1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Most of the cancer xenograft models are derived from tumor cell lines, but they do not sufficiently represent clinical cancer characteristics. Our objective was to develop xenograft models of bladder cancer derived from human tumor tissue and characterize them molecularly as well as histologically. A total of 65 bladder cancer tissues were transplanted to immunodeficient mice. Passagable six cases with clinico-pathologically heterogeneous bladder cancer were selected and their tumor tissues were collected (012T, 025T, 033T, 043T, 048T, and 052T). Xenografts were removed and processed for the following analyses: (i) histologic examination, (ii) short tandem repeat (STR) genotyping, (iii) mutational analysis, and (iv) array-based comparative genomic hybridization (array-CGH). The original tumor tissues (P 0) and xenografts of passage 2 or higher (≥P2) were analyzed and compared. As a result, hematoxylin and eosin staining revealed the same histologic architecture and degree of differentiation in the primary and xenograft tumors in all six cases. Xenograft models 043T_P2 and 048T_P2 had completely identical STR profiles to the original samples for all STR loci. The other models had nearly identical STR profiles. On mutational analysis, four out of six xenografts had mutations identical to the original samples for TP53, HRAS, BRAF, and CTNNB1. Array-CGH analysis revealed that all six xenograft models had genomic alterations similar to the original tumor samples. In conclusion, our xenograft bladder cancer model derived from patient tumor tissue is expected to be useful for studying the heterogeneity of the tumor populations in bladder cancer and for evaluating new treatments.

Bladder cancer (BCa) is also the second most common genitourinary malignancy in the United States, with an expected 69 000 newly diagnosed cases in 2008, and 14 000 deaths,[1] and it also is the second most common urologic cancer in South Korea.[2] Bladder cancer is a heterogeneous disease, with 70% of patients presenting with superficial cancers that tend to recur but are generally not life-threatening, and 30% presenting with muscle-invasive disease associated with a high risk of death from distant metastases.[1]

Animal models of BCa allow for the investigation of aspects of BCa that cannot be studied clinically, such as evaluating new chemotherapeutic agents or other treatments as well as investigating the basic mechanisms of tumor biology. Different animal models of preclinical BCa have been developed. Most of these models consist of chemically induced BCa,[3-5] implanting xenografts generated from well-established human BCa cell lines that have adapted to in vitro growth into immunodeficient mice,[6-9] or transplanting carcinogen-induced BCa in syngeneic, immunocompetent mice.[10-12] Although such models have been useful, they also have limitations. The chemically induced BCa model takes at least 8–11 months to develop tumors.[5] Models generated by planting human tumor cell lines subcutaneously in immunodeficient mice do not sufficiently represent clinical cancer characteristics, especially with regard to metastasis and drug sensitivity.[13] The cell lines are mainly undifferentiated, have only a minor relation to the tissue of origin, and show less physiological conditions in their micro-environment.[14]

Recently, a few groups have developed more relevant models based on xenografting primary human tumor tissue in immunodeficient mice, including non-small-cell lung, gastric, ovarian, and colon cancers.[14-18] These patient-derived tumor tissue xenograft models retain morphology, architecture, and molecular signatures similar to the original cancers.[17]

Therefore, the aim of our study was to develop and characterize a xenograft model of BCa using patient-derived tumor tissues with heterogeneous clinico-pathological features, which would be useful for investigating BCa biology and developing new anticancer therapeutics.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Patients and tumor tissue samples

Written informed consent was obtained from each patient and the study was approved by the Institutional Review Board of Samsung Medical Center (IRB File No. 2010-04-004). Tumor specimens were obtained at the time of surgery from 65 heterogeneous BCa patients. They were put into a medium immediately after surgical resection under sterile conditions. Samples for this study were immediately snap frozen and stored in −80°C for further analysis. For clinical data analysis, relevant clinico-pathological variables were obtained from all 65 patients. The clinical or pathologic BCa staging were determined according to the 2010 7th edition of the American Joint Committee on Cancer (AJCC) Tumor-Node-Metastasis (TNM) staging system.[19] Histologic grading of transitional cell carcinoma was determined according to the 1973 World Health Organization grading system.[20]

