Patient-Derived Xenografts from Non Small Cell Lung Cancer ... · Cancer Therapy: Preclinical Patient-Derived Xenografts from Non–Small Cell Lung Cancer Brain Metastases Are Valuable

Cancer Therapy: Preclinical

Patient-Derived Xenografts from Non–Small CellLung Cancer Brain Metastases Are ValuableTranslational Platforms for the Development ofPersonalized Targeted TherapyHye Won Lee1,2,3, Jung-il Lee1, Se Jeong Lee4,5, Hyun Jung Cho1, Hye Jin Song4,Da Eun Jeong3, Yun Jee Seo1, Sang Shin3, Je-Gun Joung6, Yong-Jun Kwon1,5,Yoon-La Choi3,5,7,Woong-Yang Park3,5,6, Hyun Moo Lee2, Ho Jun Seol1,Young Mog Shim8,Kyeung Min Joo3,4,5, and Do-Hyun Nam1,3,5

Abstract

Purpose: The increasing prevalence of distant metastases fromnon–small cell lung cancer (NSCLC) indicates an urgent need fornovel therapeutic modalities. Brain metastasis is particularlycommon in NSCLC, with severe adverse effects on clinical prog-nosis. Although themolecular heterogeneity of NSCLC and avail-ability of various targeted agents suggest personalized therapeuticapproaches for such brain metastases, further development ofappropriate preclinicalmodels is needed to validate the strategies.

Experimental Design: We established patient-derived xeno-grafts (PDX) using NSCLC brain metastasis surgical samples andelucidated their possible preclinical and clinical implications forpersonalized treatment.

Results: NSCLC brain metastases (n ¼ 34) showed a signifi-cantly higher successful PDX establishment rate than primaryspecimens (n ¼ 64; 74% vs. 23%). PDXs derived from NSCLCbrain metastases recapitulated the pathologic, genetic, and func-

tional properties of corresponding parental tumors. Furthermore,tumor spheres established in vitro from the xenografts underserum-free conditions maintained their in vivo brain metastaticpotential. Differential phenotypic and molecular responses to 20targeted agents could subsequently be screened in vitro using theseNSCLC PDXs derived from brain metastases. Although PDXestablishment from primary NSCLCs was significantly influencedby histologic subtype, clinical aggressiveness, and genetic alter-ation status, the brain metastases exhibited consistently adequatein vivo tumor take rate and in vitro tumor sphere formationcapacity, regardless of clinical and molecular conditions.

Conclusions: Therefore, PDXs from NSCLC brain metastasesmay better represent the heterogeneous advanced NSCLC popu-lation and could be utilized as preclinical models to meet unmetclinical needs such as drug screening for personalized treatments.Clin Cancer Res; 21(5); 1172–82. �2014 AACR.

IntroductionDespite efforts to develop novel personalized therapeutic strat-

egies and predictive biomarkers, non–small cell lung cancer(NSCLC) remains the leading cause of cancer-related mortality(1). Recent successful application of EGFR (2) and anaplasticlymphoma kinase (ALK)/met proto-oncogene (MET; ref. 3) tyro-sine kinase inhibitors to NSCLCs highlights the importance ofgenotype-based individualized targeted therapies. However, dueto the extensive molecular and functional heterogeneity ofNSCLCs, genomic abnormalities often are not the ideal thera-peutic efficacy predictors for various targeted agents. Moreover,new drugs targeting deregulated signaling pathways rather thanspecifically altered oncogenes have been increasingly developed.

The major cause of death from NSCLC is metastases that areresistant to conventional therapies. In particular, brainmetastasesare detected in 10% to 25% of patients with NSCLC at initialdiagnosis, and 40% to 50% of patients with lung cancer developbrain metastases during their clinical course (4, 5). The standardtreatments for NSCLC brain metastases, which include surgicalresection, radiotherapy, chemotherapy, or a combination ofthese, are rarely curative and often result in severe neurologicmorbidities (6). Nevertheless, there is a clinically urgent need forimproved therapeutic approaches to treat patients with NSCLC

1Department of Neurosurgery, Samsung Medical Center, Sungkyunk-wan University School of Medicine, Seoul, South Korea. 2Departmentof Urology, Samsung Medical Center, Sungkyunkwan UniversitySchool ofMedicine, Seoul, SouthKorea. 3SamsungAdvanced Institutefor Health Sciences and Technology (SAIHST), Samsung MedicalCenter, Sungkyunkwan University School of Medicine, Seoul, SouthKorea. 4Department of Anatomy and Cell Biology, Samsung MedicalCenter, Sungkyunkwan University School of Medicine, Seoul, SouthKorea. 5Samsung Biomedical Research Institute, Samsung MedicalCenter, Sungkyunkwan University School of Medicine, Seoul, SouthKorea. 6Samsung Genome Institute (SGI), Samsung Medical Center,Sungkyunkwan University School of Medicine, Seoul, South Korea.7Department of Pathology, Samsung Medical Center, SungkyunkwanUniversity School of Medicine, Seoul, South Korea. 8Thoracic andCardiovascular Surgery, Samsung Medical Center, SungkyunkwanUniversity School of Medicine, Seoul, South Korea.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H.W. Lee and Jung-il Lee contributed equally to this article.

Corresponding Authors: Kyeung Min Joo, Sungkyunkwan University School ofMedicine, Samsung Medical Center, 50 Irwon-Dong, Gangnam-Gu, Seoul 135-710, South Korea. Phone: 82-2-2148-9779; Fax: 82-2-2148-9829; E-mail:[email protected]; and Do-Hyun Nam, Phone: 82-2-3410-3497; Fax: 82-2-2148-9829; E-mail: [email protected]; and Young Mog Shim, Phone: 82-2-3410-3022;Fax: 82-2-3410-6986; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1589

�2014 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 21(5) March 1, 20151172

on August 19, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 30, 2014; DOI: 10.1158/1078-0432.CCR-14-1589

http://clincancerres.aacrjournals.org/

brain metastasis. Unfortunately, clinical activity data on targetedagents for the treatment of NSCLC brainmetastases remain sparse.

Previous evidence suggests that patient-derived xenografts(PDX) may be a more clinically relevant preclinical model thatwould increase the success of novel drug identification comparedwith cell line–derived xenografts (7, 8). These PDX-based pre-clinical systems accurately represent the molecular, genetic, andhistopathologic heterogeneity of the original tumors throughserial passaging in mice (9, 10). The conserved genetic diversitywithin thesemodels andmolecular/functional similarity betweenPDXs and their correspondingparental tumors provide theoppor-tunities to further develop and validate personalized approachesfor the treatment of heterogeneous NSCLCs with or withoutspecific genomic abnormalities.

Although several NSCLCPDXpanels have demonstrated a highcorrelation between preclinical and clinical results (7, 10, 11),they are mainly established from the primary tumors of treat-ment-na€�ve patients with NSCLC and thereby fail to recapitulatethe patient population with metastatic/treatment refractory dis-eases. Moreover, previous laboratory techniques often yieldedrelatively low engraftment rates of surgical specimens in immune-deficient mice (20%–40%; refs. 7, 10, 11). Recently, surgicalresection has become one of the viable therapeutic options forpatientswithNSCLCbrainmetastasis with neurologic and/or life-threatening symptoms (12). Because PDX establishment rates aremuch higher in biologically aggressive tumors (7, 10, 11), surgi-cally removed NSCLC brain metastases instead of early-stageprimary lung specimens might extend the application of PDXmodels to a wider range of patients andmeet more urgent clinicalneeds.

