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Establishment of a patient-derived Wilms' tumor xenograft model: A promising tool for individualized cancer therapy
Journal of Pediatric Urology (2014) 10, 123e129
Establishment of a patient-derived Wilms’ tumor xenograft model: A promising tool for individualized cancer therapy Mohammad-Javad Mohseni a, Saeid Amanpour b, Samad Muhammadnejad b, Shabnam Sabetkish a, Ahad Muhammadnejad b, Reza Heidari a, Mahnaz Haddadi b, Zohreh Mazaheri b, Mohammad Vasei c, Abdol-Mohammad Kajbafzadeh a,*,1 a
Pediatric Urology Research Center, Children’s Center of Excellence, Department of Pediatric Urology, Islamic Republic of Iran b Department of Experimental Research, Cancer Research Center, Iranian Cancer Institute, Islamic Republic of Iran c Department of Pathology, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran Received 5 March 2013; accepted 13 July 2013 Available online 26 August 2013
KEYWORDS Wilms’ tumor; Patient-derived tumor tissue xenograft; Individualized cancer therapy; Chemosensitivity
Abstract Objective: Lack of appropriate approaches that reliably predict response of Wilms’ tumor (WT) to anticancer agents remains a major deficiency in clinical practice of individualized cancer therapy. The aim of this study was to establish a patient-derived tumor tissue (PDTT) xenograft model of WT for individualized chemotherapeutic regimen selection in accordance with the patient’s tumor nature. Material and methods: Tumor specimens of a primary WT were orthotopically implanted into three nude mice, and after 4 weeks xenografts were harvested for serial heterotopic transplantation in 20 nude mice that were divided into three experimental groups and one control group. In vitro and in vivo chemosensitivity to doxorubicin, actinomycin-D, and vincristine were evaluated. Hematoxylin and eosin (H&E) staining and immunohistochemical examination with desmin, vimentin, myogenin, and neuron-specific enolase (NSE) were also applied to determine histological stability of the xenograft during serial transplantation compared with the original tumor tissue.
* Corresponding author. Pediatric Urology Research Center, Department of Pediatric Urology, Children’s Hospital Pediatric Center of Excellence, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran. E-mail address: [email protected] (A.-M. Kajbafzadeh). 1 Postal address: 2nd Floor, No. 32, 7th St., Saadat-Abad Ave., Tehran 1998714616, Islamic Republic of Iran. Tel.: þ98 21 2208994, þ98 91 21092241 (mobile); fax: þ98 21 22069451. 1477-5131/$36 ª 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpurol.2013.07.009
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M.-J. Mohseni et al. Results: The xenograft model was successfully established. Histopathologic characteristics of the xenograft tumors were similar to the patient’s tumor. Early passage of the PDTT showed a similar chemosensitivity pattern to the original tumor tissue. Conclusions: PDTT xenograft of WT provides an appropriate model for individualized cancer therapeutic regimen selection by means of its biological stability compared with original patient’s tumor. ª 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
Introduction
Material and methods
Wilms’ tumor (WT), or nephroblastoma, is the most common primary renal malignancy of childhood, affecting 1 in 10,000 children; typically between 2 and 4 years old [1]. It results from unregulated proliferation of embryonic blastemal cells and most frequently presents as a unilateral and sporadic tumor [2]. WT is a curable malignancy with longterm survival rate of 90% for localized disease and more than 70% for metastatic disease [3]. This therapeutic success results from a multimodality treatment approach based on the combination of cytoreductive surgery, chemotherapy with vincristine, actinomycin D, and doxorubicin, and radiotherapy [4]. However, the overall burden of treatment can lead to frequent and early severe complications of neutropenic sepsis and late complications of skeletal abnormalities, endocrinopathies, cardiac dysfunction, and secondary malignancies [5]. On the other hand, heterogeneity in response to chemotherapeutic agents is commonly seen in WT [6]. Variability in patient response to combined chemotherapy is frequently observed across the human population. These interindividual differences are due to differences in the molecular characteristics of tumors [7]. WTs are genetically heterogeneous and typically present with triphasic histology composed of blastemal, epithelial, and stromal cells; however, individual cell types may be predominant in certain tumors. This heterogeneous histology can exacerbate heterogeneity in response to chemotherapy [6]. The goal of individualized cancer therapy is to select the right chemotherapy regimen for the right patient by predicting patient response to cancer chemotherapy [8]. The use of ineffective chemotherapy can lead to unnecessary adverse drug reactions and expense, delay of more effective regimens, and the potential for the development of cross-resistance to additional chemotherapeutic agents [9]. The lack of general clinic-relevant tumor models that can reliably predict the response to chemotherapeutic agents remains a remarkable deficiency in the practice of personalized cancer therapy [10]. The fact that human tumor cells can possibly grow progressively in immunodeficient mice initiates a shift towards applying patientderived tumor tissue (PDTT) xenograft models to predict clinical response [11]. In the present pilot study, we used PDTT xenograft in athymic nude mice to assess the usefulness of the xenograft for personalized chemotherapy, and to determine the histopathologic status of the PDTT xenograft, and also to investigate the chemotherapeutic response of the PDTT xenograft of WT compared with the original tumor.
Patient’s tumor sample Following radical nephrectomy from one male patient with WT, the whole kidney was placed in a cool container (4 C). The gross anatomy of specimens (tumor sizes, weight, and the tumor capsule) was observed and recorded by a pathologist in the operating room. The tumor was immediately bivalved under supervision of the senior surgeon and observed by the clinical pathologist. The non-necrotic tumor specimen, which was not resected for pathological diagnosis, was placed in cooled Dulbecco’s modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml amphotericin B under sterile conditions and preserved for cell culture, xenograft transplantation, and in vitro chemosensitivity assay. The mass was also sent to the pathology department for final histological evaluation. Prior written informed consent was obtained from the patient’s parents. The patient had not received prior chemotherapy or radiotherapy.