Establishment of xenografts

Surgical tumor samples were cut into 1 × 1 × 1 mm3 pieces and dipped with Matrigel (BD Biosciences, Bedford, MA, USA), a matrix of a mouse basement membrane neoplasm that can induce epithelial cells to differentiate.[21] The tumor tissue-Matrigel mixture was injected subcutaneously into the flank of 6-week-old female immunodeficient mice (BALB/c-nu), which were purchased from Charles River Japan (Osaka, Japan). The mice were housed in a specific pathogen-free facility and then maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were observed daily for tumor growth. “Sufficient tumor growth” was defined as the tumor volume of 1000 to 1500 mm3. After the sufficient tumor growth was observed, tumors at a size of 1 × 1 × 1 mm3 were removed and passaged several times in nude mice (designated P1 to P6). Xenografts of P2 or higher were recognized as appropriate xenograft models for this study. Numerous samples from early passages were stored in the tissue bank (liquid nitrogen) and used for further experiments. For the present study, original tumor tissues (P0) and P2 xenograft tissues were analyzed and compared.

Histologic examination by the hematoxylin and eosin staining

Selected tumor specimens were embedded in OCT media and 3-μm sections were cut by a microtome. The sections were rehydrated for 5 min and stained in hematoxylin solution for 1 min. They were then rinsed in HCl solution and differentiated in running tap water. Eosin staining lasted 30 s, after which the sections were dehydrated with 100% EtOH, cleared with xylene, and mounted for histologic examination. All microscopic observations and comparisons between the original tumor tissues and xenografts were performed by a single pathologist (G.Y.K.).

DNA extraction and quantitative polymerase chain reaction

The genomic DNA (gDNA) was extracted from each sample using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Expression levels were measured using SYBR Green (USB, Affymetrix, Santa Clara, CA, USA) and a PRISM 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each target gene was analyzed in triplicate. For absolute quantification, we determined gene expression levels using standard curves and Ct values. The conditions for PCR were 50°C for 2 min, 95°C for 10 min, followed by 35 cycles of 95°C for 10 s and 65°C for 1 min.

Short tandem repeat genotyping

Ten nanograms of target DNA was amplified by multiplex PCR using fluorescent dye-linked primers for 16 loci: 13 autosomal short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, vWA, TPOX, D18S51, D5S818, and FGA), two additional STR loci (D2S1338 and D19S433), and the Amelogenin locus. Amplification was performed using the AmpFlSTR Identifier PCR Amplification Kit (Applied Biosystems) according to the manufacturer's instructions. The PCR products were mixed with an internal size standard (GS-500 LIZ; Applied Biosystems), electrophoresed in an ABI 3130 × L Genetic Analyzer (Applied Biosystems), and analyzed with GeneMapper 4.0 software using the supplied allelic ladders (Applied Biosystems).

Mutational analysis

Ten genes (TP53, HRAS, KRAS, NRAS, FGFR3, PIK3CA, CTNNB1, KIT, BRAF, and STK11) known to be mutated in human malignancies, including BCa, were selected.[22, 23] Ten nanograms of gDNA were amplified by single-tube, multiplex PCR using the Ion AmpliSeq Cancer Primer Pool (Life Technologies, Grand Island, NY, USA). Template DNA and unincorporated primers were removed from the amplicons by binding to Agencourt AMPure XP Reagent (Beckman Coulter, Brea, CA, USA). Amplicons were treated with FuP Reagent to partially digest primer sequences and phosphorylate the amplicons. A library was prepared with reagents from the Ion Plus Fragment Library Kit (Life Technologies). Amplicons were ligated to Ion-compatible adapters. The ligated DNA underwent nick-translation and amplification to complete the link between adapters and amplicons and to generate sufficient material to prepare the downstream template. The concentration and size were determined using an Agilent BioAnalyzer DNA High-Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA, USA). Sample emulsion PCR, emulsion breaking, and enrichment were performed using the Ion OneTouch Template Kit (Life Technologies). The sample was prepared for sequencing using the Ion Sequencing Kit (Life Technologies). The complete sample was loaded on an Ion 314 chip and sequenced on the Ion Personal Genome Machine Sequencer (Life Technologies) for 65 cycles.