Herein, we established NSCLC PDXmodels using primary andbrain metastasis specimens to compare their in vivo engraftmentrates and potentials as preclinical drug-screening platforms. Wedemonstrated that surgically removed brain metastases could beexcellent sources for the establishment of an NSCLC preclinicalplatform that closelymimics themore aggressive clinical situationand can be used for in vitro personalized drug screening.

Materials and MethodsNSCLC surgical samples and clinicopathologic parameters

This study was approved by the Samsung Medical Center(Seoul, Korea) Institutional Review Boards (IRB) and all partici-pants provided written informed consent (No. 2010-04-004).Surgical specimens and clinical records were obtained from 98patients with NSCLC who underwent surgical removal of theprimary tumors (n ¼ 64) or brain metastases (n ¼ 34) at ourinstitute from 2008 to 2012. Tumors were classified as NSCLCbased on the World Health Organization (WHO) criteria uponexamination by licensed pathologists. Synchronous brain metas-tasis was defined as brain metastatic lesions diagnosed before orwithin 3months of the primary NSCLC diagnosis. All others wereconsidered metachronous brain metastasis. Patients were catego-rized as never smokers (<100 cigarettes in their lifetime) or eversmokers (�100 cigarettes in their lifetime) according to theirsmoking status. NSCLC histologic subtypes and stages wereclassified by the pathologists according to the WHO criteria(13) and the American Joint Committee onCancer staging system(7th edition; ref. 14), respectively. The presence of a solid growthpattern (>10% solid sheets of tumor without the formation ofacini, papillae or micropapillae, or sheets or nests of tumor areaswith fibrous tissue) in the adenocarcinoma subtype was deter-mined because this pattern is an indicator of poor prognosis (15–17). Surgical specimens were divided into three portions forimplantation into immune-deficient mice, DNA/RNA extraction,and pathologic assessment within 6 hours after surgery.

PDX model establishmentAnimal experiments were conducted in accordance with the

Institute for Laboratory Animal Research Guide for the Care andUse of Laboratory Animals and following protocols approved bythe IRB at the Samsung Medical Center (Seoul, Korea). Athymicnude andNOG (IL2 receptor g-chain truncatedNOD/SCID)micewere utilized (Orient Bio). For the transplantation, 6- to 8-week-old female mice were anesthetized with 100mg/kg ketamine and10mg/kg xylazine. PrimaryNSCLC samples (n¼ 64)minced intoapproximately 1 mm3 fragments in high-concentration MatrigelBasement Membrane Matrix (BD Biosciences) were directlyimplanted into the subcutaneous space or subrenal capsule(n ¼ 4–5 for each tumor sample). NSCLC brain metastasis speci-mens (n ¼ 34) were implanted subcutaneously in high-concen-tration Matrigel Basement Membrane Matrigel after mincing orstereotactically injected into the brains (2 mm left and 1 mmanterior to the bregma, 2 mm deep from the dura) after disso-ciation into single cells according to previously published proto-cols [refs. 18, 19; 2.5 � 104–1.0 � 105 cells in 10 mL Hank'sBalanced Salt Solution (HBSS; Life Technologies), n ¼ 4–5 foreach tumor sample]. Mice were maintained and observed for 6months for the development of xenograft tumor. For subcutane-ous and subrenal capsule implantation, xenograft tumor engraft-ment was defined as a palpable mass of >1 mm in diameter andpathologic confirmation. For intracranial injection, body weightloss of >20% and pathologic confirmation were assessed assuccessful PDXmodel establishment. The origin of each xenograftwas validated by short tandem repeat DNA fingerprinting. ThePDX tumors were harvested and divided into three portions forthe generation of the second in vivo passage xenograft tumors,DNA/RNA extraction, and histopathologic examination.

Translational Relevance

The incidence ofmetastatic brain tumors is increasing due tothe development of novel therapeutic modalities controllingprimary cancers effectively. Especially, brain metastasis devel-ops early in non–small cell lung cancer (NSCLC); brain metas-tases are found in 10% to 25%of patients withNSCLC at initialdiagnosis. Although standard treatments for NSCLC brainmetastases are rarely curative, resulting in severe neurologicmorbidities, very fewpathophysiologic studieswere performedon NSCLC brain metastasis, nor are there appropriate preclin-ical models with which to study. In this study, we havedemonstrated that patient-derived xenografts (PDX)developedfrom brainmetastasis would increase the success of identifyingactive drugs for the individual patient. To the best of ourknowledge, this is the first report demonstrating that PDXsfrom NSCLC brain metastases would represent heterogeneousadvanced NSCLC population and that they have several meritsas a valuable translational research platform to elucidate keymolecular and genetic mechanisms for an individual tailoredtherapeutic approach to each patient.

Brain Metastases as Resources for NSCLC PDX Models

www.aacrjournals.org Clin Cancer Res; 21(5) March 1, 2015 1173




Histology and IHCFormalin-fixed paraffin-embedded (FFPE) samples were pro-

cessed according to conventional experimental protocols. Serial 5-mm-thick sections from each FFPE block were processed forhematoxylin and eosin (H&E) staining and were examined bya specialized pathologist. IHC was performed as previouslydescribed (20) using the following primary antibodies: thyroidtranscription factor-1 (TTF-1; Abcam; ab40880, 1:1,000), Napsin-A (Abcam; ab133249, 1:250), and p63 (Abcam; ab53039, 1:200).

Genomic characterizationArray comparative genomic hybridization (aCGH) was per-

formed using Agilent Human Whole Genome CGH 8 � 60 Kmicroarray (Agilent Technologies, Inc.). Labeling and hybridiza-tionwere performed according to themanufacturer's instructions.Briefly, 0.5 mg of test or reference genomic DNA (gDNA) wasdigested with AluI and RsaI (Promega) and purified using theQIAprep Spin Miniprep Kit (QIAGEN). DNA samples werelabeled by random priming with either Cy3-dUTP or Cy5-dUTPusing the Agilent Genomic DNA Labeling Kit PLUS (AgilentTechnologies, Inc.). Individually labeled test and reference sam-ples were combined and concentrated using Amicon Ultra-0.5centrifugal filters (Millipore). After denaturing the probe and pre-annealing with human Cot-1 DNA, samples were hybridized at65�C and 20-rpm rotation for 24 hours in a DNA MicroarrayHybridization Oven (Agilent Technologies, Inc.). Slides werescanned on an Agilent DNA microarray scanner. Data wereobtained using Agilent Feature Extraction Software 9 and ana-lyzed with Agilent CGH Analytics Version 6.5 software, using theADM-2 statistical algorithms with 6.0 sensitivity thresholds.

For gene mutational analysis, Ion Torrent sequencing wasperformed following the Ion Torrent protocol (Life Technolo-gies). gDNA (10 ng) was amplified by single-tube, multiplex PCRusing the Ion AmpliSeq Cancer Primer Pool (Life Technologies).Template DNA and unincorporated primers were removed fromthe amplicons by binding to Agencourt AMPure XP Reagent(Beckman Coulter). Amplicons were treated with FuPa reagentto partially digest primer sequences and phosphorylate the ampli-cons. A library was prepared with reagents from the Ion PlusFragment Library Kit (Life Technologies). Amplicons were ligatedto Ion-compatible adapters. The ligated DNA underwent nick-translation and amplification to complete the link betweenadapters and amplicons and to generate sufficient materials fordownstream template preparation. The concentration and sizewere determined using an Agilent BioAnalyzer DNA High-Sensi-tivity DNA Chip (Agilent Technologies, Inc.). Sample emulsionPCR, emulsion breaking, and enrichment were performed usingthe IonOneTouch Template Kit (Life Technologies). Each samplewas prepared for sequencing using the Ion Sequencing Kit (LifeTechnologies). The final template-positive Ion sphere particleswere loaded onto an Ion 314 chip and sequenced on the IonPersonal Genome Machine Sequencer (Life Technologies) for 65cycles.

