cd133þ tumor initiating cells in a syngenic murine model of … · cancer therapy: preclinical...

Cancer Therapy: Preclinical

CD133þ Tumor Initiating Cells in a Syngenic Murine Modelof Pancreatic Cancer Respond to Minnelide

Sulagna Banerjee1, Alice Nomura1, Veena Sangwan1, Rohit Chugh1, Vikas Dudeja1,Selwyn M. Vickers1,2, and Ashok Saluja1,2

AbstractPurpose: Pancreatic adenocarcinoma is the fourth leading cause for cancer-related mortality with a

survival rate of less than 5%. Late diagnosis and lack of effective chemotherapeutic regimen contribute to

these grim survival statistics. Relapse of any tumor is largely attributed to the presence of tumor-initiating

cells (TIC) or cancer stem cells (CSC). These cells are considered as hurdles to cancer therapy as no known

chemotherapeutic compound is reported to target them. Thus, there is an urgent need to develop a TIC-

targeted therapy for pancreatic cancer.

ExperimentalDesign:We isolatedCD133þ cells froma spontaneous pancreatic ductal adenocarcinoma

mouse model and studied both surface expression, molecular markers of pancreatic TICs. We also studied

tumor initiation properties by implanting low numbers of CD133þ cells in immune competentmice. Effect

of Minnelide, a drug currently under phase I clinical trial, was studied on the tumors derived from the

CD133þ cells.

Results: Our study showed for the first time that CD133þ population demonstrated all the molecular

markers for pancreatic TIC. These cells initiated tumors in immunocompetent mouse models and showed

increased expressionof prosurvival andproinvasive proteins compared to theCD133�non-TICpopulation.

Our study further showed that Minnelide was very efficient in downregulating both CD133� and CD133þ

population in the tumors, resulting in a 60% decrease in tumor volume compared with the untreated ones.

Conclusion: As Minnelide is currently under phase I clinical trial, its evaluation in reducing tumor

burden by decreasing TIC as well as non-TIC population suggests its potential as an effective therapy. Clin

Cancer Res; 20(9); 2388–99. �2014 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is currently

the fourth most frequent cause of cancer-related deaths (1).Aggressive biology, chemoresistance, and recurrence of thetumor make PDAC one of the most devastating cancers.

Since their discovery, tumor-initiating cells (TIC) havereferred to a quiescent population of cells that is capable ofcausing recurrence, both local anddistal to the site of tumor.Pancreatic TICs were first isolated as a CD24þCD44þESAþ

population from human xenografts by Li and colleagues(2). Subsequently, CD133 was identified as a TIC for PDAC

(3). Both groups demonstrated tumor-initiating propertiesin the cells enriched for thesemarkers. Pancreatic TICs havebeen further characterized using other surface markers,including CXCR4 (3, 4), Met (5), aldehyde dehydrogenaseactivity (6–8), or ABCG2 (9, 10).

CD133, a transmembrane pentaspan protein, was ini-tially described as a surface antigen specific for humanhematopoietic stem cells (11, 12). Although the biologicfunction of CD133 remains unknown, CD133 is wellrecognized as a stem cell marker for normal and canceroustissues. It has recently been demonstrated that CD133 is amarker of putative pancreatic progenitor cells in mice (13).In humans, CD133 is expressed on terminal ductal cells inthe adult pancreas and carcinoma cells in pancreatic ductaladenocarcinoma (14, 15).

TICs are hypothesized to arise by either accumulationof mutations in stem cells or progenitor cells resulting indysregulation of several self-renewal pathway genes. Con-sistent with this, pancreatic TICs have been reported toshow increased expression of developmental transcrip-tion factors such as Sox2, Nanog, Oct4 (16), and severalHedgehog and Notch pathway genes (2, 17). Apart fromthe above features, resistance to apoptosis by upregula-tion of Bcl-2 and Survivin genes has been associatedwith pancreatic TICs (18). TICs are also associated with

Authors' Affiliations: 1Division of Basic and Translational Research,Department of Surgery; and 2Masonic Cancer Center, University of Min-nesota, Minneapolis, Minnesota

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

S. Banerjee, A. Nomura, and V. Sangwan contributed equally to this article.

Corresponding Author: Ashok K. Saluja, Division for Basic and Transla-tional Research, Department of Surgery, University of Minnesota, 515Delaware Street SE, Minneapolis, MN 55455. Phone: 612-624-8108; Fax:612-624-8909; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2947

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst March 14, 2014; DOI: 10.1158/1078-0432.CCR-13-2947

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increased invasiveness. Pancreatic TIC populationCD133þ/CXCR4þ or Metþ/CD44þ has been reported tobe more invasive than either population alone.Most studies on pancreatic TICs have involved isolation

of cells from patient tumor tissue and propagation inimmunocompromised mouse models such as severe com-bined immunodeficient (SCID)mice or athymic nudemice(2, 3, 19). However, xenografts in immunocompromisedmice lack the syngenic interface between tumor and hostmicroenvironment crucial for understanding tumordynamics. One of the immunocompetent mouse modelsfor pancreatic carcinogenesis is based on the pancreas-specific expression of endogenous mutant Kras and Trp53alleles (LSL-KrasG12D, LSL-Trp53R172H, Pdx-Cre or KPCmice). These KPC mice spontaneously develop primarypancreatic tumors that recapitulate the clinical and histo-pathologic features of the human disease (20, 21).TICs often display resistance to cytotoxic cancer therapies,