Array-based comparative genomic hybridization

Array-based comparative genomic hybridization (Arry-CGH) was performed using Agilent Human Whole Genome CGH 8 × 60 K microarray (Agilent Technologies). Labeling and hybridization were performed using protocols provided by the manufacturer. Briefly, 0.5 μg of test or reference DNA was digested with Alu I and Rsa I (Promega, Madison, WI, USA) and purified with the QIAprep Spin Miniprep kit (QIAGEN). Test and reference DNA samples were labeled by random priming with either Cy3-dUTP or Cy5-dUTP using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies). Following the labeling reaction, individually labeled test and reference samples were combined and concentrated using Amicon Ultra-0.5 centrifugal filters (Millipore, Billerica, MA, USA). After denaturing the probe and preannealing with human Cot-1 DNA, samples were hybridized at 65°C and 20 rpm rotation for 24 h in a DNA Microarray Hybridization Oven (Agilent Technologies). Samples were washed in Wash Buffer 1 at room temperature for 5 min and Wash Buffer 2 at 37°C for 1 min using Agilent Oligo CGH washes. All slides were scanned on an Agilent DNA microarray scanner. Data were obtained using Agilent Feature Extraction Software 9 and analyzed with Agilent CGH Analytics Version 6.5 software, using the ADM-2 statistical algorithms with 6.0 sensitivity thresholds.

Statistical methods

Clinical variables between successful and failed xenografts were compared with χ2 and Fisher's exact tests. Clinical variables were compared among xenografts (P1 to P6) using the Kruskal–Wallis test. All analyses were performed using spss v.19.0 (SPSS Inc., Chicago, IL, USA), and P-value <0.05 was considered statistically significant.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Xenograft engraftment

A total of 65 tumor specimens were transplanted into immunodeficient mice. From these 65 samples, 10 (15.4%) xenografts were successfully passaged. Although there were no significant differences in clinical variables between successful and failed xenografts, successful xenografts tended to have a higher tumor grade and more deaths (Table 1). Also, although there were no significant differences in clinical variables among xenografts (P1 to P6), tumors with a higher pathologic T stage and tumor grade tended to be passaged further (Table 2). The medical records of the patients for all 10 successful xenografts were reviewed by two urologists (B.P. and B.C.J.) and six samples (012T, 025T, 033T, 043T, 048T, and 052T) were selected for this study based on the clinico-pathologic heterogeneity of the patients (Table 3).

Table 1. Comparison of clinical variables between successful and failed xenografts
VariablesSuccessful xenografts (n = 10)Failed xenografts (n = 55)P-value
  1. CIS, carcinoma in situ. †χ2 and Fisher's exact tests.

No. tumors (%)
16 (60.0)29 (52.7)0.810
2–71 (10.0)10 (18.2)
≥83 (30.0)16 (29.1)
Max. tumor diameter (%)
<3 cm3 (30.0)21 (38.2)0.733
≥3 cm7 (70.0)34 (61.8)
Pathologic T stage (%)
Ta1 (10.0)21 (38.2)0.599
T15 (50.0)17 (30.9)
T22 (20.0)10 (18.2)
T31 (10.0)3 (5.5)
T41 (10.0)3 (5.5)
Tis0 (0)1 (1.8)
Grade (%)
I0 (0)2 (3.6)0.240
II4 (40.0)35 (63.6)
III6 (60.0)18 (32.7)
Concomitant CIS (%)1 (10.0)7 (12.7)1.000
Micropapillary component (%)1 (10.0)2 (3.6)0.399
Recurrence (%)2 (22.2)14 (25.9)1.000
Recurrence rate
Primary7 (77.8)38 (70.4)0.147
1 per year0 (0)12 (22.2)
>1 per year2 (22.2)4 (7.4)
Death (%)1 (10.0)2 (3.6)0.399
Table 2. Comparison of clinical variables among xenografts
VariablesP1 (n = 3)P2 (n = 2)P3 (n = 2)P4 (n = 1)P5 (n = 1)P6 (n = 1)P-value
  1. CIS, carcinoma in situ. †Kruskal–Wallis test.