Primary in vitro short-term cultureXenograft tumor specimens were dissociated in to single cells

according to previously published protocols (18, 19). DissociatedNSCLC cells were cultured in neurobasal media-A supplementedwith N2 (�1/2; Life technologies), B27 (�1/2; GIBCO), basicfibroblast growth factor (bFGF; 25 ng/mL; R&D Systems), EGF

(25 ng/mL; R&D Systems), neuregulin 1 (NRG; 10 ng/mL; R&DSystems), and insulin-like growth factor 1 (IGF1; 100ng/mL;R&DSystems). Growth factors were supplemented every 3 days. Asspheres appeared in the suspension culture, they were dissociatedusing StemPro Accutase (Life Technologies) and expanded byreseeding in the aforementioned suspension culture medium.

In vivo systemic metastasis assayTo examine the in vivometastatic capacity of primarily cultured

NSCLC brainmetastasis PDX cells, 1� 104 dissociated cells in 0.1mL HBSS were injected into the left ventricle of 6-week-oldathymic nude mice. The animals' body weights were measuredevery day and autopsywas conductedwhen >25%weight losswasreached. The brain, lungs, lower leg bones, adrenal glands, andabnormal organs were examined by H&E staining.

In vitro drug sensitivity assayPrimarily cultured NSCLC brain metastasis PDX cells in the

serum-free sphere culture condition were seeded in 384-wellplates at 500 cells per well. Two hours after plating, cells weretreated with a drug library in 3-fold and 10-point serial dilutionseries (n ¼ 3 for each condition). After 6 days of incubation at37�C in a 5% CO2 humidified incubator, cell viability wasanalyzed using an adenosine triphosphate monitoring systembased on firefly luciferase (ATPlite 1step; PerkinElmer). The druglibrary was composed of 20 targeted agents that were included inthe clinical guideline or current clinical trial for the treatment ofNSCLC (gefitinib, erlotinib, lapatinib, afatinib, tivantinib, fore-tinib, crizotinib, selumetinib, temsirolimus, everolimus, cabo-zantinib, vandetanib, sunitinib, sorafenib, dovitinib, vemurafe-nib, BKM 120, pazopanib, nintedanib, and DAPT; all purchasedfrom Selleckchem). The drugs were stored and diluted accordingto the manufacturers' instructions. Test concentrations for eachdrug were empirically derived to produce a clinically relevantspectrumofdrug activity. IC50 valueswere calculated as anaverageof triplicated experiments using the Sþ Chip Analyzer (SamsungElectro-Mechanics Company, Ltd.; ref. 21).

For signal transduction assays under treatment with targetedagents, primarily cultured NSCLC brainmetastasis PDX cells weremaintained overnight in serum-free sphere culture conditionwithout growth factors, incubated for 1 hour with each inhibitor,and pulsed with original culture medium supplemented withbFGF/EGF/NRG/IGF for 15 minutes. For Western blot analysis,cells were lysed in RIPA lysis buffer supplemented with 1�phosphatase inhibitors (PhosStop; Roche Diagnostics) and a1� protease inhibitor cocktail (Complete Mini; Roche Diagnos-tics). After centrifugation at 10,000 g for 5 minutes, the super-natant was harvested. Protein concentration was determinedusing a bicinchoninic acid protein assay kit (Thermo). Equalamounts of protein were subjected to SDS-PAGE and transferredto polyvinylidene difluoridemembranes (Whatman), whichwereblocked in 5% skimmilk or BSA for 1 hour at room temperature,incubated with the indicated primary antibodies overnight, andthen blotted with the appropriate secondary antibodies. Antibo-dies against phospho-EGFR (Tyr1068), phospho-MET (Tyr1234/1235), phospho-AKT (Ser473), phospho-mTOR (Ser2448),phospho-p70S6K (Thr389), phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204), p44/42MAPK (Erk1/2; Cell Signaling Technol-ogy), EGFR, MET, AKT, mTOR, p70S6K, and GAPDH (Santa CruzBiotechnology) were used.

Lee et al.

Clin Cancer Res; 21(5) March 1, 2015 Clinical Cancer Research1174




Statistical analysisThe in vivo tumor take rates were compared by the c2 test. The

Fisher exact test was used to determine the association of clini-copathologic features with successful PDX engraftment. All Pvalues were two-sided, and a P < 0.05 was considered statisticallysignificant. All data analyses were performed with SPSS statisticalsoftware (Statistical Product and Services Solutions, version 19.0).

ResultsEstablishment ofNSCLCPDXmodels andparameters related toin vivo tumor formation

The overall clinicopathologic characteristics of all NSCLC cases(n ¼ 98) are presented in Supplementary Table S1. Although the

primary NSCLC group (n ¼ 64) had larger tumors than the brainmetastasis group (n ¼ 34, P < 0.001), age, sex, smoking status,histologic subtype, tumor differentiation, neoadjuvant chemo-treatment history, and genetic abnormality status were similar inthe twogroups. The establishmentof aPDXmodelwasmonitoredby body weight change (for intracranial injection) and palpablemass formation (for subcutaneous and subrenal capsule implan-tation) and defined as viable tumor formation (>20% bodyweight loss or >1 mm3 mass) with pathologic confirmation inat least one transplantedmouse. The overall in vivo tumor take ratewas 41% (40/98). Interestingly, the engraftment rate of NSCLCbrain metastases (25/34, 74%) was significantly higher than thatof primary NSCLCs (15/64, 23%) in the first generation of

Figure 1.Successful establishment rates ofNSCLC patient–derived xenograftmodels. A, in vivo tumor take rateswerecompared according to the sourcesof NSCLC surgical samples.B, overall survival of patients withNSCLC brain metastases whosesurgical samples had in vivotumorigenic potential (n ¼ 25), ornot (n ¼ 9), was compared. C,histologic and immunohistochemicalcomparison of patient andcorresponding xenograft tumors.ADC, adenocarcinoma.






transplantation (Fig. 1A, P < 0.001). In vivo passagability and timeto xenograft tumor formation of each NSCLC PDX model weresummarized in Supplementary Table S2. In 30 of the 40 PDXmodels (75%), in vivo passagability (>2) was confirmed.

To determine the clinicopathologic parameters related to suc-cessful in vivo tumor formation, PDX model establishment rateswere calculated and compared according to various patient char-acteristics (Supplementary Tables S3 and S4 for patients withprimary and brain metastatic NSCLC, respectively). The primary(Table 1) and brain metastatic (Table 2) NSCLC cases wereseparately analyzed, because the two groups showed significantlydifferent PDXmodel establishment rates (Fig. 1A). In the primaryNSCLC group, squamous cell carcinoma (SCC) histologic sub-type, concurrent distant metastasis, progressed overall tumorstage, presence of the solid component in adenocarcinoma his-tologic subtype, and wild-type EGFR genotype were significantlyassociatedwith successful PDXmodel establishment (Table 1 andSupplementary Table S3). Age, sex, smoking status, tumor size,pathologic tumor-node (TN) stage, implanted mouse strain,implantation route, differentiation of adenocarcinoma subtype,neoadjuvant chemotherapy, KRAS genotype, and ALK genotype

showed no significant correlation with in vivo tumor take rate(Table 1). In contrast, in the NSCLC brain metastasis group, noclinicopathologic features led to significant differences in the PDXmodel establishment result (Table 2 and Supplementary TableS4). Previously, in vivo tumorigenic potential of patient-derivedcancer cells was correlated with worse clinical outcomes ofpatients (18). The significant relationship was also observed inthe NSCLC brain metastasis samples (Fig. 1B, P ¼ 0.008),although follow-up time was not different (P ¼ 0.076).