permitting the repopulation of tumors after radiation orchemotherapy. Several groups have demonstrated that TICsfrom multiple cancer types exhibit resistance to conven-tional cancer therapies (22). PDAC is known to be resistantto most chemotherapeutic drugs. However, triptolide, a

diterpene triepoxide from the Chinese plant Trypterygiumwilfordii, downregulates heat shock genes (23, 24) andinduces apoptotic death in pancreatic cancer cells (25–27) has been an exception to this. Triptolide and its watersoluble prodrug Minnelide was recently reported to be veryeffective in tumor regression in a number ofmurinemodels(26). Other authors have demonstrated the efficacy oftriptolide by inhibition of proliferation within a numberof additional malignancies, including cholangiocarcinoma(28, 29), osteosarcoma (30), and neuroblastoma (31).However, efficacy of triptolide has not been tested on CSCs.

In this study, we have identified a population of CD133þ

cells from the tumors developed from the KPC transgenicmouse model of PDAC. We have shown that this popula-tion expresses a number of CSC markers (surface markers,transcriptionalmarkers, and developmentalmarkers); has asignificantly higher expression of prosurvival genes like theheat shock proteins, Bcl-2 and Survivin; higher NF-kBactivity and has tumor initiating properties in a syngenic,immunocompetent system. We have further shown thatthese cells, and the tumors derived from these cells, respondto Minnelide, which effectively lowers the pro-proliferativepathways and induces cell death.

Materials and MethodsMouse model, cell isolation, and establishment ofprimary tumor cell line

Moribund animals of the LSL-KrasG12D, LSL-Trp53R172H,and Pdx-Cre genetic background was sacrificed and single-cell suspension was isolated by digestion with collagenaseB and dispase II. Cells were plated in medium containinggrowth factors and 2% serum. Medium was replaced withserum-freemediumafter 48hours andcellsweremaintainedin it for 2 to 3 weeks in the absence of serum until allfibroblasts were removed. Cells were then grown in Dulbec-co’s modified Eagle medium (DMEM) with 10% serum forall experiments. The 2 cell lines derivedwere namedKPC001and KPC023. All experiments were carried out in 2 derivedcell lines and 2 to 3 primary tumors from KPC animals.

Isolation of CD133þ populationThe CD133þ population was separated from the mouse

progenitor cells and other CD133� cells using MACS sep-aration (Miltenyi Biotech) using manufacturers protocol.Single-cell suspension was generated from tumors in KPCmice according to the Li and colleagues (32).Non-epithelialprogenitor cells were removed using anti-CD31-Biotin (BDBiosciences) and anti-CD45 Biotin (BD Bioscience) usingMACS technique. The flow through free from the mouseprogenitor cells was bound to anti-mouse CD133-Microbe-ads for 10 minutes on ice and positively purified forCD133þ cells by MACS. The purity of separation was testedfor each batch by performing a FACS analysis using anti-CD133-PE antibody AC141 (Miltenyi Biotech). The sepa-rated populations were used for RNA, protein, and FACSanalysis. Cells growing in culture were scraped gently intocentrifuge tube and washed once in wash buffer (PBS, 0.5%

Translational RelevancePancreatic ductal adenocarcinoma (PDAC) is current-

ly the fourthmost frequent cause of cancer-related deathin theUnited States owing to its propensity to invade andmetastasize, and resist chemotherapy almost complete-ly. Relapse of any tumor is largely attributed to thepresence of tumor-initiating cells (TIC) or cancer stemcells (CSC). TheseCSCsor TICs arequiescent cellswithina tumor that often evade conventional chemotherapyand result in tumor recurrence either locally or in adistant organ.TICs from most cancers have been identified

and isolated from patient tumors implanted in animmune-compromised mouse model. In this study, weisolate CD133þ pancreatic TIC from both cell lines andtumors derived from the spontaneous genetic mousemodel (KPC) for pancreatic cancer.We further show thatthese CD133þ tumor cells can form tumors in C57/Bl6immune competent mice from as low as 10 cells. Ourstudy also shows that minnelide (a water soluble pro-drug of triptolide, currently under phase I clinical trial)decreases the volume of the tumors derived from theCD133þ cells. Minnelide further reduces the totalCD133þ population in KPC primary tumors.The translational significance of this study lies in the

fact that minnelide is able to decrease the CD133þ TICsin an extremely aggressive model for pancreatic cancer.Furthermore, because this model is based on animmune-competent animal model, it is more clinicallyrelevant compared to the immune-compromised TICmodels reported earlier.

Pancreatic TICs in a Syngenic Murine Model

www.aacrjournals.org Clin Cancer Res; 20(9) May 1, 2014 2389




BSA, 2 mmol/L EDTA) before binding to anti-mouseCD133 microbeads and proceeded as described earlier.

Flow cytometric analysis of isolated samplesFor flow cytometric analysis of tumor tissues, single-cell

suspensionwas prepared according to the protocol of Li andcolleagues (32). Cells isolated from tumor, as well as thosegrowing in culture, were stained with directly conjugatedmonoclonal antibodies in the presence of FcR blockingreagent (Miltenyi Biotech) with anti-CD133-PE (MiltenyiBiotech), anti-CD44-FITC (BD Biosciences), ESA-APC (BDBioscience), and CD24-PE (BD Biosciences). The Aldefluorassay was performed per manufacturer’s instructions (StemCell Technologies) complete with DEAB controls. Immu-noglobulin G (IgG) isotype controls corresponding to eachdirectly conjugated fluorophore were utilized to identify,quantify, and positively select desired cell populations. AllFACS analyses were performed on a BD FACSCanto II (BDBiosciences) using FACSDiva (BD Biosciences) and FlowJo(Tree Star) software. Debris and cell clusters were excludedduring side- and forward-scatter analyses.

