Abstract
Background/Aim: The aim of the present study was to establish a patient-derived xenograft (PDX) mouse model of non–small cell lung cancer (NSCLC) and investigate the anti-tumor efficacy of silencing of TUG1 and LCAL6 long non-coding RNA in the PDX model. Materials and Methods: PDXs were established by subcutaneously implanting NSCLC surgical tumor fragments into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical and real-time polymerase chain reaction (RT-PCR) analyses for NSCLC subtype-specific markers and expression of LCAL6 and TUG1. Anti-tumor efficacy of siRNA silencing of TUG1 and LCAL6 was also investigated in the PDX model. The effect of TUG1 and LCAL6 silencing on protein expression of proliferation marker Ki67 and HOX-gene family HOXB7 in the tumors was assessed by immunohistochemical staining and Western blotting. Results: Establishment of NSCLC PDX models resulted in 9 of 26 cases (34.6%). Lung squamous cell carcinomas (SCC) had a higher engraftment rate (58.3%) than lung adenocarcinomas (ADC) (18.2%) (p<0.05). Comparative analysis indicated these established PDX models of NSCLC closely resembled the original tumors with regard to NSCLC subtype-specific markers TTF-1, napsin A, p63 and expression of LCAL6 and TUG1. The tumor volume and weight were significantly reduced in the TUG1-silenced group as compared to the control group (p<0.05). However, no significant tumor growth inhibition was found in the LCAL6-silenced group (p>0.05). Expression of both TUG1and LCAL6 was reduced by siRNA treatment. Expression of Ki67 and HOXB7 was significantly suppressed in both the TUG1- and LCAL6-silenced groups compared to the control group (p<0.01). The TUG1-silenced group showed more reduced Ki67 expression than the LCAL6-silenced group (p<0.05). Conclusion: PDX NSCLC models were established with a high degree of similarity with the original tumor with regard to histological, immunohistochemical features and RNA expression of TUG1 and LCAL6. Silencing of TUG1 inhibited both tumor growth and expression of the proliferation marker Ki67 and HOX-gene family HOXB7 in the PDX model of NSCLC.
Non–small cell lung cancer (NSCLC) accounts for over 80% of lung cancer deaths (1, 2). The prognosis of NSCLC is still poor (3-5).
Taurine up-regulated gene 1 (TUG1) is induced by taurine. TUG1 is over-expressed and can promote cell proliferation in some cancers (6-9). Lung-cancer-associated transcript 6 (LCAL6) is up-regulated and correlated with tumor progression in lung adenocarcinoma (10). Silencing of TUG1 and LCAL6 inhibited tumor growth and metastasis in some cancer models (10, 11). However, the understanding for the roles of TUG1 and LCAL6 in lung cancer is still very limited.
In the present study, we established a panel of patient-derived xenografts (PDXs) of NSCLC (12-18) and characterized these xenograft models for expression of TUG1 and LCAL6. Furthermore, the anti-tumor efficacy of silencing of TUG1 and LCAL6 was investigated in a PDX model of NSCLC.
Materials and Methods
Patient tumor samples. A total of 26 primary NSCLC tissues were collected from patients who had undergone surgery at the Jinling Hospital (the Second Affiliated Hospital of Nanjing Medicine University, Nanjing, China) between 2016 and 2017. Tumors were classified as NSCLC based on the World Health Organization (WHO) criteria upon examination by licensed pathologists. None of the patients received chemotherapy or radiotherapy before surgery. Written informed consent was obtained from each patient and the study was approved by the Institutional Review Board of Nanjing Medical University.
Animals. Animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and approved by the Animal Center of Jinling Hospital (Nanjing, P.R. China). BALB/C male nude mice, 6-8 weeks, 20-25 g, were purchased from the Beijing Vital River Laboratory and Animal Technology Co., Ltd. (Beijing, P.R. China). All mice were maintained in a HEPA-filtered environment at 24-25°C and humidity was maintained at 50-60%. All animals were fed with in autoclaved laboratory rodent diet.