Preservation of the parental NSCLCs' biologic characteristics inPDX models

The utility of PDX models as a model system for humanNSCLCs depends on the precise reflection of the parental tumors'pathologic and molecular characteristics. Parental NSCLCs andcorresponding xenograft tumors (n ¼ 15 and 25 for primary andbrain metastatic NSCLCs, respectively) were determined whetherthe engraftment of NSCLC tissues in immunodeficient micemaintained the key features of the parental tumors. Pathologiccomparison revealed a high degree of similarity in the subtype-specific morphologic features and differentiation status between

Table 1. In vivo tumor take rate of primary NSCLCs according to clinicopathologic characteristics

Parameters Class Tumor take rate (%) P

Age <60 years 9/28 (32.1) 0.15�60 years 6/36 (16.7)

Sex Male 13/47 (27.7) 0.19Female 2/17 (11.8)

Smoking status Ever 11/46 (23.9) 0.89Never 4/18 (22.2)

Histologic subtypea ADC 6/35 (17.1) 0.020b

SCC 9/22 (40.9)Others 0/7 (0)

Tumor size <3 cm 2/14 (14.3) 0.36�3 cm 13/50 (26)

Pathologic T stage 1–2 11/47 (23.4) 0.623–4 4/17 (23.5)

Pathologic N stage 0 5/33 (15.2) 0.11þ 10/31 (32.3)

Distant metastasis 0 12/60 (20) 0.012b

þ 3/4 (75)Overall tumor stage (I–IV) I–II 6/36 (16.7) 0.031b

III 6/24 (25)IV 3/4(75)

Mouse strain NOG 7/23 (30.4) 0.32Nude 8/41 (19.5)

Implantation route Subcutaneous 10/44 (22.7) 0.84Subrenal 5/20 (25)

Differentiationc Well 2/4 (50) 0.50Moderate 6/28 (21.4)Poor 7/26 (26.9)

Histologic growth patternd With solid component 5/14 (35.7) 0.017b

Without solid component 1/21 (4.8)History of neoadjuvant chemotherapy No 13/49 (26.5) 0.29

Yes 2/15 (13.3)EGFR genotype WTe 10/41 (24.4) 0.049b

Mutated 0/13 (0)KRAS genotype WT 7/47 (14.9) 0.076

Mutated 3/7 (42.9)ALK genotype WT 10/53 (18.9) 0.63

Translocated 0/1 (0)aADC, adenocarcinoma.bP < 0.05.cCases with available pathologic reports on differentiation status were analyzed (n ¼ 58).dWith solid growth pattern in the adenocarcinoma (n ¼ 35) subtype was determined as >10% solid sheets of tumor without the formation of acini, papillae ormicropapillae, or sheets or nests of tumor areas with fibrous tissue.eWT, wild type.

Lee et al.





the xenografts and corresponding parental tumors (Fig. 1C andSupplementary Fig. S1). Moreover, the preferential expression ofNSCLC histologic subtype-specific markers such as TTF-1, NapsinA (for adenocarcinoma), andp63 (for SCC)was reproduced in thexenograft tumors (Fig. 1C). When xenograft tumors were seriallytransplanted into immunodeficient mice, the parental tumorcharacteristics were maintained at least until the third in vivopassage (data not shown). Genomic similarities (gene copy-number alterations and mutations) between parental and corre-sponding xenograft tumorswere also confirmedby aCGHand IonAmpliSeq Cancer Panel sequencing results (data not shown).

Application of NSCLC brain metastasis PDX models topersonalized anticancer agent screening

PDX model establishment experiments demonstrated that invivo tumor take rate was significantly higher when surgical sam-ples from NSCLC brain metastases were utilized (74% vs.23%, Fig. 1A). Moreover, PDX models could be consistentlygenerated by NSCLC brain metastasis samples regardless of vary-ing NSCLC clinicopathologic characteristics (Table 2). Similarly,in vitroprimary culture ofNSCLC cells fromPDX tumors in serum-free conditions was more successful in the NSCLC brain metas-tasis group. We had attempted to make tumor spheres in vitrousing 15 and 25 PDX tumors from primary and brain metastatictumors, respectively. In 1 (1/15, 6.6%) and 9 (9/25, 36%) cases,tumors sphere was formed (P ¼ 0.038). Among the 9 brainmetastasis cases, 5 cases showed in vitro passagability (P > 3). Inthe other cases (n ¼ 4), we confirmed tumor sphere formation inthe second in vitro passage, whereas further subculture was nottried. These data indicated that PDX models using surgical sam-ples of NSCLC brain metastases could be better suited as thepreclinical platform for NSCLC in terms of technical feasibilityand unmet clinical needs.

Accordingly, we further validated the applicability of NSCLCbrain metastasis PDX models for preclinical screening of person-alized anticancer therapeutics using five representative cases, LC-MBT-15, LC-MBT-17, LC-MBT-19, LC-MBT-33, and LC-MBT-34(Fig. 2A and Supplementary Table S4). LC-MBT-15 was a meta-chronous brain metastasis obtained from a 56-year-old womanwith a smoking history and confirmed lung adenocarcinomadiagnosis with extracranial lung, liver, and bone metastases. Thispatient experienced treatment resistance to multiple chemother-apy regimens including erlotinib, an agent targeting EGFR. LC-MBT-17 was a metachronous brain metastasis of a 68-year-oldman with a smoking history and confirmed lung SCC diagnosis.Erlotinib also showed no clinical benefit in this case. LC-MBT-19was a 65-year-old female nonsmoker who initially presentedwitha mass in the left upper lobe (adenocarcinoma) and a synchro-nous brain metastasis. No extracranial metastases were identifiedvia imaging studies, including PET-CT. This patient did not receiveany treatment before surgical resection. LC-MBT-33 was a 68-yearold male smoker who was initially diagnosed as a lung SCC.During clinical managements, this patient experienced chemore-sistance and a brain metastasis. LC-MBT-34 was a metachronousbrain metastasis of a 68-year-old male with a smoking history.This patient with lung SCC had chemotherapy, which did notshow significant clinical efficacy to drawmultiple systemicmetas-tases, including brain, bone, liver, and adrenal gland. Whengenomic alterations of EGFR, ALK, and KRAS were examined forthese cases, a duplication of EGFR exon 20 and a KRASmutation(12G>C) was found in LC-MBT-15 and LC-MBT-34, respectively(Supplementary Table S4).

PDX models were successfully established from the surgicalsamples of the five cases. NSCLC cancer cells were primarilycultured in serum-free condition from the five PDX tumors,demonstrating in vitro sphere-forming capacity (Fig. 2B).

Table 2. In vivo tumor take rate of brain metastatic NSCLCs according to clinicopathologic characteristics

Parameters Class Tumor take rate (%) P

Age <60 years 9/13 (69.2) 0.66�60 years 16/21 (64.0)

Sex Male 16/21 (64.0) 0.66Female 9/13 (69.2)

Smoking status Never 8/11 (72.7) 0.94Ever 17/23 (73.9)

Tumor size <3 cm 10/17 (58.8) 0.052�3 cm 15/17 (88.2)

Histologic subtype ADC 17/23 (73.9) 0.96SCC 6/8 (75.0)Others 2/3 (66.7)

Pattern of brain metastasis Synchronous 9/14 (64.3) 0.31Metachronous 16/20 (80.0)

Extracranial metastasis Yes 10/13 (76.9) 0.72No 15/21 (71.4)

Multiple chemoresistance Yes 6/7 (85.7) 0.41No 19/27 (70.4)

Mouse strain NOG 7/9 (77.8) 0.74Nude 18/25 (72.0)

Implantation route Subcutaneous 14/20 (70.0) 0.57Intracerebral 11/14 (78.6)

EGFR genotype WT 12/17 (70.6) 0.44Mutated 5/9 (55.6)

KRAS genotype WT 15/24 (62.5) 0.28Mutated 2/2 (100)

ALK genotype WT 16/25 (64) 0.46Translocated 1/1 (100)

Abbreviations: ADC, adenocarcinoma; WT, wild-type.