Migration assayMigration assay for different cell lines was conducted by

Electric Cell–Substrate Impedance Sensing (ECIS; AppliedBiophysics). In ECIS, the cells are grown on the surface ofsmall and planar gold-film electrodes and the AC imped-ance of the cell-covered electrode is measured continuouslyat a frequency of 64 kHz.A total of 4�104pancreatic cancercells (MiaPACA-2, S2VP10, S2013, AsPC-1, and KPC001)were seeded into an 8E1W ECIS array (Applied Biophysicscontaining a 250 mmol/L electrode. Upon confluence, ahigh field current with frequency 48 kHz and amplitude 5was applied for 10 seconds, killing cells overgrowing theelectrode, creating "wounds" in the wells devoid of cells.

Themigrationwas compared between each cell line at theend of each experiment, which was presented as a measureof changing impedance over time after normalizing to thetime for wounding.

ImmunohistochemistryFor immunohistochemistry, paraffin tissue sections were

deparaffinized in xylene and hydrated through graded eth-anol. Slides were steamed with a Reveal Decloaker (BiocareMedical) to minimize background staining. Sniper Univer-sal Blocking Sera (Biocare Medical) were used throughoutthe protocol. The slides were stained using anti-Ki67, anti-HSP 70 antibody (Abcam), and anti-GRP78 antibody(Cell Signaling Technology). Staining was detected usinganti-rabbit secondary antibody conjugated to horseradishperoxidase followed byDiaminobenzidine Peroxidase Sub-strate Kit (Vector Laboratories). The tissue sections werecounterstained with Gill hematoxylin (Vector Laborato-ries). The primary antibody was omitted for the negativecontrols. TUNELwas performed using the In SituCellDeathDetermination Kit (Roche Diagnostics) according to man-ufacturer’s protocol. For immunofluorescence, fluorescentantibody conjugates were used after primary antibody

staining. Slides were counterstained with 40,6-diamidino-2-phenylindole and visualized in a Nikon fluorescentmicroscope. Tissue samples were incubated with mouseIgG1 isotype controls (BD Biosciences) and did not dem-onstrate any specific staining.

NF-kB assayNF-kB activity assay was performed on CD133þ sorted

cells andminnelide-treatedCD133þ generated tumors.DNAbinding and downstream activation of NF-kB pathway wasassessed using a transcription factor binding ELISA (ThermoScientific) according to manufacturer’s instruction.

Illumina microarray for gene expression study ofCD133þ and CD133�

Microarray studies were performed at the University ofMinnesota Biomedical Genomics Center. RNA samplesfrom 3 independent cell sorts were submitted to the facilityfor Illumina microarray analysis. Results were analyzedusing GeneData Expressionist software at the MinnesotaSupercomputing Institute.

Animal experimentAll animal experiments were performed according to

the University of Minnesota Animal Care Committeeguidelines.

Subcutaneous animal model. Sorted cells were washedwith serum-freeHBSS and suspended in serum-freeDMEM/Matrigel mixture (1:1 volume). A total of 10, 50, 100, and500 cells (CD133þ/CD133�/unsorted) were injected sub-cutaneously into the right and left flank of age and gendermatched C57BL/6 mice (Jackson Laboratories). A total of 8to 10mice were used in each group. Tumors weremeasuredweekly and tumor volume was documented. Tumors wereallowed to grow until they reached a volume of 1 cm3, atwhich the mice were sacrificed and the tumor tissue washarvested and processed for other experiments.

For serial transplantation, tumormeasuring�700 to 800mm3was choppedup into approximately 1 to2mm.Tumorpieces were implanted subcutaneously into right and leftflank of age matched 10 C57BL/6 mice. Tumors weremeasured weekly to monitor their progress.

Treatment with Minnelide. Minnelide treatment wasstarted 5 days after tumor implantation following random-ization of animals. 0.42 mg Minnelide/kg body weight wasadministered intraperitoneally every day for 25 days.Tumors were measured as stated above. At the end of study,animals were sacrificed according to the University of Min-nesota Animal Care guidelines.

Orthotopic animal model. For orthotopic model, 500CD133þ cells or 5,000unsortedKPC cellswere injected intothe pancreas of 24C57BL/6mice. A total of 5 to 6mice weresacrificed every 10 days to monitor development of tumorin each group.

Statistical analysisValues are expressed as the mean � SEM. All in vitro

experiments were performed at least 3 times. Statistical

Banerjee et al.

Clin Cancer Res; 20(9) May 1, 2014 Clinical Cancer Research2390




significance of results was calculated using the Studentt test. Columns represent mean; bars represent SE (n ¼ 4;�P < 0.05).