In vivo experiment PDTT xenograft transplantation Animals were treated according to guidelines outlined by Institutional Ethical Committee. All animals were kept under optimized hygienic conditions in an individually ventilated cage system. The animals were fed an autoclaved commercial diet and water ad libitum. After preliminary washing of the tumor specimen in Hank’s balanced salt solution (HBSS); supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml amphotericin B, necrotic areas were removed from the tumor sample. The tumor tissue was cut into multiple 1 3 3 mm3 explants for grafting under the renal capsules of three athymic nude mice, as previously described [12]. Four weeks later, tumor-bearing animals with distended abdomen were killed using CO2 gas. The mice were then submerged in 70% ethanol for 2 min and transferred to a laminar flow hood for dissection. Tumors were minced under sterile conditions. Tumor explants were implanted into 20 athymic nude mice (2 grafts per mouse) under balanced anesthesia with 100 mg/kg ketamine and 10 mg/kg xylazine by a small incision and subcutaneous pocket made on flanks in which one explant had been deposited in the pocket. One drop of 100 penicillinestreptomycin solution was placed into the pocket before suturing the incision. Measurement of tumor
Establishment of a patient-derived Wilms’ tumor xenograft model volume was done every 3 days. Tumor volume was determined by caliper measurement in two perpendicular diameters of the tumor and calculated using the formula a b2 0.52, in which a and b stand for long and short diameter, respectively [13].
In vivo treatment protocol Xenografts from mouse-to-mouse passage were allowed to grow to a size of 100 mm3. Seven days after xenografting, mice were randomized in the following four groups of treatment with five mice in each group: (a) doxorubicin (20 mg/kg); (b) actinomycin D (125 mg/kg); (c) vincristine (415 mg/kg); and (d) control (100 ml of saline). The applied dosages were based on appropriate translation of the dose from children to mice. The mouse equivalent dose (MED) was calculated using the formula suggested by the Food and Drug Administration of the United States [14]. MEDZchild0 s dose child0 skm =mouse km : In this formula, km refers to the body weight (kg) divided by body surface area (mm2). The km is considered to be 25 and 3 in children and mice, respectively according to the study of Reagan-Shaw S. et al. [14]. The drugs were administered intraperitoneally on a schedule based on the mass doubling time of the tumor. All groups were treated every 4 days for three consecutive sessions (q4d 3). The experiment was terminated when the tumor volume of control group reached 1000 mm3. Relative tumor growth inhibition (TGI) was calculated by relative tumor growth of the treated group divided by relative tumor growth of the control group (T/C). According to the criteria of the Division of Cancer Treatment (NCI), we defined a response as 0e20% TGI; stability, 21e50% TGI; and tumor progression >50% TGI [15].
In vitro experiment Primary cultures for the in vitro experiment To obtain a sufficient number of cells for the in vitro chemosensitivity assay, the primary explant technique was initiated from fresh tumor specimen. Briefly, tumor tissue was minced with scalpels into 1-mm3 explants, washed three times with HBSS supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml amphotericin B, and plated on 25-cm2 culture flasks in 1 ml of 50:50 DMEM:F12 medium supplemented with 20% FBS. After adhesion of tumor material, medium was added to reach a volume of 5 ml. The medium was changed every 2 days. When a substantial outgrowth was observed, the cells were subcultivated using trypsin-EDTA. To reach relatively purified neoplastic cells, subcultivation was performed three times. During cell culture, isolated cells were observed using an inverted phase contrast microscope and photographed by a pathologist who was totally blind to the study (Olympus, Tokyo, Japan).
In vitro chemosensitivity assay For chemosensitivity evaluation, a modification of the clonogenic assay was used as described by Hamburger and
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Salmon [16]. The bottom layer which consisted of 0.2 ml/ well DMEM was supplemented with 20% FBS and 0.75% agar; 20 104 cells were added to 0.2 ml of the medium containing 0.4% agar and were plated in 24-multiwell dishes above the bottom layer. Doxorubicin (0.1 mg/ml), actinomycin D (0.01 mg/ml), or vincristine (0.1 mg/ml) was added as drug overlays in 0.2 ml of medium in triplicate. Cultures were incubated at 37 C under 5% CO2 in a humidified atmosphere for up to 21 days and were monitored closely for colony formation using an inverted microscope. On day 21, colonies more than 50 mm in diameter were counted. TGI was calculated by colony count of the treated group divided by colony formation of the control group (T/C). We defined response, stability, and resistance, with T/C < 30%, 30% < T/C < 50%, and T/C > 50%, respectively [17].
Pathological examination Tumor specimens from the patient, orthotopic, and heterotopic models were fixed by 4% formaldehyde in 0.1 M phosphate-buffered saline solution. The fixed tissues were dehydrated using graded concentrations of ethanol, embedded in paraffin wax, and stained with H&E. Then a pathologist blinded to the study reviewed each slide. Immunohistochemical staining of tumor specimens was performed using desmin, vimentin, myogenin, and NSE markers.
Statistical analysis Data were represented as mean SD. Statistically significant differences were calculated with one-way analysis of variance (ANOVA) followed by the Bonferroni t-test for multiple comparisons. All statistical analyses were performed with BioStat 2008.
Results Establishment of WT xenograft model in athymic nude mice As shown in Fig. 1, the orthotopic model of WT from PDTT was successfully established with a 67% take rate. After 20 days, the abdominal wall of tumor-bearing animals was distended unilaterally, with blood vessels visible from the outside. At day 28, one of the animals appeared moribund. Urinary retention was obvious in gross examination because of tumor pressure on the bladder. Heterotopic generation of WT from orthotopic PDTT xenografts was also established successfully, with a take rate of 75%.