No. tumors
Max. tumor diameter
<3 cm3000000.109
≥3 cm022111
Pathologic T stage
Concomitant CIS0010000.549
Micropapillary component0100000.549
Recurrence rate
1 per year000000
>1 per year200000
Table 3. Clinical and pathologic characteristics of all 6 bladder cancer patients
  1. †The patient had 20% micropapillary component in the specimen pathology. ‡The patient had concurrent right ureter cancer with periureteral fat invasion (pT3). LRC, laparoscopic radical cystectomy with ileal conduit; ORC, open radical cystectomy with ileal conduit; TURB, transurethral resection of bladder tumor.

Tumor stagepT3bpT1pT4pT1pT1pT2, cT3
Additional treatmentsNoneNoneAdjuvant chemotherapyAdjuvant chemotherapyIntravesical BCGAdjuvant chemotherapy

Histologic examination by the hematoxylin and eosin staining

A comparison of the hematoxylin and eosin (H&E) staining of original samples and xenografted tumors after few passages revealed similar architectural pattern of nesting configuration and comparable cytologic atypia in all six xenograft models (Fig. 1).


Figure 1. Histologic examination with hematoxylin and eosin staining, which revealed similar architectural pattern of nesting configuration and comparable cytologic atypia between the primary and xenograft tumors (a) 048T_P0, ×400; (b) 048T_P2, ×400; (c) 048T_P0, ×100; (d) 048T_P2, ×100).

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Human-mouse quality control testing

To confirm that the genomic status of the xenografts was appropriate, without cross-contamination, we conducted a quality control testing for the xenografts. We determined the levels of expression of human and mouse albumin (ALB) genes using the standard curve methods and Ct values. As a result, the mean Ct values for human ALB genes were lower than those of mouse ALB genes for all six xenografts, indicating that human DNA was more abundant than mouse DNA in the xenografts (Table 4, Fig. 2). Furthermore, the mean Ct values for human ALB genes were lower than 29, indicating a strong positive reaction and sufficient human DNA within xenografts.

Table 4. Results of human-mouse quality control testing
IDHuman ALB (Mean Ct values)Mouse ALB (Mean Ct values)
  1. ALB, albumin; N/A, not applicable.


Figure 2. Results of human-mouse quality control tests using human and mouse albumin (ALB) genes in the 012T_P0 human sample and 012T_P2 xenograft.

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Short tandem repeat genotyping and array-based comparative genomic hybridization

Xenograft models 043T_P2 and 048T_P2 had STR profiles completely identical to the original human samples at all STR loci. The other models (012T_P2, 025T_P2, 033T_P2, and 052T_P2) had nearly identical STR profiles, with only 1 to 3 minor variations (Table 5, Fig. 3). An array-CGH analysis revealed that all six xenografts had genomic alternation patterns similar to the original samples (Fig. 4).

Table 5. Short tandem repeat profiles of patient tumor tissues and xenografts
LocusChromosome location012T_P0012T_P2025T_P0025T_P2033T_P0033T_P2043T_P0043T_P2048T_P0048T_P2052T_P0052T_P2
D8S1179810, 1210, 1212, 151214, 1714, 1713, 1513, 1513, 1613, 161515
D21S1121q11.2-q2128, 2928, 2929, 3029, 3028, 3028, 3029, 3029, 3028, 3028, 3029, 3029, 30
D7S8207q11.21-22101010109, 129, 128, 108, 108, 118, 118, 128, 12
CSF1PO5q33.3-3411, 121111, 1211, 1211, 1211, 12111112, 1312, 1310, 1210, 12
D3S13583p15, 1815, 1815, 1615, 161414151514, 1514, 1514, 1514, 15
TH0111p15.58, 98, 97, 97, 96, 96997, 97, 96, 96
D13S31713q22-311212121288889, 129, 1211, 1211, 12
D16S53916q24-qter10, 1310, 1310, 1210, 129, 139, 139, 119, 119910, 1110, 11
D2S13382q35-37.1232317, 2317, 2318, 2318, 232424191919, 2419
D19S43319q12-13.113, 15.213, 15.212, 15.212, 15.212, 1312, 1313, 1413, 1412, 1412, 141414
vWA12p12-pter14, 1814, 1814, 2014, 2018, 1918, 19181817, 1917, 1917, 1817, 18
TPOX2p23-pter8, 98, 98, 118, 118, 118, 1111118, 118, 118, 118, 11
D18S5118q21.313, 1513, 1517, 191915, 1615181814, 1714, 1716, 1816, 18
AmelogeninX: p22.1-22.3 Y: p11.2X, YX, YX, YXX, YX, YXXX, YX, YX, YX
D5S8185q21-3110, 1110, 119, 119, 119, 109, 10121211, 1211, 121212
FGA4q2823, 2523, 2521, 2521, 2522, 232318, 2318, 2321, 2321, 2321, 2221, 22