ResultsKPC tumors and cells exhibit a population of CD133þ

TICsWe analyzed 3 primary KPC tumors and 2 cell lines

derived from KPCmouse tumors for PDAC for the differentstem cell markers. A flow cytometric analysis showed thesecells had 6% to 9% of CD133þ population (Fig. 1A andSupplementary Table S1). A population of CD24þ/CD44þ/ESAþ cells were also present but to a much lesser percent(3% to 4%) compared with the CD133þ population (Fig.1B). A minor population of these cells (�2%) also showedhigher Aldh1 activity (Fig. 1C). To put our study in per-spective, we studied TICmarkers froma classical TICmodel:human tumors transplanted in mice as well as one that wasfreshly isolated tumor. These tumors showed 3% to 4%CD133þ cells whereas 1% to 2% CD24þCD44þESAþ cells.To see if thepercent ofCD133þ cells differed inmetastatic

tumors or in accumulated ascitic fluid inKPC tumorbearinganimals, we analyzed cells from accumulated ascitic fluidfrom these animals. Our analysis showed �2% CD133þ

cells were present in ascitic fluid from 3 animals tested.Similarly, cells from a liver metastasis nodule from a pan-creatic tumor bearing KPC animal also showed �2%CD133þ cells. The relative abundance of CD133þ cells

from primary tumor, malignant ascites, or metastaticnodules is summarized in Table 1.

Our results showed that CD133þ population was higher(6%–9%) in the primary tumor whereas it was �2% to 3%in malignant ascitic cells or metastasizing cells. We nextlooked for expression of genes involved in development ofthe pancreas and known to be upregulated in pancreaticTICs. KPC cells showed several hundred fold overexpressionof CD133, Sox2, Nanog, Aldh1, CXCR4, SHH, Gli, andNotch genes (Fig. 1D) compared with the normal pancre-atic ductal cells. These data indicated that KPC cells harbor apopulation of TICs characterized by the same markers aswas previously reported from the human tumor xenograftsand pancreatic cancer cell lines (2, 3).

CD133þ population in KPC cells includes ALDH1þ andCD24þ/CD44þ/ESAþ subpopulations

Because�6% to 9% of cells derived from the KPC tumorwere found to be CD133þ, we used magnetic cell sorting(MACS) with anti-mouse CD133 (AC133) coupledmicrobeads to isolate this population. The enrichment forCD133 was verified by flow-cytometry using CD133 anti-body (AC141) against a different epitope after purification.Approximately, 97% purity was achieved consistently(Fig. 2A) by this cell separation technique. Sorted cells werelabeled with CD24, CD44, and ESA antibody and assayedfor Aldh1 activity. It was observed that CD133þ populationfrom KPC001, KPC023 as well as cells isolated from KPC

Figure 1. KPC cells showexpression pancreatic TICmarkers. A, representative CD133expression; B, CD24þ/CD44þ/ESAþ expression; and C, Aldh1activity in KPC cells. D, fold changeinmRNA expression of a number ofTICmarkers inKPCcells comparedwith normal ductal cells.






primary tumors, included the CD24þCD44þESAþ popula-tion, whereas the CD133� did not (Fig. 2B and Supple-mentary Fig. S1A).It was also seen that CD133þ populationharbored cells with maximum Aldh1 activity whereas theCD133� population lacked any Aldh1 activity (Fig. 2C andSupplementary Fig. S1A). This indicated that both CD24þ

CD44þESAþ population and Aldh1þ population were sub-populations of CD133þ cells. This was further confirmedwith immunofluorescence, which showed that onlyCD133þ cells costained with Aldh1 (Fig. 2D).

Expression of CD133 in pancreatic cancer cell linescorrelate with their invasiveness

CD133 expression has been correlated with poor prog-nosis in hepatocellular carcinoma (33). To evaluate ifhigher levels of CD133þ cells correlated with increasedaggressiveness in pancreatic cancer cells, CD133 expres-sion in normal pancreatic ductal cells was compared withcancer cell lines (MIA-PaCa2, BxPC3, Capan1, S2013, andS2VP10, AsPC1), human patient tumor xenografts, andKPC tumor–derived cells. KPC001, KPC023 derived fromthe tumor had increased (6%–9%) expression of CD133compared with 1%–4% in all the other cells (Supplemen-tary Fig. S1B). No significant expression of CD133 wasseen in normal pancreatic ductal cells (0.01%) and 0.5%to 1% expression of CD133 was observed in the lessaggressive pancreatic cancer cells such as MIA-PaCa2. TheKPC001 cells (with 8% CD133) showed increased rate ofmigration (assayed as a measure of wound healing abil-ity) compared with the other pancreatic cancer cell lines(Supplementary Fig. S1C).Migrationwasmeasuredby ECISand the data were represented as changing impedence overtime. Because KPC cells showed increased invasiveness andbecause they had higher CD133 expressing cells, we studiedthe expression of genes involved in EMT in CD133þ andCD133� cells isolated from theKPCcell lines (bothKPC001and KPC023). Consistent with the functional assay, expres-sion of EMT-associated transcription factors such as SNAI1,

SLUG, and Twist were 10- to 80-fold higher in CD133þ cells(Supplementary Fig. S1D).

CD133þ cells show tumor-initiating property in asyngenic model at low cell numbers

Tumorigenicity of CD133þ population was next testedby injecting 100, 50, and 10 MACS sorted cells subcuta-neously in both flanks of C57BL/6 mice. Tumor volumewas measured weekly to monitor tumor growth (Fig. 3A).Tumors formed from 10 CD133þ cells in these micewithin 3 weeks of cell injection. Neither CD133� cellsnor unsorted cell (Fig. 3B) population formed tumors upto 2 months post-inoculation at these cell numbers.Unsorted KPC cells did not show tumor formation until20,000 cells were injected. Tumorigenicity of CD133þ

cells is summarized in Supplementary Table S1. To putour study in perspective, we isolated CD133þ cells from 3human tumor xenografts propagated in mice andimplanted these at low numbers in immunocompro-mised SCID mice. CD133þ cells showed tumor initiationat as low as 100 cells whereas CD133� cells did not evenat 1,000 cells (Supplementary Fig. S2A–S2D).