Furthermore, when 1 � 104 primarily cultured LC-MBT-15, LC-MBT-17, or LC-MBT-19 cancer cells were injected into the leftventricle of nudemice, systemicmetastatic tumorswere formed invarious organs including the brain (Fig. 2B) to effectively confirmthe in vivo brain metastatic potential. However, correlationbetween extracranial metastatic sites of parental tumors andcorresponding animal models was not observed (Fig. 2A). Thegenetic properties of patients' surgical samples, PDX tumor tis-sues, and dissociated PDX tumor cells were analyzed by aCGHand the Ion Personal Genome Machine Sequencer using the IonAmpliSeq Cancer Panel (22). EGFR exon-20 duplication wasconsistently detected in LC-MBT-15 (Fig. 2C). LC-MBT-17, LC-MBT-19, LC-MBT-33, and LC-MBT-34 harbored ERBB4

(1058T>A), TP53 (839G>C), KRAS (12G>A), and TP53(1015T>C) mutations, respectively (Fig. 2C). No other geneticmutations were detected. The genetic mutations and copy-num-ber variations of patients' tumors were preserved in both xeno-grafts and primarily cultured cancer cells in all five cases (Fig. 2Cand D).

The IC50 values for 20 targeted agents were obtained in vitrousing the primarily cultured cells in sphere culture condition(Table 3). The drug library was composed of targeted agentsincluded in the clinical guideline or current clinical trials for thetreatment of NSCLC. Cells were considered resistant to an agentwhen the IC50 of that agent was greater than 1,000 nmol/L,according to the previous studies (23). Interestingly, the efficacy

Figure 2.Establishment and genetic validationof brain metastatic NSCLC patient–derived xenograft models. A, clinicalproperties of five brain metastaticNSCLCs (LC-MBT-15, 17, 19, 33, and 34)and metastatic sites of correspondinganimal models. Cancer cells (1 � 104)primarily cultured from patient-derived xenograft models of LC-MBT-15, 17, and 19 were injected into thelateral ventricle of immune-deficientmice. B, NSCLC cells were primarilycultured from established xenografttumors in serum-free sphere culturecondition (left plot). In vivo brainmetastases were formed in the brainsof immunodeficient mice by injectingprimarily culture cancer cells into theleft ventricle (right plot). In vivohematogenous brain metastaticpotential of LC-MBT-33 and LC-MBT-34 was not accessed. C, geneticmutations of patients' tumors,xenograft tumors, and primarilycultured cancer cells were screenedand compared. D, gene amplificationsand deletions of patients' tumors,xenograft tumors, and primarilycultured cancer cells were analyzed byaCGH and compared. Data wereobtained from LC-MBT-15 samples.ADC, adenocarcinoma.

Lee et al.





of these 20 therapeutic agents varied dramatically in five casesexamined (>100-fold differences in IC50, Table 3), indicating thepossibility of personalized approach.

Elucidation of case-specific oncogenic signaling using NSCLCbrain metastasis PDX models

As shown in Table 3, the proliferation of LC-MBT-15 cells wasnot affected by EGFR inhibitors such as gefitinib (IC50 ¼ 7,900nmol/L), erlotinib (IC50 > 20,000 nmol/L), and lapatinib (IC50¼4,100 nmol/L). Only afatinib, an irreversible EGFR and HER2inhibitor, showed marked inhibitory effects on LC-MBT-015 cellproliferation (IC50¼ 270 nmol/L, Table 3). Patients with NSCLCwith EGFR mutations in exons 18, 19, or 21, which encompassmost of the tyrosine kinase domain of EGFR (24), have beenshown to respond to EGFR-targeted agents including erlotinib(25, 26). However, EGFR in-frame duplication and/or insertionmutations in exon20 (27) often result in poorer responsiveness tothese EGFR inhibitors (28). The resistance to erlotinib of LC-MBT-15 cellswas consistentwith the clinical treatment outcomes of thispatient whose tumors harbored EGFR exon-20 duplicationmuta-tion (Fig. 2A). However, afatinib reportedly exhibits therapeuticeffects on patients with NSCLC with EGFR exon-20 duplication(insertion) mutations (29), which was reproduced in vitro in thisstudy. In addition, tivantinib, an MET-targeted agent, showed asimilar IC50 (270nmol/L) for the proliferationof LC-MBT-15 cellsto that of afatinib. Interestingly, the sensitivity was correlatedwithdecreased phosphorylation of ERK regardless of the primarytherapeutic targets (Fig. 3A), suggesting the downstream ERKsignaling pathway is important in the proliferation of LC-MBT-15 cells. Accordingly, an ERK inhibitor, SCH772984, significantlyreduced ERK phosphorylation of LC-MBT15 cells and showedinhibitory effects on proliferation (Fig. 3B).

LC-MBT-19, an adenocarcinoma with wild-type EGFR, showedbetter response toEGFR-targeted therapeutics (gefitinib, erlotinib,lapatinib, and afatinib) than LC-MBT-15 (Table 3). Treatmentwith EGFR-targeted agents reduced the phosphorylation of EGFR;however, AKT phosphorylation, a downstream signaling compo-nent of EGFR, was decreased by only afatinib, which showed thelowest IC50 for the proliferation of LC-MBT-19 cells (Table 3

and Fig. 3C). Although EGFR wild-type adenocarcinomas havebeen reported to be resistant to EGFR-targeted agents clinically,afatinib had therapeutic activities against this patient population(30, 31), which could draw the lowest IC50. Interestingly, theactivation status of ERK was not affected by any of the EGFR-targeted agents. Crizotinib, a specific inhibitor of ALK and MET,dramatically suppressed the phosphorylation of MET but notAKT, and had a relatively high IC50 (3,100 nmol/L). Accordingly,the proliferation of LC-MBT-19 cells was decreased by an AKTinhibitor, AZD5363, which significantly reduced the phosphor-ylationof AKTdownstream signaling components, such asGSK3band PRAS (Fig. 3B). These results indicated that AKT signalingpathway rather than ERK is important for LC-MBT-19 cell survivaland proliferation.

The proliferation of LC-MBT-17, a case of SCC, cells was notaffected by EGFR and MET inhibitors (Table 3), while mTORinhibitors (temsirolimus and everolimus) showed low IC50

values (43 and 65 nmol/L, respectively) by totally abolishing thephosphorylation of p70S6K, a downstream signaling componentofmTOR (Fig. 3B). In contrast, erlotinib effectively suppressed thephosphorylation of both AKT and ERK (Fig. 3B), but could notsuppress the proliferation of LC-MBT-17 cells. Therefore, themTOR-p70S6K pathway, rather than the AKT and ERK pathways,was thought to be important in the proliferation of LC-MBT-17cells. Another SCC case, LC-MBT-33, showed differential sensi-tivity toMET inhibitors (tivantinib, foretinib, and crizotinib; IC50

¼ 260, 260, and 930 nmol/L, respectively), whereas LC-MBT-34SCC cells did not respond to most of tested agents except temsir-olimus (Table 3).