Figure 3. Short tandem repeat (STR) profiles of the 043T_P0 human sample (a) and the 043T_P2 xenograft (b) showing identical STR profiles. Short tandem repeat loci are indicated above the electropherogram. Numbers of repeats are indicated below the peaks.

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Figure 4. Array-based comparative genomic hybridization results of all six human samples and xenografts (a) 012T and 025T; (b) 033T and 043T; (c) 048T and 052T; (d) 043T_P0 and 043T_P2 combined). Note the similar genomic profiles between the original samples and the xenografts.

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Mutational analysis

Four (012T, 025T, 033T, and 052T) of six xenografts had mutations. The original sample 012T_P0 and xenograft 012T_P2 had identical HRAS mutations, Q61R (182A>G) (Fig. 5a). 025T_P0 and 025T_P2 had identical CTNNB1 mutations, S37F (100C>T) (Fig. 5b), and BRAF mutations, V600E (1799T>A) (Fig. 5c). Moreover, 033T_P0 and 033T_P3 had identical TP53 mutations, E258K (772G>A) (Fig. 5D) and H193Y (577C>T) (data not shown). Finally, 052T_P0 and 052T_P2 had identical TP53 mutations, Q167* (499C>T; glutamine converted to a stop codon) (data not shown). There were no mutations in KRAS, NRAS, FGFR3, PIK3CA, KIT, or STK11 in either the original samples or the xenografts.


Figure 5. Results of mutational analysis. (a) The original sample 012T_P0 and xenograft 012T_P2 have an identical HRAS mutation: Q61R. (b) 025T_P0 and 025T_P2 have an identical CTNNB1 mutation: S37F. (c) 025T_P0 and 025T_P2 have an identical BRAF mutation: V600E. (d) 033T_P0 and 033T_P3 have an identical TP53 mutation: E258K.

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Xenograft models increasingly use patient-derived tumor tissues implanted subcutaneously in immunodeficient mice to reflect the biological characteristics of human tumors more accurately than tumor cell lines.[17] Fichtner and colleagues mentioned that cell lines used for preclinical studies as xenografts mainly represented poorly differentiated carcinomas, which lacked similarity to the original tumor and therefore the clinical situation.[24] There are several inherent problems with cell line xenograft models. To survive, tumor cells in culture adjust to a microenvironment devoid of stromal and endothelial elements. This adjustment likely occurs gradually over serial passages and involves genetic changes that make these tumor cells differ from the original tumor. In addition, cell lines do not reflect tumor heterogeneity, as xenografts derived from a cell line typically have homogenous histologies lacking architectural organization.[25] A study by Johnson et al. using 39 anticancer drugs reported that, with the exception of lung, histological matches were not found between cell line xenograft models and clinical response. Moreover, they also showed that the relationship between chemo-response levels of various cancer cell line xenografts and those in the clinical trials was limited.[26] On the other hand, studies of colon cancer and sarcoma have shown that the drug responsiveness between the early-passage tissue xenografts and the original cancers was closely correlated.[24, 27] Our study definitely showed that early passages (P2) of xenografts almost preserved the characteristics of the original human cancer tissues, especially with referring the array-CGH results. Therefore, although there has been no study that directly compared between the tissue and cell line xenografts, it is suggested that tissue-based xenografts may be more representative of the original tumor than the cell line xenografts. Further comparative analysis is required to clarify this issue.