Because one of the hallmarks of cancer stem cells istheir ability to generate serially transplantable tumorsthat recapitulate the cellular and histologic structure ofthe parent tumor (3, 34), we injected 100 CD133þ cellssubcutaneously into the flanks of the C57BL/6 mice.Tumors formed in an average of 21 days. 1 mm3 tumorpieces were implanted into a new set of C57BL/6 miceand observed for a month once the parental tumorreached a volume of 700 to 800 mm3. Similar transplan-tation was done with the second-generation tumors, oncethey reached a size of 700 to 800 mm3 (SupplementaryFig. S3A and S3B). The tumors from all the 3 generationswere histologically identical as seen by H&E staining(Supplementary Fig. S3C), and showed identical prolif-eration pattern as seen by Ki67 staining (SupplementaryFig. S3D). This indicated that tumor-initiating CD133þ

Table 1. TIC markers cells in different pancreatic tumors and cell lines

Nature of sample CD133 (% positive) CD44/24/ESA (% positive) Aldh activity (net) (% positive)

KPC001 Cell line 8% (�1.8%) 4.3% (�1.6%) 2.2% (�1.1%)KPC023 Cell line 7.1% (�1.2%) 3.8% (�1.2%) 1.8% (�0.8%)KPC883 Tumor 6.9% 4.1% 0.9%KPC820 Tumor 8.2% 3.2% 1.1%KPC793 Tumor 7.8% 2.6% 0.8%AsKPC883 Ascitis 2.2% 0 0AsKPC820 Ascitis 2.8% 0 0AsKPC793 Ascitis 2.1% 0 0Liver001 Cell line 2.4% (�0.7%) 0.5% (�0.2%) 0.8 (�0.4%)hPDX1 Tumor xenograft 4.2% (�1.6%) 1.2% (�0.3%) 2.8% (�1.2%)hPDX2 Tumor xenograft 3.5% (�1.4%) 0.8% (�0.3%) 1.1% (�0.9%)hPDX3 Tumor xenograft 2.8% (�0.9%) 0.3% (�0.2%) 0.9% (�0.4%)hPDX4 Tumor (fresh) 4.5% 0.2% 0.8%

Banerjee et al.





cells had tumorigenic properties that were sustained withserial transplantation in animals.

CD133þ cell population promoted early tumorigenesisin an orthotopic mouse modelFive hundred CD133þ cells were implanted in the tail of

the pancreas in 20 mice and tumorigenesis was comparedwith 5,000 unsorted KPC cells. Groups of 5 to 7 mice weresacrificed at days 10, 20, and 40 after implantation andtumor development was assessed upon necropsy.Mice withCD133þ cells developed visible and palpable tumors byday 20 whereas the mice with 5,000 unsorted KPC cellsshowed a delayed tumor growth (Fig. 3C). By day 40 ofimplantation, tumors derived from CD133þ cells had an

average volume of �900 mm3, whereas tumors derivedfrom unsorted KPC cells had an average volume of�300mm3. The experimentwas terminated at day 40owingto morbidity in the CD133þ group. CD133þ mice alsoshowed increased ascites volume (0.978 � 0.234 mL)compared with unsorted KPC cells (0.098 � 0.05 mL),indicating increased aggressiveness of the CD133þ derivedtumor (Supplementary Fig. S3E). Histology of the pancreasin the 2 groups at day 20 and day 40 after tumor implan-tation showed early onset of tumor in CD133þ inoculatedanimals compared to the unsorted KPC cells (Fig. 3D).These data indicated that CD133þ cells are responsible forearly tumor formation resulting in increased accumulationof ascitic fluid.

Figure 2. CD133þ subset in KPCcells expressed TIC markersassociated with other cell types.A, enrichment of KPC CD133þ

population by MACS; B, CD44þ

CD24þESAþ population withinCD133� cells and CD133þ

population. C, Aldh1 activitywithin CD133� cells and CD133þ

population. D, coexpressionof CD133þ/Aldh1þ byimmunofluorescence.






CD133þ cells have higher expression of HSPs andantiapoptotic genes and show increased NF-kB activity

We next analyzed the expression of proinvasion andsurvival genes in the CD133þ population of KPC cells.Antiapoptotic and prosurvival genes Bcl-2 and Survivinwere 4- to 5-fold higher in the CD133þ population com-paredwith theCD133�population (Fig. 4A). Similarly 3- to4-fold higher expression of TIC-specific genes such asNanog (4-fold) and Sox2 (2-fold) were also observed inCD133þ population versus the CD133� population (Fig.4B). CD133þ cells also showed �4-fold increased expres-sion of CXCR4 compared with CD133� cells. Heat shockproteins are overexpressed in pancreatic cancer allowingincreased survival. HSP70, GRP78, HSP27, HSF1 werefound to be at least 3- to 4-fold higher in CD133þ cellscompared with CD133� cells (Fig. 4C). This increase inmRNA expression corresponded to an increase in proteinexpression of GRP78, HSP70, and Bcl-2 in CD133þ cellsversus CD133�, unsorted KPC or normal pancreatic ductalcells (Fig. 4E). BothGRP78 andHSP70 (Supplementary Fig.S4A–S4C) showed increased staining in immunohis-tochemistry CD133þ cell–derived tumor.