DiscussionThe morbidity of NSCLC brain metastasis is rising, and the

prognosis of patients remains poorwith amedian survival of onlya few weeks to months despite intensive multimodal therapy(32). Nevertheless, very few pathophysiologic studies have beenperformed on NSCLC brain metastasis, and the appropriatepreclinical models for such studies remain to be defined. Sincethe 1980s, surgery has become a standard therapeutic option for

Table 3. In vitro sensitivity of primarily cultured PDX tumor cells to a panel of targeted therapeutic agentsa

IC50 (nmol/L)Drug Target LC-MBT-15 LC-MBT-17 LC-MBT-19 LC-MBT-33 LC-MBT-34

Gefitinib EGFR 7,900 11,000 620 1,700 3,100Erlotinib EGFR >20,000 8,700 2,900 20,000 3,200Lapatinib EGFR/HER2 3,400 6,400 720 2,000 1,300Afatinib EGFR/HER2 270 2,100 46 1,700 2,500Tivantinib MET 370 690 210 260 6,100Foretinib MET/KDR 1,200 790 1,600 260 1,600Crizotinib MET/ALK 1,900 2,000 3,100 930 4,600Selumetinib MEK 1,500 8,500 1,400 5,200 >20,000Temsirolimus mTOR 570 43 300 13,000 270Everolimus mTOR 4,100 65 620 20,000 >20,000Cabozantinib KDR >20,000 7,000 12,000 2,900 8,300Vandetanib KDR 5,700 9,500 2,000 1,900 3,100Sunitinib PDGFR/KDR 3,900 3,900 3,800 1,500 3,100Sorafenib BRAF/PDGFR/KDR 4,000 2,600 4,400 2,000 2,900Dovitinib c-kit/FGFR/VEGFR 1,700 1,100 1,200 220 3,900Vemurafenib BRAF 9,200 8,600 6,200 5,700 >20,000BKM120 PI3K 1,000 1,100 570 600 1,300Pazopanib VEGFR/PDGFR/c-kit 14,000 2,300 >20,000 870 15,000Nintedanib VEGFR/FGFR/PDGFR 2,100 >20,000 3,700 720 10,000DAPT g-secretase >20,000 >20,000 >20,000 >20,000 >20,000aIC50 is an average value derived from triplicated experiments.






patients with solitary and symptomatic or life-threatening mul-tiple brain metastases (12). Moreover, surgical resection couldimprove both patients' survival and quality of life (33, 34).Therefore, surgically obtained NSCLC brain metastasis samplescould be utilized to establish NSCLC PDXs without ethical issues.In this study, NSCLCbrainmetastasis samples demonstrated highengraftment efficiency in immune-deficient mice (74%), and theresulting PDXs closely resembled the original tumors morpho-logically and molecularly. In addition, they recapitulated thecorresponding patients' functional characteristics such as in vivobrain metastatic potential and differential drug responses, indi-cating their potential as in vitro and in vivo preclinical models foradvanced NSCLCs.

Preclinical drug development has traditionally relied on estab-lished human cancer cell lines that are cultured in serum-contain-ing media in adherent conditions for extended periods of time.However, the reliability of such cell lines has provoked consid-erable debates due to the high failure rate of newly developedtargeted agents in subsequent clinical trials (35). Xenograftsderived from cell lines generally show a homogeneous histologywith a lack of human stroma and immune cells, which areimportant for tumor growth and metastatic processes (36). Incontrast, xenografts directly derived from patient biopsies orsurgical specimens with minimal in vitro manipulations appear

to possess better retention of the morphologic and molecularcharacteristics of the original tumors (37). Therefore, PDXs maybe an informative preclinical model for the development of noveltherapeutics as they can more accurately predict the subsequentclinical success and allow various mechanistic studies that are notpossible in patients (37).

The elucidation of target populations is a prerequisite for theclinical success of molecularly targeted agents. Because of thelimited intra- and intertumoral heterogeneity of conventional celllines, it is very difficult to develop reliable personalized strategiesin the preclinical stage. Combining PDXs with high-throughputdrug screening technologies would be a promising solution,because PDXs recapitulate the functional properties of corre-sponding individual tumors and a PDX library would representheterogeneous patient populations. Indeed, drug screeningexperiments in this study using five independent NSCLC brainmetastasis samples showed dramatically different responses totargeted agents, highlighting the importance of personalizedapproaches. Although several drug screening tests have beenperformed on resected NSCLC specimens, including in vitro testsusing short-term cell cultures (38) and in vivo tests using first-generation tissue xenografts in immune-deficient mice (39), earlyattempts to use primary lung tumors as experimental models hadlimited success. In particular, primary NSCLC-derived PDXs

Figure 3.Molecular response of primarily culturedNSCLC cells to various targeted agents.A, alterations in the expression andphosphorylation of related downstreamsignaling components as the results oftreatment with targeted agents wereassessed by Western blot analysis.B, IC50 of the ERK and AKT inhibitoragainst primarily cultured LC-MBT-15and LC-MBT-19 cells was determined,respectively (left plot). Alterations in theexpression and phosphorylation ofrelated downstream signalingcomponents as the results of treatmentwith the targeted agents were assessedby Western blot analysis (right plot).

Lee et al.





showed low in vivo take rate (30%–40%), which is similar to ourresults (23%; refs. 7, 10). While specific factors associated withsuccessful in vivo engraftment of primary lung tumors, such assquamous histology, the presence of distant metastasis, solidgrowth pattern, and wild-type EGFR genotype, were elucidatedby this and previous studies (10, 16, 40), those factors wouldinevitably limit applicable NSCLC patient populations. In con-trast, no significant factor was found in cases of NSCLC brainmetastases. In spite of those merits of using surgical samples ofNSCLC brain metastasis, their availability would limit the exper-imental and/or clinical applications of them because surgicalmanagements are not usually primary medical treatments forpatients with NSCLC brain metastasis.

The fact that preclinical drug sensitivity data are obtained fromprimary NSCLC models instead of systemic metastatic advancedNSCLC models could be another reason for the mismatchbetween preclinical and clinical treatment results (10). Indeed,aCGH analyses or single nucleotide polymorphism arrays utiliz-ing primary and paired metastasis tissues showed considerablediscrepancies (41, 42). Furthermore, functional analyses revealedsignificant differences in the proliferation, motility, and chemo-sensitivity of cancer cells (43). Especially, chemosensitivity isstrongly affected by the biologic aggressiveness of tumor cells,such as themetastatic potential (39, 44, 45). Therefore, concertedefforts to utilize surgical samples obtained from advanced casesare necessary to represent clinical situations withmore treatment-resistant or metastatic diseases. The significant associationbetween PDX formation capacity and clinical aggressiveness ofthe parental NSCLCs (16, 40) has important preclinical andclinical implications. In this study, as NSCLC brain metastasissamples were efficiently engrafted in vivo, and established PDXsrecapitulated the histopathologic, genomic, and biologic prop-erties of parental NSCLCs in situ, our strategy of using surgicallyresected metastatic tissues would provide more reliable transla-tional models representing the aggressive phenotypes and geno-types of NSCLC for rationale-driven clinical trials to overcomeNSCLC treatment resistance or metastasis.

Recent attention has focused on the need for better models thatpreserve the integrity of cancer stem cell (CSC) populations.Manystudies have reported the increased invasiveness of CSCs, impli-cating the involvement of CSCs in the progression and/or devel-opment of metastases (46, 47). However, most previous in vitrodrug screenings on lung cancer patient–derived cancer cells wereperformed in serum-containing medium (48), whereas tumorspheres in serum-free condition enrich CSC-like populations(49). Herein, PDXs derived from NSCLC brain metastases gave

rise to numerous tumor spheres in a few days after mechanicaldissociation ex vivo in a reproducible manner, whereas PDXs fromNSCLC primary tumor demonstrated poor viability and sphere-forming ability in vitro. In previous study, the ability to formtumor spheres was also correlated with tumor aggressiveness, andthe chemosensitivity of tumor spheres was similar to that of the invivo original xenografts (50). Therefore, ex vivo tumor sphereswould be one of the potential preclinical tools for the validationof new drugs in a setting that more closely resembles patients'tumors but also for the investigation of patient-tailored thera-peutics with high-throughput drug screen.