Histopathologic characterization The patient’s tumor depicted a classical triphasic nephroblastoma containing hypercellular blastemal cells arranged in sheets and nests with a spindle cell stroma. In the blastemal component, a glandular configuration, representative of tubule formation, was noted. There was no evidence of marked nuclear enlargement and hyperchromasia or aberrant tissue formation. Mitotic figures were easily seen
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Figure 1 Patient-derived tumor tissue (PDTT) xenograft models of Wilms’ tumor. Patient’s tumor tissues were first implanted into the subrenal capsule of athymic nude mice. After 4 weeks, orthotopic xenografts were harvested for serial heterotopic transplantation: (A) orthotopic PDTT xenograft tumor; (B) heterotopic PDTT xenograft tumor.
in each high power field, with no abnormalities in any of the samples. Histopathologic characterization of xenograft tumors showed a triphasic WT with favorable histology resembling the original tumor. Immunohistochemistry illustrated that both the original and the xenograft tumors expressed desmin, vimentin, myogenin and NSE (Fig. 2).
Chemosensitivity of PDTT xenograft model of WT Heterotopic xenptransplantation gives us the ability to monitor the growth kinetic properties of transplanted tumors by measuring the volume in a real-time manner. But in orthotopic transplantation, real-time monitoring is not feasible, except with imaging systems. So, in the present study, growth kinetic analysis was carried out using heterotopic transplantation. The growth kinetic curves of the
control and treatment groups are shown in Fig. 3. Briefly, the mean latency period of tumor appearance was determined as 9.33 4.99 days. The mean tumor volume reached 100 mm3 and 1000 mm3 at 16.74 6.63 days and 46.63 17.10 days, respectively. Statistically significant differences were shown in TGI at the cut-off time as determined by one-way ANOVA (p < 001). Xenograft tumors were responsive to vincristine (TGI Z 16.03%). Doxorubicin led to tumor growth stability (TGI Z 35.12%). However, these xenograft tumors were resistant to actinomycin-D (TGI Z 66.18%).
In vitro chemosensitivity assay A statistically significant difference was shown in the number of formed colonies between different treatment groups, as determined by one-way ANOVA (p < 001) (Fig. 4). Neoplastic cells were responsive to vincristine (T/
Figure 2 Histopathologic features of patient-derived tumor tissue (PDTT) heterotopic xenograft model of Wilms’ tumor (WT). (A) In this photomicrograph, triphasic histology of WT including blastemal, epithelial and stromal cells is shown (H&E staining; original magnification, 200). (B) Presence of hyperchromatin cells and observation of mitotic divisions, indicate the activity of blastemal component, which can differentiate to epithelial-like and fibroblast-like cells (H&E staining; original magnification, 400). (CeF) Immunohistochemical photomicrographs illustrating the expression of desmin (C), vimentin (D), myogenin (E), and neuron-specific enolase (F) (original magnification, 200).
Establishment of a patient-derived Wilms’ tumor xenograft model
Figure 3 Growth kinetic curves of heterotopic patientderived tumor tissue (PDTT) xenografts of Wilms’ tumor after treatment with vincristine, doxorubicin, and actinomycin-D. Treatments were begun when mean tumor volume was reached to around 200 mm3. These PDTT xenografts showed response to Vincristine, stability to Doxorubicin, and resistance to actinomycin D.
C Z 13.24%), stable with doxorubicin (T/C Z 46/73%), and resistant to actinomycin-D (T/C Z 72.9%). Fig. 5 summarizes the correlation between in vivo and in vitro assays. Similar results were observed in patients and children experiencing an uneventful postchemotherapy period after 3 years of follow-up.
Discussion Innovation of models that can retain the histopathologic, genetic and phenotypic features of the original tumor can play a crucial role in identifying new therapeutic targets and studying various aspects of tumor progression [18]. During the past decades, treatment of cancer has slowly begun to change from a “one-size-fits-all” to a more personalized approach [19], and xenograft models have been considered as a major preclinical screen to develop
Figure 4 In vitro chemosensitivity assay on primary Wilms’ tumor (WT). Single-cell suspensions of WT were treated with vincristine (0.1 mg/ml), doxorubicin (0.1 mg/ml), actinomycin-D (0.01 mg/ml) and medium as placebo in soft agar. In day 21, counting of colonies with more than 50 mM in diameter was carried out. Error bars represent SD.
Figure 5
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The correlation between in vivo and in vitro assays.
cancer therapeutic approaches. Many investigators have tried to develop an assay system that would predict the response of an individual patient’s tumor to a particular chemotherapeutic drug. One of the proposed systems developed to perform in vivo chemosensitivity prediction is to use transplantable subcutaneous tumors which is established from patients’ tumor specimens [17]. Although this technique has a good predictive value, it suffers from low take rates of around 30%. However, establishment of subcutaneous xenograft models may take as long as 10 months [20], which severely limits the application of this method for individualized chemotherapy. The results of the current study demonstrated that primary orthotopic xenotransplantation of primary WT and secondary heterotopic passage can resolve these problems by increasing the take rate and allowing assay of chemotherapeutic agents within a short time frame to corroborate the timely initiation of chemotherapy. It is worth emphasizing that if the chemo response profile of the tumor is obvious, in cases of recurrence, patients can be treated according to personalized medicine instead of a one-size-fits-all strategy. Rapid graft microvasculature development and high tissue perfusion are among the advantages of the subrenal capsule (SRC) site for engraftment and drug response evaluation [21]. In the study by Beppino C. Giovanella et al. [22], three human tumor types (melanomas, colorectal carcinomas, and breast carcinomas) were grown as serially transplanted xenografts under the kidney capsules of 60e100 adult nude mice. The mice received the chemotherapy regimen for 8 days (adriamycin, 5-fluorouracil, methotrexate, cytoxan, vincristine, methyl-CCNU, alkeran and vinblastine, and BCNU) with one control group that was injected daily with saline. The results were then compared with same tumor types that had been treated in human patients, which supported the validity of this model as a predictive scheme, evaluating the effectiveness of different anticancer drugs. The present study aimed to establish a PDTT xenograft model of WT, useful for prediction of response to chemotherapy. This study demonstrated the feasibility of establishing the PDTT xenograft model of WT at a suitable time by primary orthotopic xenotransplantation of a patient’s tumor and secondary heterotopic passage. Our results highlighted the maintenance of histopathologic
128 characteristics of the primary neoplastic WT during the establishment and passage of the xenograft model. This work clearly showed that there is a correlation between the results of in vitro clonogenic assay and in vivo activity in response to chemotherapy. Our data supported the usefulness of the PDTT xenograft of WT for individualized chemotherapy. As the cancer cells develop after derivation from aggressive and advanced neoplasms, they lack their natural environment, which plays a crucial role in their progression and development [12]. We can assess a successful engraftment by the application of highly advanced cancers and extremely low take rate of the tumor [23]. Preservation of xenograft tumor tissue histology and recapitulation of the phenotypic and genotypic characteristics compared with the original sample, expansion of the natural tumor stroma by the collaboration of host stromal elements, and potential sources of nutrition are among the most notable advantages of the SRC xenografts of human tumor tissue models [12]. In one study in 2006 [12], SRC grafting methodology was applied to immunodeficient mice in order to establish human lung cancer tissue xenografts. The result of H&E staining during the first 2 months authenticated the retention of xenograft histologic features compared with original tumors. In the study of Xin Dong et al. [20] using immunodeficient mice, subrenal capsule xenografts were generated from primary patients’ non-small-cell lung cancers. The chemotherapeutic response rates of the xenografts were estimated as 28% in regimen A (cisplatin þ vinorelbine), 42% in regimen B (cisplatin þ docetaxel), and 44% in regimen C (cisplatin þ gemcitabine). In one study in 2010 [15], fresh surgical specimens of ependymoma, a malignant brain tumor, were directly injected intracerebrally in five severe complex immune deficiency mice. The results demonstrated that histopathologic features of the xenograft tumors were nearly identical to the original patient tumors and also CD133þ was maintained in xenografts during serial passaging. Xenograft models of human WT in athymic nude mice reproduced the histopathology of the original primary neoplasm, as mentioned by Li et al. [24], and Pinthuset et al. [5]. The results of the present study showed that the PDTT xenograft model of WT retained its similarity to the original relevant tumor in histologic features. The case in this study did not receive preoperative chemotherapy. This technique was applied to a The National Wilms Tumor Study (NWTS)-treated case in which an up-front, surgery-based system was developed. It seems that PDTT xenografts can be established from specimens obtained by needle biopsies carried out prior to neoadjuvant chemotherapy. Good predictive value of the in vivo chemosensitivity assay for PDTT xenograft models has been indicated by this study. The correlation of the chemo response profile obtained by this method with the clinical response cannot be estimated from only one patient, which is a limitation of the current study. Fiebig et al. [17] reported predictive correlations of 97% and 90% for resistance and sensitivity assays, respectively, to chemotherapeutic agents using PDTT xenograft tumors. Our data demonstrated a reliable correlation between in vitro assay of the primary patient’s tumor specimens as a surrogate to clinical response, and in vivo assay of PDTT xenograft
M.-J. Mohseni et al. tumors after passage in response to chemotherapy. This finding highlights the preservation of the response profile to chemotherapy from the patient’s body to a first-passage heterotopic PDTT xenograft model of WT.
Conclusions We conclude that the PDTT xenograft system may be a promising approach to predict the clinical response of WT to chemotherapeutic agents if similar results can be obtained from more patients. This system seems to be suitable for reliable assessment of the chemotherapeutic response to an individual patient’s tumor. It holds promise to study different aspects of tumor progression and select the most effective regimen and successful targeted treatment strategies for patients affected by WT. However, more studies are required to confirm the clinical applicability of the present pilot study. In subsequent studies, attempts will be made to reduce the time needed for the chemosensitivity test.
Conflict of interest None of the authors has direct or indirect commercial financial incentive associating with publishing the article and does not have any conflict of interest, and will sign the Disclosure Form.
Funding None.
Acknowledgments We are grateful to Mrs. S. Lotfi for her precise final linguistic revision of the manuscript.
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Establishment of a patient-derived Wilms’ tumor xenograft model [8] Meric-Bernstam F, Mills GB. Overcoming implementation challenges of personalized cancer therapy. Nat Rev Clin Oncol 2012;9:542e8. [9] Brower SL, Fensterer JE, Bush JE. The ChemoFx assay: an ex vivo chemosensitivity and resistance assay for predicting patient response to cancer chemotherapy. Methods Mol Biol 2008;414:57e78. [10] Jin K, He K, Han N, Li G, Wang H, Xu Z, et al. Establishment of a PDTT xenograft model of gastric carcinoma and its application in personalized therapeutic regimen selection. Hepatogastroenterology 2011;58:1814e22. [11] Jin K, Teng L, Shen Y, He K, Xu Z, Li G. Patient-derived human tumour tissue xenografts in immunodeficient mice: a systematic review. Clin Transl Oncol 2010;12:473e80. [12] Cutz JC, Guan J, Bayani J, Yoshimoto M, Xue H, Sutcliffe M, et al. Establishment in severe combined immunodeficiency mice of subrenal capsule xenografts and transplantable tumor lines from a variety of primary human lung cancers: potential models for studying tumor progressionerelated changes. Clin Cancer Res 2006;12:4043e54. [13] Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148e54. [14] Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J 2008;22:659e61. [15] Teicher BA, Andrews PA. Anticancer drug development guide: preclinical screening, clinical trials, and approval. 2nd ed. Humana Press, Totowa; 2004. [16] Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science 1977;197:461e3.
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[17] Fiebig H, Maier A, Burger A. Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery. Eur J Cancer 2004;40:802e20. [18] Kerbel RS. Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceiveddbut they can be improved. Cancer Biol Ther 2003;2(Suppl. 1):S134e9. [19] Van’t Veer LJ, Bernards R. Enabling personalized cancer medicine through analysis of gene-expression patterns. Nature 2008;452:564e70. [20] Dong X, Guan J, English JC, Flint J, Yee J, Evans K, et al. Patientderived first generation xenografts of non-small cell lung cancers: promising tools for predicting drug responses for personalized chemotherapy. Clin Cancer Res 2010;16:1442e51. [21] Bogden A, Haskell P, LePage D, Kelton D, Cobb W, Esber H. Growth of human tumor xenografts implanted under the renal capsule of normal immunocompetent mice. Pathobiology 1979;47:281e93. [22] Giovanella BC, Stehlin Jr JS, Shepard RC, Williams Jr LJ. Correlation between response to chemotherapy of human tumors in patients and in nude mice. Cancer 1983;52: 1146e52. [23] Wang Y, Revelo MP, Sudilovsky D, Cao M, Chen WG, Goetz L, et al. Development and characterization of efficient xenograft models for benign and malignant human prostate tissue. Prostate 2005;64:149e59. [24] Li MH, Yamase H, Ferrer F. Characterization of a WiT49 cell line derived orthotopic model of Wilms tumor. Pediatr Blood Cancer 2010;54:316e8.
Establishment of a patient-derived Wilms’ tumor xenograft model: A promising tool for individualized cancer therapy Mohammad-Javad Mohseni a, Saeid Amanpour b, Samad Muhammadnejad b, Shabnam Sabetkish a, Ahad Muhammadnejad b, Reza Heidari a, Mahnaz Haddadi b, Zohreh Mazaheri b, Mohammad Vasei c, Abdol-Mohammad Kajbafzadeh a,*,1 a
Pediatric Urology Research Center, Children’s Center of Excellence, Department of Pediatric Urology, Islamic Republic of Iran b Department of Experimental Research, Cancer Research Center, Iranian Cancer Institute, Islamic Republic of Iran c Department of Pathology, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran Received 5 March 2013; accepted 13 July 2013 Available online 26 August 2013
KEYWORDS Wilms’ tumor; Patient-derived tumor tissue xenograft; Individualized cancer therapy; Chemosensitivity
Abstract Objective: Lack of appropriate approaches that reliably predict response of Wilms’ tumor (WT) to anticancer agents remains a major deficiency in clinical practice of individualized cancer therapy. The aim of this study was to establish a patient-derived tumor tissue (PDTT) xenograft model of WT for individualized chemotherapeutic regimen selection in accordance with the patient’s tumor nature. Material and methods: Tumor specimens of a primary WT were orthotopically implanted into three nude mice, and after 4 weeks xenografts were harvested for serial heterotopic transplantation in 20 nude mice that were divided into three experimental groups and one control group. In vitro and in vivo chemosensitivity to doxorubicin, actinomycin-D, and vincristine were evaluated. Hematoxylin and eosin (H&E) staining and immunohistochemical examination with desmin, vimentin, myogenin, and neuron-specific enolase (NSE) were also applied to determine histological stability of the xenograft during serial transplantation compared with the original tumor tissue.
* Corresponding author. Pediatric Urology Research Center, Department of Pediatric Urology, Children’s Hospital Pediatric Center of Excellence, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran. E-mail address: [email protected] (A.-M. Kajbafzadeh). 1 Postal address: 2nd Floor, No. 32, 7th St., Saadat-Abad Ave., Tehran 1998714616, Islamic Republic of Iran. Tel.: þ98 21 2208994, þ98 91 21092241 (mobile); fax: þ98 21 22069451. 1477-5131/$36 ª 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpurol.2013.07.009
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M.-J. Mohseni et al. Results: The xenograft model was successfully established. Histopathologic characteristics of the xenograft tumors were similar to the patient’s tumor. Early passage of the PDTT showed a similar chemosensitivity pattern to the original tumor tissue. Conclusions: PDTT xenograft of WT provides an appropriate model for individualized cancer therapeutic regimen selection by means of its biological stability compared with original patient’s tumor. ª 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
Introduction
Material and methods
Wilms’ tumor (WT), or nephroblastoma, is the most common primary renal malignancy of childhood, affecting 1 in 10,000 children; typically between 2 and 4 years old [1]. It results from unregulated proliferation of embryonic blastemal cells and most frequently presents as a unilateral and sporadic tumor [2]. WT is a curable malignancy with longterm survival rate of 90% for localized disease and more than 70% for metastatic disease [3]. This therapeutic success results from a multimodality treatment approach based on the combination of cytoreductive surgery, chemotherapy with vincristine, actinomycin D, and doxorubicin, and radiotherapy [4]. However, the overall burden of treatment can lead to frequent and early severe complications of neutropenic sepsis and late complications of skeletal abnormalities, endocrinopathies, cardiac dysfunction, and secondary malignancies [5]. On the other hand, heterogeneity in response to chemotherapeutic agents is commonly seen in WT [6]. Variability in patient response to combined chemotherapy is frequently observed across the human population. These interindividual differences are due to differences in the molecular characteristics of tumors [7]. WTs are genetically heterogeneous and typically present with triphasic histology composed of blastemal, epithelial, and stromal cells; however, individual cell types may be predominant in certain tumors. This heterogeneous histology can exacerbate heterogeneity in response to chemotherapy [6]. The goal of individualized cancer therapy is to select the right chemotherapy regimen for the right patient by predicting patient response to cancer chemotherapy [8]. The use of ineffective chemotherapy can lead to unnecessary adverse drug reactions and expense, delay of more effective regimens, and the potential for the development of cross-resistance to additional chemotherapeutic agents [9]. The lack of general clinic-relevant tumor models that can reliably predict the response to chemotherapeutic agents remains a remarkable deficiency in the practice of personalized cancer therapy [10]. The fact that human tumor cells can possibly grow progressively in immunodeficient mice initiates a shift towards applying patientderived tumor tissue (PDTT) xenograft models to predict clinical response [11]. In the present pilot study, we used PDTT xenograft in athymic nude mice to assess the usefulness of the xenograft for personalized chemotherapy, and to determine the histopathologic status of the PDTT xenograft, and also to investigate the chemotherapeutic response of the PDTT xenograft of WT compared with the original tumor.
Patient’s tumor sample Following radical nephrectomy from one male patient with WT, the whole kidney was placed in a cool container (4 C). The gross anatomy of specimens (tumor sizes, weight, and the tumor capsule) was observed and recorded by a pathologist in the operating room. The tumor was immediately bivalved under supervision of the senior surgeon and observed by the clinical pathologist. The non-necrotic tumor specimen, which was not resected for pathological diagnosis, was placed in cooled Dulbecco’s modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml amphotericin B under sterile conditions and preserved for cell culture, xenograft transplantation, and in vitro chemosensitivity assay. The mass was also sent to the pathology department for final histological evaluation. Prior written informed consent was obtained from the patient’s parents. The patient had not received prior chemotherapy or radiotherapy.
In vivo experiment PDTT xenograft transplantation Animals were treated according to guidelines outlined by Institutional Ethical Committee. All animals were kept under optimized hygienic conditions in an individually ventilated cage system. The animals were fed an autoclaved commercial diet and water ad libitum. After preliminary washing of the tumor specimen in Hank’s balanced salt solution (HBSS); supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml amphotericin B, necrotic areas were removed from the tumor sample. The tumor tissue was cut into multiple 1 3 3 mm3 explants for grafting under the renal capsules of three athymic nude mice, as previously described [12]. Four weeks later, tumor-bearing animals with distended abdomen were killed using CO2 gas. The mice were then submerged in 70% ethanol for 2 min and transferred to a laminar flow hood for dissection. Tumors were minced under sterile conditions. Tumor explants were implanted into 20 athymic nude mice (2 grafts per mouse) under balanced anesthesia with 100 mg/kg ketamine and 10 mg/kg xylazine by a small incision and subcutaneous pocket made on flanks in which one explant had been deposited in the pocket. One drop of 100 penicillinestreptomycin solution was placed into the pocket before suturing the incision. Measurement of tumor
Establishment of a patient-derived Wilms’ tumor xenograft model volume was done every 3 days. Tumor volume was determined by caliper measurement in two perpendicular diameters of the tumor and calculated using the formula a b2 0.52, in which a and b stand for long and short diameter, respectively [13].
In vivo treatment protocol Xenografts from mouse-to-mouse passage were allowed to grow to a size of 100 mm3. Seven days after xenografting, mice were randomized in the following four groups of treatment with five mice in each group: (a) doxorubicin (20 mg/kg); (b) actinomycin D (125 mg/kg); (c) vincristine (415 mg/kg); and (d) control (100 ml of saline). The applied dosages were based on appropriate translation of the dose from children to mice. The mouse equivalent dose (MED) was calculated using the formula suggested by the Food and Drug Administration of the United States [14]. MEDZchild0 s dose child0 skm =mouse km : In this formula, km refers to the body weight (kg) divided by body surface area (mm2). The km is considered to be 25 and 3 in children and mice, respectively according to the study of Reagan-Shaw S. et al. [14]. The drugs were administered intraperitoneally on a schedule based on the mass doubling time of the tumor. All groups were treated every 4 days for three consecutive sessions (q4d 3). The experiment was terminated when the tumor volume of control group reached 1000 mm3. Relative tumor growth inhibition (TGI) was calculated by relative tumor growth of the treated group divided by relative tumor growth of the control group (T/C). According to the criteria of the Division of Cancer Treatment (NCI), we defined a response as 0e20% TGI; stability, 21e50% TGI; and tumor progression >50% TGI [15].
In vitro experiment Primary cultures for the in vitro experiment To obtain a sufficient number of cells for the in vitro chemosensitivity assay, the primary explant technique was initiated from fresh tumor specimen. Briefly, tumor tissue was minced with scalpels into 1-mm3 explants, washed three times with HBSS supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml amphotericin B, and plated on 25-cm2 culture flasks in 1 ml of 50:50 DMEM:F12 medium supplemented with 20% FBS. After adhesion of tumor material, medium was added to reach a volume of 5 ml. The medium was changed every 2 days. When a substantial outgrowth was observed, the cells were subcultivated using trypsin-EDTA. To reach relatively purified neoplastic cells, subcultivation was performed three times. During cell culture, isolated cells were observed using an inverted phase contrast microscope and photographed by a pathologist who was totally blind to the study (Olympus, Tokyo, Japan).
In vitro chemosensitivity assay For chemosensitivity evaluation, a modification of the clonogenic assay was used as described by Hamburger and
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Salmon [16]. The bottom layer which consisted of 0.2 ml/ well DMEM was supplemented with 20% FBS and 0.75% agar; 20 104 cells were added to 0.2 ml of the medium containing 0.4% agar and were plated in 24-multiwell dishes above the bottom layer. Doxorubicin (0.1 mg/ml), actinomycin D (0.01 mg/ml), or vincristine (0.1 mg/ml) was added as drug overlays in 0.2 ml of medium in triplicate. Cultures were incubated at 37 C under 5% CO2 in a humidified atmosphere for up to 21 days and were monitored closely for colony formation using an inverted microscope. On day 21, colonies more than 50 mm in diameter were counted. TGI was calculated by colony count of the treated group divided by colony formation of the control group (T/C). We defined response, stability, and resistance, with T/C < 30%, 30% < T/C < 50%, and T/C > 50%, respectively [17].
Pathological examination Tumor specimens from the patient, orthotopic, and heterotopic models were fixed by 4% formaldehyde in 0.1 M phosphate-buffered saline solution. The fixed tissues were dehydrated using graded concentrations of ethanol, embedded in paraffin wax, and stained with H&E. Then a pathologist blinded to the study reviewed each slide. Immunohistochemical staining of tumor specimens was performed using desmin, vimentin, myogenin, and NSE markers.
Statistical analysis Data were represented as mean SD. Statistically significant differences were calculated with one-way analysis of variance (ANOVA) followed by the Bonferroni t-test for multiple comparisons. All statistical analyses were performed with BioStat 2008.
Results Establishment of WT xenograft model in athymic nude mice As shown in Fig. 1, the orthotopic model of WT from PDTT was successfully established with a 67% take rate. After 20 days, the abdominal wall of tumor-bearing animals was distended unilaterally, with blood vessels visible from the outside. At day 28, one of the animals appeared moribund. Urinary retention was obvious in gross examination because of tumor pressure on the bladder. Heterotopic generation of WT from orthotopic PDTT xenografts was also established successfully, with a take rate of 75%.
Histopathologic characterization The patient’s tumor depicted a classical triphasic nephroblastoma containing hypercellular blastemal cells arranged in sheets and nests with a spindle cell stroma. In the blastemal component, a glandular configuration, representative of tubule formation, was noted. There was no evidence of marked nuclear enlargement and hyperchromasia or aberrant tissue formation. Mitotic figures were easily seen
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Figure 1 Patient-derived tumor tissue (PDTT) xenograft models of Wilms’ tumor. Patient’s tumor tissues were first implanted into the subrenal capsule of athymic nude mice. After 4 weeks, orthotopic xenografts were harvested for serial heterotopic transplantation: (A) orthotopic PDTT xenograft tumor; (B) heterotopic PDTT xenograft tumor.
in each high power field, with no abnormalities in any of the samples. Histopathologic characterization of xenograft tumors showed a triphasic WT with favorable histology resembling the original tumor. Immunohistochemistry illustrated that both the original and the xenograft tumors expressed desmin, vimentin, myogenin and NSE (Fig. 2).
Chemosensitivity of PDTT xenograft model of WT Heterotopic xenptransplantation gives us the ability to monitor the growth kinetic properties of transplanted tumors by measuring the volume in a real-time manner. But in orthotopic transplantation, real-time monitoring is not feasible, except with imaging systems. So, in the present study, growth kinetic analysis was carried out using heterotopic transplantation. The growth kinetic curves of the
control and treatment groups are shown in Fig. 3. Briefly, the mean latency period of tumor appearance was determined as 9.33 4.99 days. The mean tumor volume reached 100 mm3 and 1000 mm3 at 16.74 6.63 days and 46.63 17.10 days, respectively. Statistically significant differences were shown in TGI at the cut-off time as determined by one-way ANOVA (p < 001). Xenograft tumors were responsive to vincristine (TGI Z 16.03%). Doxorubicin led to tumor growth stability (TGI Z 35.12%). However, these xenograft tumors were resistant to actinomycin-D (TGI Z 66.18%).
In vitro chemosensitivity assay A statistically significant difference was shown in the number of formed colonies between different treatment groups, as determined by one-way ANOVA (p < 001) (Fig. 4). Neoplastic cells were responsive to vincristine (T/
Figure 2 Histopathologic features of patient-derived tumor tissue (PDTT) heterotopic xenograft model of Wilms’ tumor (WT). (A) In this photomicrograph, triphasic histology of WT including blastemal, epithelial and stromal cells is shown (H&E staining; original magnification, 200). (B) Presence of hyperchromatin cells and observation of mitotic divisions, indicate the activity of blastemal component, which can differentiate to epithelial-like and fibroblast-like cells (H&E staining; original magnification, 400). (CeF) Immunohistochemical photomicrographs illustrating the expression of desmin (C), vimentin (D), myogenin (E), and neuron-specific enolase (F) (original magnification, 200).
Establishment of a patient-derived Wilms’ tumor xenograft model
Figure 3 Growth kinetic curves of heterotopic patientderived tumor tissue (PDTT) xenografts of Wilms’ tumor after treatment with vincristine, doxorubicin, and actinomycin-D. Treatments were begun when mean tumor volume was reached to around 200 mm3. These PDTT xenografts showed response to Vincristine, stability to Doxorubicin, and resistance to actinomycin D.
C Z 13.24%), stable with doxorubicin (T/C Z 46/73%), and resistant to actinomycin-D (T/C Z 72.9%). Fig. 5 summarizes the correlation between in vivo and in vitro assays. Similar results were observed in patients and children experiencing an uneventful postchemotherapy period after 3 years of follow-up.
Discussion Innovation of models that can retain the histopathologic, genetic and phenotypic features of the original tumor can play a crucial role in identifying new therapeutic targets and studying various aspects of tumor progression [18]. During the past decades, treatment of cancer has slowly begun to change from a “one-size-fits-all” to a more personalized approach [19], and xenograft models have been considered as a major preclinical screen to develop
Figure 4 In vitro chemosensitivity assay on primary Wilms’ tumor (WT). Single-cell suspensions of WT were treated with vincristine (0.1 mg/ml), doxorubicin (0.1 mg/ml), actinomycin-D (0.01 mg/ml) and medium as placebo in soft agar. In day 21, counting of colonies with more than 50 mM in diameter was carried out. Error bars represent SD.
Figure 5
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The correlation between in vivo and in vitro assays.
cancer therapeutic approaches. Many investigators have tried to develop an assay system that would predict the response of an individual patient’s tumor to a particular chemotherapeutic drug. One of the proposed systems developed to perform in vivo chemosensitivity prediction is to use transplantable subcutaneous tumors which is established from patients’ tumor specimens [17]. Although this technique has a good predictive value, it suffers from low take rates of around 30%. However, establishment of subcutaneous xenograft models may take as long as 10 months [20], which severely limits the application of this method for individualized chemotherapy. The results of the current study demonstrated that primary orthotopic xenotransplantation of primary WT and secondary heterotopic passage can resolve these problems by increasing the take rate and allowing assay of chemotherapeutic agents within a short time frame to corroborate the timely initiation of chemotherapy. It is worth emphasizing that if the chemo response profile of the tumor is obvious, in cases of recurrence, patients can be treated according to personalized medicine instead of a one-size-fits-all strategy. Rapid graft microvasculature development and high tissue perfusion are among the advantages of the subrenal capsule (SRC) site for engraftment and drug response evaluation [21]. In the study by Beppino C. Giovanella et al. [22], three human tumor types (melanomas, colorectal carcinomas, and breast carcinomas) were grown as serially transplanted xenografts under the kidney capsules of 60e100 adult nude mice. The mice received the chemotherapy regimen for 8 days (adriamycin, 5-fluorouracil, methotrexate, cytoxan, vincristine, methyl-CCNU, alkeran and vinblastine, and BCNU) with one control group that was injected daily with saline. The results were then compared with same tumor types that had been treated in human patients, which supported the validity of this model as a predictive scheme, evaluating the effectiveness of different anticancer drugs. The present study aimed to establish a PDTT xenograft model of WT, useful for prediction of response to chemotherapy. This study demonstrated the feasibility of establishing the PDTT xenograft model of WT at a suitable time by primary orthotopic xenotransplantation of a patient’s tumor and secondary heterotopic passage. Our results highlighted the maintenance of histopathologic
128 characteristics of the primary neoplastic WT during the establishment and passage of the xenograft model. This work clearly showed that there is a correlation between the results of in vitro clonogenic assay and in vivo activity in response to chemotherapy. Our data supported the usefulness of the PDTT xenograft of WT for individualized chemotherapy. As the cancer cells develop after derivation from aggressive and advanced neoplasms, they lack their natural environment, which plays a crucial role in their progression and development [12]. We can assess a successful engraftment by the application of highly advanced cancers and extremely low take rate of the tumor [23]. Preservation of xenograft tumor tissue histology and recapitulation of the phenotypic and genotypic characteristics compared with the original sample, expansion of the natural tumor stroma by the collaboration of host stromal elements, and potential sources of nutrition are among the most notable advantages of the SRC xenografts of human tumor tissue models [12]. In one study in 2006 [12], SRC grafting methodology was applied to immunodeficient mice in order to establish human lung cancer tissue xenografts. The result of H&E staining during the first 2 months authenticated the retention of xenograft histologic features compared with original tumors. In the study of Xin Dong et al. [20] using immunodeficient mice, subrenal capsule xenografts were generated from primary patients’ non-small-cell lung cancers. The chemotherapeutic response rates of the xenografts were estimated as 28% in regimen A (cisplatin þ vinorelbine), 42% in regimen B (cisplatin þ docetaxel), and 44% in regimen C (cisplatin þ gemcitabine). In one study in 2010 [15], fresh surgical specimens of ependymoma, a malignant brain tumor, were directly injected intracerebrally in five severe complex immune deficiency mice. The results demonstrated that histopathologic features of the xenograft tumors were nearly identical to the original patient tumors and also CD133þ was maintained in xenografts during serial passaging. Xenograft models of human WT in athymic nude mice reproduced the histopathology of the original primary neoplasm, as mentioned by Li et al. [24], and Pinthuset et al. [5]. The results of the present study showed that the PDTT xenograft model of WT retained its similarity to the original relevant tumor in histologic features. The case in this study did not receive preoperative chemotherapy. This technique was applied to a The National Wilms Tumor Study (NWTS)-treated case in which an up-front, surgery-based system was developed. It seems that PDTT xenografts can be established from specimens obtained by needle biopsies carried out prior to neoadjuvant chemotherapy. Good predictive value of the in vivo chemosensitivity assay for PDTT xenograft models has been indicated by this study. The correlation of the chemo response profile obtained by this method with the clinical response cannot be estimated from only one patient, which is a limitation of the current study. Fiebig et al. [17] reported predictive correlations of 97% and 90% for resistance and sensitivity assays, respectively, to chemotherapeutic agents using PDTT xenograft tumors. Our data demonstrated a reliable correlation between in vitro assay of the primary patient’s tumor specimens as a surrogate to clinical response, and in vivo assay of PDTT xenograft
M.-J. Mohseni et al. tumors after passage in response to chemotherapy. This finding highlights the preservation of the response profile to chemotherapy from the patient’s body to a first-passage heterotopic PDTT xenograft model of WT.
Conclusions We conclude that the PDTT xenograft system may be a promising approach to predict the clinical response of WT to chemotherapeutic agents if similar results can be obtained from more patients. This system seems to be suitable for reliable assessment of the chemotherapeutic response to an individual patient’s tumor. It holds promise to study different aspects of tumor progression and select the most effective regimen and successful targeted treatment strategies for patients affected by WT. However, more studies are required to confirm the clinical applicability of the present pilot study. In subsequent studies, attempts will be made to reduce the time needed for the chemosensitivity test.
Conflict of interest None of the authors has direct or indirect commercial financial incentive associating with publishing the article and does not have any conflict of interest, and will sign the Disclosure Form.
Funding None.
Acknowledgments We are grateful to Mrs. S. Lotfi for her precise final linguistic revision of the manuscript.
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