A gene expression profile of CD133þ cells as comparedwith CD133� cells was performed using an Illuminamicro-array on RNA isolated from these cells. One of the genes

overexpressed in CD133þ subset was IDH1, a metabolicenzyme that has been as a potential oncogene in a numberof cancers (35, 36). Increased expression of IDH1 inCD133þ was confirmed by quantitative real-time (qRT)-PCR (Supplementary Fig. S5).

BecauseNF-kB signalingpathway regulates survival, inva-sion, and apoptosis in tumor cells, we analyzed DNAbinding activity of CD133þ cells and compared it to theCD133� cells. CD133þ cells had increased (�2.5-fold) NF-kB DNA binding activity compared with CD133� cells(Fig. 4D).

Minnelide induced apoptosis and decreasedproliferation in CD133þ pancreatic cancer cells andcaused CD133þ cell–derived tumor regression

To study if CD133þ cells were resistant to other cytotoxicchemotherapeutic agents such as paclitaxel, 5FU or currentstandard of care gemcitabine, KPC cells were sorted forCD133 and both positive and negative cells were treatedwith different concentration of 5FU, paclitaxel, triptolide,and gemcitabine. 5FU and paclitaxel reduced viability ofCD133� cells but did not have any effect on CD133þ cells.Gemcitabine did not affect either positive or negative pop-ulation at a lower dose and reduced the viability of CD133�

cells at a higher concentration. Interestingly, triptolide was

Figure 3. CD133þ cells formed serially transplantable tumors in immune-competent mice. A, tumor progression with different numbers of CD133þ cells.B, representative pictures of C57BL6 mice showing tumor from 10 CD133þ cells, whereas CD133� and unsorted KPC did not form tumors with thisnumber. CD133þ cells showed early tumorigenesis in an orthotopic model: tumor progression in an orthotopic model of pancreatic cancer showedtumor formation inCD133þ cells as early as 20days as comparedwith unsorted cells that did not form tumors (C). H&E staining showing histology of tumors inan orthotopic model (D).

Banerjee et al.





seen to reduce viability of CD133þ (0.4þ/0.02) similar toCD133� cells (0.32 � 0.04; Fig. 5A).To test the effect of Minnelide on CD133þ-derived

tumors, CD133þ cells were implanted in flanks ofC57BL/6 mice. Treatment with therapeutic dose of Minne-lide (0.42 mg/kg body weight) was started once implanted

tumors reached a volume of 300 mm3. Tumor progressionas well as endpoint measurements of tumor volumes oftreated tumors showed�60% decrease in tumor volume inminnelide-treated tumors compared with untreated ones(Fig. 5B). Minnelide-treated tumors also showed decreasednumber of Ki-67 positive (�30/field; Supplementary

Figure 4. CD133 cells have higherexpression of prosurvival proteins.Fold change in mRNA expressionof (A) Bcl-2 and Survivin, (B)developmental markers, and (C)heat shock genes of CD133þ

compared with CD133� KPC cells.NF-kB activity in CD133þ andCD133� cells (D).Wild-type andmutant oligos were used asspecificity controls for NF-kBbinding assay.

Figure 5. KPC cells respond totriptolide. A, viability of CD133þ

and CD133� cells with triptolide,paclitaxel, 5FU, and gemcitabine.B, tumor progression afterMinnelide treatment comparedwith untreated tumor. Minnelidereduces TIC population in vivo.C, expression of CD133 in primaryKPC tumors and CD133þ

implanted tumors after treatmentwithMinnelide, (D)NF-kBactivity inMinnelide-treated and -untreatedtumors from KPC primary tumorsandCD133þcell implanted tumors.A total of 4 to 5 tumors wereanalyzed for each experiment.






Fig. S6A–S6C) indicating decreased proliferation andincreased TUNEL staining (�60/field; Supplementary Fig.S6D–S6F) indicating apoptosis.

To test the efficacy ofMinnelide inhumanpatient tumor–derived xenograft, we sorted CD133þ cells from a humantumor and generated subcutaneous tumors on the flanks ofNOD/SCID mice (n ¼ 12 tumors). Treatment with Minne-lide (0.42mg/kg bodyweight) was started when the tumorsreached �1,000 mm3 in volume. Tumor regression (871 �102 mm2) was apparent after 2 weeks of Minnelide treat-ment (Supplementary Fig. S6G).

Minnelide reduced tumor-initiating population andinhibited prosurvival genes

Minnelide reduced the expressionof theTICmarkers in theCD133þ cell–generated tumors as studiedbyflowcytometry.Minnelide-treated tumors were seen to express less CD133(�4%) comparedwith the untreated tumors (�8%; Fig. 5C).A similar decrease (1.2% � 0.32 compared with 3.5 � 1.2)was observed in the expression of CD24þ/CD44þ/ESAþ andin Aldh1 activity (0.3 � 0.02 in Minnelide compared with1.43 � 0.78 in untreated) in tumors treated with Minnelideas compared with control (data not shown).

To see if similar effects of this drug were obtained inprimary KPC tumor–bearingmice, we analyzed the primarytumors treated with minnelide. Our analysis showed thatminnelide treated animals has 3% to 4% CD133þ cellscompared with the untreated animals that had 6% to 9%CD133þ cells (Fig. 5C).