In this study, we demonstrated that brain metastases fromNSCLC could be a valuable resource for the establishment ofPDXs as a powerful preclinical platform to further develop per-sonalized approaches for the treatment of patients with advancedNSCLC. Moreover, a platform library representing heterogeneousadvanced NSCLCs would be a valuable modality for the compre-hensive understanding of NSCLC progression and metastasis aswell as mechanisms of drug resistance.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H.W. Lee, Y.M. Shim, K.M. Joo, D.-H. NamDevelopment of methodology: H.W. Lee, S.J. LeeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.W. Lee, J.-il Lee, S.J. Lee, H.J. Cho, H.J. Song,D.E. Jeong, S. Shin, Y.-J. Kwon, Y.-L. Choi, H.J. Seol, Y.M. ShimAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.W. Lee, H.J. Cho, H.J. Song, S. Shin, J.-G. Joung,Y.-J. Kwon, Y.-L. Choi, W.-Y. Park, K.M. JooWriting, review, and/or revision of the manuscript: H.W. Lee, H.M. Lee, Y.M.Shim, K.M. JooAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.J. Seo, H.J. SeolStudy supervision: W.-Y. Park, H.M. Lee, Y.M. Shim, K.M. Joo, D.-H. Nam

Grant SupportThis research was supported by a grant from the Korea Health Technology

R&D Project through the Korea Health Industry Development Institute(KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea(HI14C3418).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 20, 2014; revised November 3, 2014; accepted December 7,2014; published OnlineFirst December 30, 2014.

References1. Siegel R, NaishadhamD, Jemal A. Cancer statistics, 2012. CA Cancer J Clin

2012;62:10–29.2. Uramoto H, Mitsudomi T. Which biomarker predicts benefit from EGFR-

TKI treatment for patients with lung cancer? Br J Cancer 2007;96:857–63.3. Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al.

Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med 2010;363:1693–703.

4. Steeg PS, Camphausen KA, Smith QR. Brain metastases as preventive andtherapeutic targets. Nat Rev Cancer 2011;11:352–63.

5. Palmieri D, Chambers AF, Felding-Habermann B, Huang S, Steeg PS. Thebiology ofmetastasis to a sanctuary site. ClinCancer Res 2007;13:1656–62.

6. MehtaMP, Rodrigus P, TerhaardCH,RaoA, Suh J, RoaW, et al. Survival andneurologic outcomes in a randomized trial of motexafin gadolinium and

whole-brain radiation therapy in brain metastases. J Clin Oncol2003;21:2529–36.

7. Fichtner I, Rolff J, Soong R, Hoffmann J, Hammer S, Sommer A, et al.Establishment of patient-derived non-small cell lung cancer xenografts asmodels for the identification of predictive biomarkers. Clin Cancer Res2008;14:6456–68.

8. Marangoni E, Vincent-Salomon A, Auger N, Degeorges A, Assayag F, deCremoux P, et al. A new model of patient tumor-derived breastcancer xenografts for preclinical assays. Clin Cancer Res 2007;13:3989–98.

9. Cutz JC, Guan J, Bayani J, Yoshimoto M, Xue H, Sutcliffe M, et al.Establishment in severe combined immunodeficiency mice of subrenalcapsule xenografts and transplantable tumor lines froma variety of primary






human lung cancers: potential models for studying tumor progression-related changes. Clin Cancer Res 2006;12:4043–54.

10. John T, Kohler D, Pintilie M, YanagawaN, PhamNA, LiM, et al. The abilityto form primary tumor xenografts is predictive of increased risk of diseaserecurrence in early-stage non-small cell lung cancer. Clin Cancer Res2011;17:134–41.

11. Zhang XC, Zhang J, Li M, Huang XS, Yang XN, Zhong WZ, et al. Estab-lishment of patient-derived non-small cell lung cancer xenograft modelswith genetic aberrations within EGFR, KRAS and FGFR1: useful tools forpreclinical studies of targeted therapies. J Transl Med 2013;11:168.

12. Narita Y, Shibui S. Strategy of surgery and radiation therapy for brainmetastases. Int J Clin Oncol 2009;14:275–80.

13. BeasleyMB, Brambilla E, Travis WD. The 2004World HealthOrganizationclassification of lung tumors. Semin Roentgenol 2005;40:90–7.

14. Groome PA, Bolejack V, Crowley JJ, Kennedy C, KrasnikM, Sobin LH, et al.The IASLC Lung Cancer Staging Project: validation of the proposals forrevision of the T, N, and M descriptors and consequent stage groupings inthe forthcoming (seventh) edition of the TNM classification of malignanttumours. J Thorac Oncol 2007;2:694–705.

15. Motoi N, Szoke J, Riely GJ, Seshan VE, Kris MG, Rusch VW, et al. Lungadenocarcinoma: modification of the 2004 WHO mixed subtype toinclude the major histologic subtype suggests correlations between pap-illary and micropapillary adenocarcinoma subtypes, EGFR mutations andgene expression analysis. Am J Surg Pathol 2008;32:810–27.

16. YoshizawaA,MotoiN, RielyGJ, SimaCS,GeraldWL, KrisMG, et al. Impactof proposed IASLC/ATS/ERS classification of lung adenocarcinoma: prog-nostic subgroups and implications for further revision of staging based onanalysis of 514 stage I cases. Mod Pathol 2011;24:653–64.

17. ChaMJ, LeeHY, Lee KS, Jeong JY, Han J, ShimYM, et al.Micropapillary andsolid subtypes of invasive lung adenocarcinoma: clinical predictors ofhistopathology and outcome. J Thorac Cardiovasc Surg 2014;147:921–8. e2.

18. Joo KM, Kim J, Jin J, Kim M, Seol HJ, Muradov J, et al. Patient-specificorthotopic glioblastoma xenograft models recapitulate the histopathologyand biology of human glioblastomas in situ. Cell Rep 2013;3:260–73.

19. Joo KM, Kim SY, Jin X, Song SY, Kong DS, Lee JI, et al. Clinical andbiological implications of CD133-positive and CD133-negative cells inglioblastomas. Lab Invest 2008;88:808–15.

20. Lee HW, Park YM, Lee SJ, Cho HJ, Kim DH, Lee JI, et al. Alpha-smoothmuscle actin (ACTA2) is required for metastatic potential of human lungadenocarcinoma. Clin Cancer Res 2013;19:5879–89.

21. Lee DW, Choi YS, Seo YJ, Lee MY, Jeon SY, Ku B, et al. High-throughputscreening (HTS)of anticancer drug efficacy onamicropillar/microwell chipplatform. Anal Chem 2014;86:535–42.

22. KimHS, Sung JS, Yang SJ, KwonNJ, Jin L, Kim ST, et al. Predictive efficacy oflow burden EGFR mutation detected by next-generation sequencing onresponse to EGFR tyrosine kinase inhibitors in non-small-cell lung carci-noma. PLoS One 2013;8:e81975.

23. Hatzivassiliou G, Liu B, O'Brien C, Spoerke JM, Hoeflich KP, Haverty PM,et al. ERK inhibition overcomes acquired resistance toMEK inhibitors. MolCancer Ther 2012;11:1143–54.

24. Kobayashi S, BoggonTJ,DayaramT, Janne PA,KocherO,MeyersonM, et al.EGFRmutation and resistance of non-small-cell lung cancer to gefitinib. NEngl J Med 2005;352:786–92.

25. TaronM, Ichinose Y, Rosell R,Mok T, Massuti B, Zamora L, et al. Activatingmutations in the tyrosine kinase domain of the epidermal growth factorreceptor are associated with improved survival in gefitinib-treated chemor-efractory lung adenocarcinomas. Clin Cancer Res 2005;11:5878–85.

26. Takano T, Ohe Y, Sakamoto H, Tsuta K, Matsuno Y, Tateishi U, et al.Epidermal growth factor receptor gene mutations and increased copynumbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005;23:6829–37.

27. Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II,et al. Clinical and biological features associated with epidermal growthfactor receptor gene mutations in lung cancers. J Natl Cancer Inst2005;97:339–46.

28. Wu JY,Wu SG, Yang CH, GowCH, Chang YL, Yu CJ, et al. Lung cancer withepidermal growth factor receptor exon 20 mutations is associated withpoor gefitinib treatment response. Clin Cancer Res 2008;14:4877–82.

29. Nguyen KS, Kobayashi S, Costa DB. Acquired resistance to epidermalgrowth factor receptor tyrosine kinase inhibitors in non-small-cell lung

cancers dependent on the epidermal growth factor receptor pathway. ClinLung Cancer 2009;10:281–9.

30. Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, Chirieac LR, et al.BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in pre-clinical lung cancer models. Oncogene 2008;27:4702–11.

31. Solca F, Dahl G, Zoephel A, Bader G, Sanderson M, Klein C, et al. Targetbinding properties and cellular activity of afatinib (BIBW 2992), anirreversible ErbB family blocker. J Pharmacol Exp Ther 2012;343:342–50.

32. PreusserM, Capper D, Ilhan-Mutlu A, Berghoff AS, Birner P, Bartsch R, et al.Brain metastases: pathobiology and emerging targeted therapies. ActaNeuropathol 2012;123:205–22.

33. Read RC, Boop WC, Yoder G, Schaefer R. Management of nonsmall celllung carcinoma with solitary brain metastasis. J Thorac Cardiovasc Surg1989;98:884–90; discussion 90-1.

34. I H, Lee JI, Nam DH, Ahn YC, Shim YM, Kim K, et al. Surgical treatment ofnon-small cell lung cancer with isolated synchronous brain metastases.J Korean Med Sci 2006;21:236–41.

35. Kerbel RS. Human tumor xenografts as predictive preclinical models foranticancer drug activity in humans: better than commonly perceived-butthey can be improved. Cancer Biol Ther 2003;2:S134–9.

36. De Wever O, Mareel M. Role of tissue stroma in cancer cell invasion.J Pathol 2003;200:429–47.

37. Rubio-Viqueira B, Jimeno A, Cusatis G, Zhang X, Iacobuzio-Donahue C,Karikari C, et al. An in vivo platform for translational drug development inpancreatic cancer. Clin Cancer Res 2006;12:4652–61.

38. Gout PW, Wang Y. Drug sensitivity testing for personalized lung cancertherapy. J Thorac Dis 2012;4:17–8.

39. Dong X, Guan J, English JC, Flint J, Yee J, Evans K, et al. Patient-derived firstgeneration xenografts of non-small cell lung cancers: promising tools forpredicting drug responses for personalized chemotherapy. Clin Cancer Res2010;16:1442–51.

40. Warth A,Muley T,MeisterM, Stenzinger A, ThomasM, Schirmacher P, et al.The novel histologic International Association for the Study of LungCancer/American Thoracic Society/European Respiratory Society classifi-cation system of lung adenocarcinoma is a stage-independent predictor ofsurvival. J Clin Oncol 2012;30:1438–46.

41. Petersen S, Aninat-Meyer M, Schluns K, Gellert K, Dietel M, Petersen I.Chromosomal alterations in the clonal evolution to themetastatic stage ofsquamous cell carcinomas of the lung. Br J Cancer 2000;82:65–73.

42. Takahashi K, Kohno T, Matsumoto S, Nakanishi Y, Arai Y, Yamamoto S,et al. Clonal and parallel evolution of primary lung cancers and theirmetastases revealed bymolecular dissection of cancer cells. Clin Cancer Res2007;13:111–20.

43. Gottschling S, Jauch A, Kuner R, Herpel E, Mueller-Decker K, Schnabel PA,et al. Establishment and comparative characterization of novel squamouscell non-small cell lung cancer cell lines and their corresponding tumortissue. Lung Cancer 2012;75:45–57.

44. Higashiyama M, Oda K, Okami J, Maeda J, Kodama K, Imamura F, et al.Prediction of chemotherapeutic effect on postoperative recurrence by invitro anticancer drug sensitivity testing in non-small cell lung cancerpatients. Lung Cancer 2010;68:472–7.

45. Higashiyama M, Okami J, Maeda J, Tokunaga T, Fujiwara A, Kodama K,et al. Differences in chemosensitivity between primary and paired meta-static lung cancer tissues: In vitro analysis based on the collagen gel dropletembedded culture drug test (CD-DST). J Thorac Dis 2012;4:40–7.

46. Croker AK, Goodale D, Chu J, Postenka C, Hedley BD, Hess DA, et al. Highaldehyde dehydrogenase and expression of cancer stem cell markers selectsfor breast cancer cells with enhanced malignant and metastatic ability.J Cell Mol Med 2009;13:2236–52.

47. Liu H, Patel MR, Prescher JA, Patsialou A, Qian D, Lin J, et al. Cancer stemcells from human breast tumors are involved in spontaneousmetastases inorthotopic mouse models. Proc Natl Acad Sci U S A 2010;107:18115–20.

48. Zheng C, Sun YH, Ye XL, Chen HQ, Ji HB. Establishment and character-ization of primary lung cancer cell lines from Chinese population. ActaPharmacol Sin 2011;32:385–92.

49. Nolte SM, Venugopal C, McFarlane N, Morozova O, Hallett RM, O'FarrellE, et al. A cancer stem cell model for studying brain metastases fromprimary lung cancer. J Natl Cancer Inst 2013;105:551–62.

50. Weiswald LB, Richon S, Massonnet G, Guinebretiere JM, Vacher S, Laur-endeau I, et al. A short-term colorectal cancer sphere culture as a relevant toolfor human cancer biology investigation. Br J Cancer 2013;108:1720–31.


Lee et al.




2015;21:1172-1182. Published OnlineFirst December 30, 2014.Clin Cancer Res Hye Won Lee, Jung-il Lee, Se Jeong Lee, et al. Development of Personalized Targeted TherapyMetastases Are Valuable Translational Platforms for the

Small Cell Lung Cancer Brain−Patient-Derived Xenografts from Non

Updated version

10.1158/1078-0432.CCR-14-1589doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2015/01/06/1078-0432.CCR-14-1589.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/21/5/1172.full#ref-list-1

This article cites 50 articles, 17 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/21/5/1172.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/21/5/1172To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-14-1589

http://clincancerres.aacrjournals.org/content/suppl/2015/01/06/1078-0432.CCR-14-1589.DC1

http://clincancerres.aacrjournals.org/content/21/5/1172.full#ref-list-1

http://clincancerres.aacrjournals.org/content/21/5/1172.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/21/5/1172


Documents

Patient-Derived Xenografts from Non Small Cell Lung Cancer ... · Cancer Therapy: Preclinical Patient-Derived Xenografts from Non–Small Cell Lung Cancer Brain Metastases Are Valuable