNRs and GNRs solution with different Au concentrations including 21, 42, and 84 µg/mL were radiated with an 808-nm laser at 0.75 W/cm2 for 5 min and cooling down naturally to room temperature. In vitro PTT studies


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The in vitro cytotoxicity of the MGNRs upon near infrared (NIR) irradiation was assessed with MTT assays. Briefly, Capan-2 cells were seeded into a 96-well plate at the density of approximately 104 cells/well, incubated for 24 h until cells reached approximately eighty percent confluence. After being washed twice with PBS, cells were treated with varying concentration of MGNRs and GNRs at 37 ºC for 3 h. Afterwards, cells were radiated at 0.75 W/cm2 with an 808-nm laser for 5 min. For MTT assay, cells were treated with 0.5 mg/mL of MTT in 1640 medium for 4 h and dissolved with dimethyl sulfoxide after removing the medium. Relative cell viability was determined by monitoring the optical densities at 570 nm with a microplate reader (BioTeK SynergyTM, America). For fluorescent imaging, cells were stained by Calcein-AM (2 µmol/L) and PI (4 µmol/L) and photographed using an inverted fluorescence microscope (LEICA DMI 4000 B, Germany). Calcein-AM is a green fluorescence probe labeling living cells, whereas PI is a red fluorescence probe labeling cells with compromised membranes. In order to find out whether cyclopamine enhances the cancer cell-killing effect of PTT treatment, the in vitro cytotoxicity of the MGNRs upon NIR irradiation in the presence of cyclopamine was also assessed with MTT assays. Briefly, Capan-2 cells were seeded into a 96-well plate and incubated as described above. Afterwards, cells were incubated with GNRs or MGNRs solution with Au content 42 µg/mL in the presence of varying concentration of cyclopamine at 37 ºC for 3 h. Then cells were radiated at 0.75 W/cm2 with an 808 nm laser for 5 min and subjected to MTT assay as described above. Cyclopamine treatment of tumor xenograft-bearing mouse models To develop PDA-bearing mouse models, Capan-2 cells (5×106 cells/100 µl) were subcutaneously implanted into the right hind flank region of nude mice. 22 Tumor diameters were determined



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using a caliper, and tumor volumes (V) were approximated using the following formula: ܸ = 0.5 × ܽ × ܾ ଶ, where a represents the maximum diameter, and b represents the minimum diameter. When the tumor diameter reached 4-6 mm, mouse models were subjected to cyclopamine treatment. Cremophor EL, a FDA-approved surfactant with low toxicity used in oral preparations, injections, and external preparations was used to solubilize water-insoluble cyclopamine for oral administration. Cyclopamine was dissolved in the mixture of ethanol, Cremophor EL and deionized water with the cyclopamine concentration of 10 mg/ml and orally administrated to mouse models once a day at the dose of 50 mg/kg for three weeks. The control group received blank vehicles containing an equal dose of ethanol and Cremophor EL. ECM modulation by cyclopamine treatment When cyclopamine treatment ended, mouse models were sacrificed, and tumor xenografts were sliced for immunofluorescence staining of fibronectin, a marker of ECM, 8, 23 as previously described. 22. To evaluate the fibronectin disruption effect of cyclopamine treatment, the fluorescence intensity of fibronectin in tumor slices from six randomly-assigned regions in each tumor (n=3) was quantified by the ZEN 2012 software 22. Tumor perfusion improvement by cyclopamine treatment When cyclopamine treatment ended, DyLight® 488-lectin-labeling experiment was performed to evaluate the effect of cyclopamine treatment on tumor perfusion as previously described. 24 Briefly, DyLight® 488-lectin was intravenously injected at the dose of 5 mg/kg one hour before heart perfusion. Tumor xenografts were collected and sliced for tumor vessel staining by CD31 goat polyclonal primary antibody. After the nuclei were stained with Hoechst 33342, the slices were mounted in Dako flurescent mounting medium. The co-localization of CD31 signals and DyLight®


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488-lectin signals in the tumor sections from six randomly-assigned regions in each tumor (n=3) were captured at 200× magnification and further analyzed using the ImageJ software (NIH) to assess tumor perfusion. In vivo ultrasound imaging of tumor xenografts When cyclopamine treatment ended, tumor xenografts of mouse models were subjected to in vivo ultrasound imaging with a Vevo LAZR imaging system (VisualSonics Inc., Toronto, Canada) to directly visualize the effect of cyclopamine treatment on tumor blood perfusion. 22 Briefly, after anesthetization with 5% chloral hydrate, mouse models were placed for ultrasound scanning. Videos and images of the tumor region were acquired before, during, and after a bolus injection of 107 microbubbles into the mice through tail vein. Parameters associated with the perfusion model function from tumor xenografts were acquired for analysis of tumor perfusion by VevoLAB software. 25 MGNR pharmacokinetics and biodistribution studies For pharmacokinetics studies, male Balb/c mice aged 6-8 weeks were randomly divided into MGNRs group and GNRs group. Each mouse was intravenously injected 150 µL of MGNRs or GNRs suspended in PBS (Au content of 2.5 mg/mL). 50 µL of blood were collected by cheek pouch puncture at varying time points (1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h) after injection and subjected to Au content determination with ICP-MS as described above. The GNP concentration in the blood was expressed as the percentage of injected dose per ml (% ID /ml). The pharmacokinetics parameters including area under the concentration-time curve (AUC0-t), mean residence time (MRT0-t), elimination rate constant (k), clearance (Cl), and the half-life (t1/2) were calculated by DAS 3.0 software.