Minnelide also decreased the expression of genes involvedinHedgehog signaling (3%),Notch signaling, stemness suchas Nanog (19% of untreated), ALDH1 (2%), CXCR4 (23%)prosurvival genes such as HSP70 (26%) and GRP78 (16%)and antiapoptotic genes such as Bcl2 (8%) and Survivin(21%; Supplementary Fig. S6H and S6I) compared withsaline-treated animals. In additional, NF-kB activity wasdecreased in minnelide-treated tumors (�500 RLU/mg pro-tein) compared with untreated (2,670 RLU/mg protein; Fig.5D). Thus, minnelide seemed to downregulate pro-prolifer-ative pathways and decrease the CD133þ tumor-initiatingpopulation in pancreatic cancer cells.

Similarly, Minnelide also decreased expression of thegenes involved in the Hedgehog signaling (9% of untreat-ed), Notch signaling (18% of untreated), and stemnessgenes (21% to 24% of untreated) in KPC animals bearingprimary tumors. Consistent with our results obtained fromimplanted cells in C57BL/6 mice, minnelide also decreasedexpression of HSP70 (40% of untreated), GRP78 (32% ofuntreated), and other antiapoptotic genes such as Survivin(41% of untreated) and Bcl-2 (26% of untreated). Alongwith this, minnelide also downregulated NF-kB activity inthe primary tumors (876 RLU/mg protein) compared withuntreated tumor (3712RLU/mg protein; Fig. 5D).

DiscussionTICs are a population of quiescent cells within a tumor

that evade apoptosis and cell death induced by standard

chemotherapy, often resulting in tumor recurrence. As aresult, there is a need to develop therapy that can reducethis population and contribute to tumor regression. Inpancreatic cancer, TICs have been isolated using a numberof markers, including surface expression of CD133(2, 3, 5, 10, 32).

However, all of these studies have been performed ineither immunocompromised, athymic nude mice, or inSCIDmice. Although transplantation into these mice is thebest way of assessing tumorigenicity, such studies oftencannot address the extent to which these cells might bepositively or negatively regulated by the actual physiologyof animal. As development of solid tumors is a complexphenomenon involving intricate interaction with the hostsystem, use of a syngenic model is a closer representation toactual tumor development of PDAC from tumor initiatingcells. In this study, we report isolation and characterizationof CD133-expressing TIC from a cell line derived from theKPC genetic mouse model for pancreatic cancer in a syn-genic host.

As reported by other groups, genetically engineered KPCmice are the closest clinical representation of pancreaticadenocarcinoma (20). Our study revealed that 6% to 9% ofthe cells in primary pancreatic tumors from KPC mice aswell as in the cell lines isolated from the KPC tumors areCD133þ. The CD133þ population was also responsible forinitiation of a fresh tumor in both subcutaneous andorthotopic models (Fig. 3). The CD133þ cells showed earlytumorigenesis and increased invasiveness in both thesemodels. Particularly, in the orthotopic mouse model,CD133-derived tumors showed increased accumulation ofabdominal ascites compared with unsorted KPC cell–derived tumor-bearing animals (Supplementary Fig. S3E).The CD133þ cells derived from the KPC model formedtumors from as low as 10 cells, which reflected theirincreased tumorigenicity when compared with that ofCD133þ cells isolated from human tumors (that formedtumors from a minimum of 100 cells). In all these studies,the CD133� population did not form tumors in almosttwice the amount of time.

The CD133þ population in KPC cells also included asmaller subset of Aldhþ population, previously described asa TIC marker in SCID mice (Fig. 2C and D; ref. 8). Further-more, the CD133þ population also contained an indepen-dent subset of CD24þ/CD44þ/ESAþ cells, shown previous-ly in human pancreatic cancer xenografts as a population ofTICs (Fig. 2B; ref. 2).

Although pancreatic TICs have been well characterized,there has been limited study on the dysregulation ofmolecular pathways in these cell populations. Biologicalfunction of CD133 is currently unclear. Our studyrevealed that in addition to an expected increase inexpression of Bcl2 and Survivin genes, the CD133þ pop-ulation had increased expression of heat shock proteins;HSP27, HSP70, and GRP78. Previous studies from ourgroup have shown that HSP70 is overexpressed in pan-creatic cancer cells and downregulation of HSP70 resultsin loss of viability in PDAC cells (24, 25).

Banerjee et al.





HSP70 thus functions as an inhibitor of apoptosis inpancreatic cancer. Similarly, HSP27has been shown tohavean increased expression in breast cancer stem cells (37).GRP78, aheat shockprotein family that is a gatekeeper of ERstress in eukaryotic cells, has been found to be overex-pressed in PDAC and involved in its cell survival. GRP78was found to be crucial for embryonic stem cell precursorsurvival and also highly expressed in hematopoietic stemcells (38).In addition, GRP78 has been shown to be upregulated in

TICs in other cancers (39, 40). Our studies show anincreased expression of HSP27, HSP70, and GRP78 inCD133þ cells isolated from the tumor, as well as in immu-nohistochemical studies on tumors derived from these cells(Supplementary Fig. S4A–S4C). This may be of significantclinical relevance as therapy targeted toward these proteinscan be used for complete eradication of pancreatic TICs.Along with overexpression of heat shock proteins, the

prosurvival NF-kB pathway was also overexpressed inCD133þ KPC cells (Fig. 4D). The NF-kB pathway has beenreported to also regulate multiple phenomena in TICs fromother cancers (41). In the KPC tumor–derived pancreaticcancer cell line, increased expression of NF-kB in theCD133þTICs indicate the role of this pathway inpromotingtumorigenesis. Minnelide also reduced the expression ofgenes that define tumor initiating cells like Hedgehog andNotch signaling pathway genes in CD133þ-derived tumors(Supplementary Fig. S6H and S6I).As expression of CD133 in different cell lines correlated

with increased invasion, we studied the expression level ofEMT genes such as SNAI1, SLUG, Twist, and Vimentin.CD133þ cells showed �5- to 10-fold increased expressionof SLUG and Twist genes whereas almost 50- to 80-foldincreased expression of SNAI1 and Vimentin genes com-pared with CD133� cells (Supplementary Fig. S1D).Overexpression of the IDH1 gene was seen in the gene

expression microarray for CD133þ cells, as compared withCD133� (Supplementary Fig. S5). However, there is noreport of IDH1 overexpression in this disease. IDH1 is aNADPþ-dependent isocitrate dehydrogenase, which cata-lyzes the oxidative decarboxylation of isocitrate to a keto-glutarate. Overexpression of IDH1 in CD133þ cells ofpancreatic cancer may indicate increased metabolic activi-ties in TICs.It is now well-established standard chemotherapy targets

rapidly proliferating cells as a result of which the quiescentTICs evade the cytotoxic effect of the drug. Our studiesshowed that CD133þ cells were indeed very slow prolifer-ating compared with the CD133� cells (Supplementary Fig.S1E). Our lab has previously demonstrated that minnelidetreatment prevents tumor recurrence and causes tumorregression in a number ofmousemodels (26). In this study,minnelide was effective in decreasing CD133þ populationas well. Our in vitro study showed that the CD133þ pop-ulation responded equally to triptolide (the activecompound) as the CD133� population, although bothpopulations remained relatively resistant to gemcitabine,the current first line of therapy for pancreatic cancer. Our

study also showed that cytotoxic agents 5FU and paclitaxeldecreased the viability of CD133� cells whereas CD133þ

cells were unaffected (Fig. 5A).Consistent with our in vitro studies, a 50% to 60%

decrease tumor volumewithin theMinnelide-treated groupwas observed in our animal studies along with almost 50%decrease in the expression of stem cell marker CD133þ (Fig.5C). Similar regression was also observed on tumorsderived from CD133þ population of the human tumors(Supplementary Fig. S6G). Analysis of the CD133þ-derivedtumors showed a decrease in tumor volume and less pro-liferation in minnelide-treated animals and increased celldeath by the drug (Fig. 5A and Supplementary Fig. S6).Treatment withMinnelide also showed a similar decrease inexpression of prosurvival genes (HSP70, GRP78, Bcl-2,Survivin) in tumors (Supplementary Fig. S6H and S6I).NF-kB activity, which was upregulated in CD133þ popu-lation, was decreased in minnelide-treated tumors (Fig.5D). Effect of other compounds targeting TICs have beenstudied in pancreatic cancer (Supplementary Table S2).However, all of these previous studies have been done inimmunocompromised models. This study is the first reportshowing the effectiveness of minnelide in an immunocom-petent syngenic system.

ConclusionOur study on TIC in a syngenic animal system show

that cells derived from KPC tumors have a high percent-age of CD133þ cells forming tumors from very low cellnumbers. This study shows that minnelide regressesCD133þ-derived tumor volume and reduces the numberof TICs in tumor. It also downregulates all the prosurvivalproteins overexpressed in these cells. The observation thatminnelide can reduce the TIC population strengthens ourearlier observation that it can prevent recurrence of pan-creatic tumor. This study is the first report of pancreaticTIC characterization in an immunocompetent syngenicsystem as minnelide is being evaluated in a phase Iclinical trial.

Disclosure of Potential Conflicts of InterestR. Chugh has ownership interest in a patent. Selwyn Vickers has owner-

ship interest (including patents) in Minneamrita. A.K. Saluja has ownershipinterest (including patents) inMinneamrita therapeutics and is a consultant/advisory board member for Minneamrita Therapeutics. No potential con-flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Banerjee, V. Dudeja, S.M. Vickers, A.K. SalujaDevelopment of methodology: S. Banerjee, V. Sangwan, V. Dudeja, S.M.VickersAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S. Banerjee, A. Nomura, V. Sangwan, R. Chugh,A.K. SalujaAnalysis and interpretation of data (e.g., statistical analysis, biostatist\ics, computational analysis): S. Banerjee, A. Nomura, V. Sangwan,R. Chugh, V. Dudeja, S.M. Vickers, A.K. SalujaWriting, review, and/or revision of the manuscript: S. Banerjee,A. Nomura, V. Sangwan, R. Chugh, V. Dudeja, A.K. SalujaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V. Dudeja, S.M. Vickers, A.K. SalujaStudy supervision: A.K. Saluja






Grant SupportThis study was funded by NIH grants R01-CA170946 and CA124723 (to

A.K. Saluja).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 29, 2013; revised February 10, 2014; accepted February17, 2014; published OnlineFirst March 14, 2014.

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2014;20:2388-2399. Published OnlineFirst March 14, 2014.Clin Cancer Res Sulagna Banerjee, Alice Nomura, Veena Sangwan, et al. Pancreatic Cancer Respond to Minnelide

Tumor Initiating Cells in a Syngenic Murine Model of+CD133

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