Tumor engraftment and PDX maintenance. Immediately after surgery, fresh tumor tissues were placed in RPMI 1640 culture medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1 mmol/1 L-glutamine, 10% fetal bovine serum (FCS) (Life Technologies Inc., Burlington, Canada) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin) and transferred to the animal facilities in Jinling Hospital within 1-2 h after resection. Necrotic tissues were removed and viable tissues were cut with scissors and minced into 2-3 mm3 pieces. Animals were anesthetized by injection of 0.02 ml of a solution comprising 50% ketamine, 38% xylazine, and 12% acepromazine maleate. Tumor fragments were subcutaneously engrafted on the flank of nude mice. The exponentially-growing tumors were harvested and passaged subcutaneously to other mice. After 3-5 consecutive passages, the xenografts were stabilized and subsequently subjected to model characterization. Fresh xenograft tumor fragments were cryopreserved in a medium containing 10% DMSO in liquid nitrogen, using standard techniques, for future model recovery. All surgical procedures and animal manipulations were conducted under HEPA-filtered laminar-flow hoods.
Histology and immunohistochemistry. For histological examination, tumor tissue specimens were fixed in 4% formalin, embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin (H&E). Tumor histology was evaluated by a pathologist who was blinded to the experimental groups. For immunohistochemical analysis, formalin-fixed, paraffin-embedded tumor tissues were sectioned at 4 μm thickness. All sections were deparaffinized using 100% xylene, dehydrated with an ethanol gradient. Antigen retrieval was performed by autoclaving (120°C for 2 min in 1 mmol/l EDTA, pH 8.0). Incubation with primary antibodies against thyroid transcription factor-1 (TTF-1) (1:100, ab40880, Abcam, Cambridge, U.K.); napsin-A (1:250, ab133249, Abcam); p63 (1:100, ab53039, Abcam; Ki67 (1:300, VP-RM04, Vector Laboratories, Burlingame, CA, USA); and homeobox B7 (HOXB7) (1:100, ab196007, Abcam) was performed overnight at 4°C. After washing with pH 7.4 phosphate-buffered saline (PBS), the sections were then incubated with a secondary antibody (Dako REAL EnVision Detection System, Dako, Stockport, UK) for 30 min at room temperature. Color development was performed with 3, 3’-diaminobenzidine (DAB). Nuclei were lightly counter stained with hematoxylin. The slides were viewed at ×400 magnification and positive cells were recognized by the appearance of brown staining. Expression levels were quantified by counting the number of positive cells in at least 3 fields/sample and expressing the results as percentage of the total number of cells in the same field.
Treatment. siRNA targeting TUG1 (siTUG1) (sense 5’-CUACAACUAUCUUCCUUUACCACCG-3’; antisense 5’-CGGUGGUAAAGGAAGAUAGUUGUAGCA-3’) and LCAL6 (siLCAL6) (sense 5’-CCUGCAGAUAGAUGCUCAAGU-3’; antisense 5’-UUGAGCAUCUAUCUGCAGGUG-3’) was obtained from Gene Pharma (Shanghai, P.R. China). Treatments were initiated when the average tumor size reached 100 mm3. The mice with tumors were randomly divided into three groups of 6 mice each: Group 1 served as normal control and received saline only. Group 2 (siTUG1) received siTUG1 treatment at 1.5 mg/kg/dose. Group 3 (siLCAL6) received siLCAL6 treatment at 1.5 mg/kg/dose. All treatments were administered by intraperitoneal injection (ip) twice per week for 2 weeks. Tumor growth was measured twice a week with calipers. Tumor volume was calculated using the formula (L × W2) × ½, where W and L represent the perpendicular minor dimension and major dimension, respectively. All animals were sacrificed four weeks after treatment initiation. At autopsy the tumors were removed and weighed.