Historically, we are not the first group to directly implant a patient-derived BCa tissue into mice. A few studies have developed BCa xenograft models by implanting patient-derived tumor tissues, some even more than 30 years ago.[28-31] Successful passage rates were 9–15%, which are similar to those of our study (15.4%). Although these studies successfully developed passagable BCa xenograft models, the characterization was limited to phenotypic analyses, such as H&E or immunocytochemical staining, without detailed molecular analyses. Although histological analyses are the first step in assuring that xenografts mimic the original tumor, they are not sufficient, as molecular changes could occur in the absence of histological changes. To investigate the molecular alterations in xenografts, further genomic analyses should be performed. Short tandem repeat genotyping, mutational analysis, and array-CGH are three well-established genomic analyses used in many other studies.[32] Thus, to our knowledge, this is the first study to develop a BCa xenograft model from patient-derived tumor tissue and characterize it using these three genomic analyses as well as conventional histologic analysis.

In the present study, we developed six BCa xenograft models with heterogeneous clinico-pathological features that can be routinely passaged. The consistency of the histological patterns between the original tumors and serial passages of the xenografts support the validity of this model. Four of the six xenograft models had a mutation in HRAS, CTNNB1, BRAF, or TP53, with none having mutations in other genes. These results are possibly related to the mutation frequency of each gene. The frequency of TP53 mutation is over 40% in human BCa,[33] whereas that of the RAS family is 13% and PIK3CA is 13 to 27%.[34] One unique finding in our study is the BRAF mutation, which is infrequent in human BCa.[35] Finally, our STR genotyping and array-CGH results confirmed that our models accurately represented their respective donors, because the original samples and xenografts had almost identical results.

Two of the most significant reasons to establish animal models of human cancer are to evaluate new chemo- or other therapeutic agents or treatments, and to investigate the basic mechanisms of tumor biology. Using patient-derived tumor tissue xenograft models for these two purposes assumes that xenografts closely resemble the original tumor. In our study, we demonstrated that early passages of BCa xenograft models are highly similar to the original cancer with regard to histology, mutation status, and genomic alterations.

Our xenograft models not only retain the histopathological features and molecular signatures of the original tumors, but also show the clinico-pathological heterogeneity of BCa. Therefore, our models are pertinent to investigate an anticancer drug response or resistance, or to evaluate the biology of BCa expressing specific clinico-pathological feature. In fact, an experiment to establish a cisplatin-resistant BCa model using our xenografts is ongoing.

Our study has a few limitations. First, although our xenograft models almost preserved phenotypic and genotypic characteristics of the primary human BCa tissues, it has not been proven whether they show the similarity in ‘clinical’ aspects, for example, drug or radiation sensitivity. Further clinical relevance study using our xenografts is necessary. Second, since our models lacked grade I urothelial carcinoma (Table 3), they cannot generalize the BCa as a whole. Although there were a few grade I tumor cases which were passaged to P1, P1 is not defined as a successful xenograft model in our study. Third, reporting array-CGH results was limited to showing the similarity of genetic profile patterns. For further investigations, it is required to analyze specific genetic loci where amplifications or deletions have occurred, and this may reveal novel genes causing mutations in the BCa. Finally, we selected P2 xenografts as a role of indicator models representing patients arbitrarily. But we don't know the exact passage number to prove the superiority of our tissue xenograft model over cell line xenografts. In general, studies for anticancer drug response using tumor tissue xenografts used early-passage models such as our xenografts in other cancers.[24, 27]

In conclusion, our BCa xenograft models derived from human tumor tissue not only retain histopathological features and molecular signatures similar to the original tumors, but also have the clinico-pathological heterogeneity of BCa. Therefore, we expect our model to be useful for studying the heterogeneity of BCa populations and for evaluating new treatments.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This study was supported by a grant from the Korea Healthcare Technology R&D project, Ministry for Health & Welfare Affairs, Republic of Korea (A092255).


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
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