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For biodistribution studies, mouse models with or without cyclopamine treatments were intravenously injected with 150 µL of MGNRs suspended in PBS (Au content of 2.5 mg/mL). Twenty-four hours post injection; the mice were sacrificed followed by heart perfusion with saline. Tumors and major organs including hearts, livers, spleens, lungs and kidneys were harvested and digested with 4 mL of aqua regia for Au content determination with ICP-MS. The GNP concentration in tissues was expressed as the percentage of injected dose per gram of tissue (% ID/g). In vivo photoacoustic (PA) imaging of tumor-bearing mice Due to the specific photoacoustic character of MGNRs, the concentration of MGNRs at the tumor site can be indirectly determined by PA imaging using a Vevo LAZR system equipped with a linear array transducer (LZ400, 30Hzs center frequency). To determine the specific absorption wave length of MGNR, the spectral scanning of MGNR was performed after subcutaneous injection of 150 µL of MGNR dispersion on the back of mice. To reduce the signal disturbance caused by hemoglobin in circulation, PA signals at the wavelength of 685 nm, 705 nm, 750 nm, 780 nm, 808 nm, 850 nm, and 865 nm were all collected for spectral unmixing process. For PA imaging, Capan-2 tumor-bearing mice with or without cyclopamine treatments were intravenously injected with 150 µL of MGNR dispersion in PBS (Au content of 125 µg). After being anesthetized using isoflurane at a concentration of 1.5-2%, the mice were placed on the working plate with continuous isoflurane inhalation anesthesia for further PA imaging. PA signals of MGNR in tumors were obtained at different time points (2, 8, 12, 24, and 48 h) post injection and the data were analyzed by Vevo LAB software. In vivo PTT efficacy


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Capan-2 tumor bearing nude mouse models were randomized into 4 treatment groups (n=5): control group, cyclopamine group, laser only group, and cyclopamine + laser group. The cyclopamine group and cyclopamine + laser group were orally treated with cyclopamine solution while control group and laser only group received blank vehicles as described above. After cyclopamine or vehicle treatment for 3 weeks, the laser only group and cyclopamine+laser group were intravenously injected with 150 µL of MGNRs dispersion (Au content of 125 µg). Two hours post injection; tumor xenografts were radiated at 2 W/cm2 with an 808-nm laser (Changchun New Industries Optoelectronics Technology, Changchun, China) for 5 min. The temperature was recorded using an infrared camera thermographic system (InfraTec, vario CAM® hr research, German) during the irradiation. After the irradiation ended, mouse models were returned to the animal house and maintained under standard housing condition. Tumor size and body weight of each mouse were measured every two days for 3 weeks. When tumor volume reached above 1500 mm3, all mouse models were sacrificed and the tumors were collected for photography and wet weight recording. Tumor growth inhibition rates based on tumor size (TGIRV) and tumor weight (TGIRW) were calculated by the following formulas, respectively: ܶ‫= ݒܴܫܩ‬ ௐ௖ିௐ௧ ௐ௖

௏௖ି௏௧ ௏௖

; ܶ‫= ݓܴܫܩ‬

. In these formulas, Vc and Wc indicated tumor volume and tumor weight in the control

group, respectively; Vt and Wt indicated tumor volume and tumor weight in the treatment group, respectively. Tumors were imbedded in paraffin and sectioned into slices of 5-µm thickness for hematoxylin-eosin (H&E) staining as previously described. 26 For in vivo toxicity assay, the body weight of mouse models were monitored every three days during the whole experiment. Major organs including hearts, livers, spleens, lungs and kidneys were obtained from mouse models at the study end point and sectioned for H&E staining for histological analysis. The H&E staining



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tumor slices and normal organ slices were observed under the fluorescence microscope (LEICA DMI4000B, Germany). Statistical analysis Statistical differences were analyzed with an unpaired Student's t-test for two groups’ comparison and one-way analysis of variance (ANOVA) for multiple-group comparison. Data are presented as mean ± standard deviation, and a P values

Tumor Microenvironment Modulation by Cyclopamine Improved

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