Real-time polymerase chain reaction (RT-PCR). Total RNA was extracted from tissues with trizol reagent (Life Technologies, Scotland, UK) according to the manufacturer's protocol. Total RNA (1.5 μg) was reverse transcribed in a final volume of 20 μl using random primers under standard conditions using the PrimeScript RT Master Mix (Takara, Cat: RR036A). After the RT reaction, quantitative RT-PCR was performed using the SYBR Select Master Mix (cat: 4472908, Applied Biosystems, Foster City, CA, USA) with 0.5 μl complementary DNA (cDNA) on an ABI 7300 system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The primer sequences were as follows: GAPDH (sense, 5’-CGCTCTCTGCTCCTCCTGTTC-3’, and antisense, 5’-ATCCGTTGACTCCGACCTTCAC-3’); TUG1(sense, 5’-TAGCAGTTCCCCAATCCTTG-3’, and antisense, 5’-CACAAATTCCCATCATTCCC-3’); LCAL6 (sense, 5’-GTCCTCAAGACCACAACCCT-3’, and antisense, 5’-ACCATCCACGAGTCAGTTCA-3’). The RT-PCR reaction included an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 92°C for 15 s and 60°C for 1 min. The Ct value for each sample was calculated with the ΔΔCt method, and expression-fold changes (tumor vs. normal) were calculated using 2−ΔΔCT methods.
Western blotting. Protein from tumor tissue was extracted with RIPA buffer (Roche Diagnostics, Indianapolis, IN, USA). Total proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membranes were incubated with 5% nonfat milk solution for blocking non-specific binding and then with primary antibodies to Ki67 (1:300, VP-RM04, Vector Laboratories) HOXB7 (1:100, ab196007, Abcam, Cambridge, USA) and tubulin (Sigma, St. Louis, MO, USA. After washing twice with Tris buffered saline, the membranes were incubated with an appropriate secondary antibody (1:5000) conjugated with HRP for 2 h at room temperature. Analysis of electrochemiluminescence was performed according to the manufacturer's instructions using a Bio-Rad imaging system. Quantity One version software (Bio-Rad, Hercules, CA, USA) was used to quantify the density of bands.
Statistical analysis. The in vivo tumor take rates were compared with the X2 test. The significant difference between control and treated groups was analyzed using the Student's t-test. Analyses were performed using SPSS 20.0 statistical software. All statistical tests were two-sided with p<0.05 indicating statistical significance.
Results and Discussion
Establishment of NSCLC PDX nude mouse models. Twenty-six NSCLC surgical specimens from primary tumors were subcutaneously implanted into immunodeficient mice leading to the establishment of 9 PDX models, representing a tumor take rate of 34.6%.
To determine clinic-pathologic parameters related to PDX establishment success rates, tumor take rates were compared according to various patient characteristics. As shown in Table I, the tumor histological type was the only parameter that had a significant impact on the success rate of PDX establishment (X2=3.884, p<0.05). Lung squamous cell carcinomas (SCC) were much more tumorigenic in nude mice (7/12; 58.3%) compared to lung adenocarcinomas (ADC) (2/11; 18.2%). Other factors, including age, sex, smoking status, tumor size, and overall tumor stage did not correlate with the success rate of PDX establishment (p>0.05).
Preservation of parental NSCLC's biologic characteristics in PDX. The application of PDX models as a model system for human NSCLCs depends on the precise retention of the parental tumors' pathologic and molecular characteristics. As shown in Figure 1, histologic comparison revealed a high degree of similarity in the subtype-specific morphologic features and differentiation status between the parental tumors and corresponding xenografts. The expression of NSCLC histologic subtype-specific markers TTF-1, napsin A and p63 was compared by immuno histochemical analysis between patient tumors and corresponding PDXs. As shown in Table II, the expression of TTF-1 and napsin A protein in ADC patient tumors was not significantly changed in corresponding xenografts (p>0.05). No significant difference in the expression level of p63 protein was found between SCC patient tumors and corresponding PDXs (p>0.05).
Moreover, we detected the expression level of TUG1 and LCAL6 in normal lung tissues, patient tumors and PDX tumors by RT-PCR. We found that both TUG1 and LCAL6 were strongly over-expressed in NSCLC patient tumors and PDXs as compared to normal lung tissues (Figure 2A). No significant change in expression level of TUG1 and LCAL6 was found between the patient tumors and corresponding PDX tumors (Figure 2B).
Effect of silencing of TUG1 and LCAL6 on expression of TUG1and LCAL6 in the NSCLC PDX model. The expression of TUG1and LCAL6 was detected using RT-PCR in the tumor tissues from the mice treated with siTUG1 and siLCAL6. As shown in Figure 3, the expression of TUG1 in the siTUG1-treated group was significantly reduced compared to the control group (p<0.01). The expression of LCAL6 in the siLCAL6-treated group was also significantly lower than the control group (p<0.01). This result indicated that the expression of TUG1and LCAL6 in PDX models of NSCLC was suppressed by siTUG1 and siLCAL6 treatment, respectively.
Effect of silencing of TUG1 and LCAL6 on tumor growth in the NSCLC PDX model. The anti-tumor efficacy of siTUG1 and siLCAL6 was evaluated in a PDX model of lung adenocarcinoma. Tumor images, tumor growth curves and mean tumor weight in each group are shown in Figure 4. Tumor volume and weight were significantly reduced in the siTUG1-treated group compared to the control group (p<0.05). However, no significant tumor-growth inhibition was found in the siLCAL6-treated group although lower mean tumor volume and weight was observed compared to the control group (p>0.05).
Effect of silencing of TUG1 and LCAL6 on expression of Ki67 and HOXB7 in the NSCLC PDX model. Immunohistochemical staining and Western blotting were performed to assess the effects of siTUG1 and siLCAL6 treatment on protein expression of proliferation markers Ki67 and HOX-gene-family HOXB7 in the tumors. As shown in Figures 5A and B, immunohistochemical staining demonstrated positive protein expression of Ki67 and HOXB7 in the tumors of the control group. Ki67 expression was significantly suppressed in the siTUG1- and siLCAL6-treated groups compared to the control group (p<0.01). The siTUG1 group showed more reduced Ki67 expression than the siLCAL6 group (p<0.05). HOXB7 expression was significantly reduced in the siTUG1- and siLCAL6-treated groups compared to the control group (p<0.05). Western blotting analysis further confirmed significantly-reduced expression of Ki67 and HOXB7 protein in the siTUG1- and siLCAL6-treated groups compared to the control group (p<0.01) (Figure 5C). As observed by immunohistochemical staining, the siTUG1 group also showed more reduced Ki67 expression than the siLCAL6 group (p<0.05). This result suggests siTUG1 can inhibit tumor Ki67 expression more than siLCAL6.
Numerous new long non-coding RNA (lncRNA) molecules have been shown to be involved in tumorigenesis and progression of (19). It was found that knockdown of TUG1 and LCAL6 inhibited cancer cell growth both in vitro and in vivo (8, 10).
In the present study, we explored the potential function of TUG1 and LCAL6 in NSCLCs by siRNA-mediated silencing in NSCLC PDX models. siTUG1 and siLCAL6 significantly reduced TUG1 and LCAL6 expression, respectively. However, only siTUG1 treatment resulted in significant tumor growth inhibition, suggesting possible greater therapeutic potential of targeting TUG1 than LCAL6 in NSCLC.
Ki67, a cell proliferation marker, was significantly suppressed by silencing of TUG1 and LCAL6 in the NSCLC PDX. The silencing with siTUG1 showed a more suppressive effect on Ki67 expression than siLCAL6, which may explain the greater anti-tumor efficacy of siTUG1 than siLCAL6 on the NSCLC PDX. Our result showed that HOXB7 expression was significantly reduced by silencing of TUG1 and LCAL6 in the NSCLC PDX model, indicating association of HOX-gene with regulation of TUG1 and LCAL6.
PDX models are very important for the discovery and individualization of cancer therapy (20). The present study indicates that NSCLC histotype is most important in establishment of PDX models with squamous cell carcinoma having a greater establishment rate than adenocarcinoma. Our studies in other cancer types have suggestd that tumor size (21) and tumor grade and therapeutic pretreatment (16), are very important for establishment.
Future experiments will focus on patient-derived orthotopic xenograft (PDOX) of NSCLC (13) in order to study metastasis and its therapeutic inhibition.
Acknowledgements
This research was supported by the Research Project of Jinling Hospital (No. 2016044).
Footnotes
↵* These Authors contributed equally to this work.
Conflicts of Interest
None of the Authors have a conflict of interest in regard to this study.
- Received October 10, 2017.
- Revision received October 31, 2017.
- Accepted November 1, 2017.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved