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Highly tumorigenic lung cancer CD133 cellsdisplay stem-like
features and are sparedby cisplatin treatmentGiulia Bertolinia,1,
Luca Roza,1, Paola Peregob, Monica Tortoretob, Enrico Fontanellac,
Laura Gattib, Graziella Pratesib,Alessandra Fabbrid, Francesca
Andriania, Stella Tinellib, Elena Roze, Roberto Caserinia,
Salvatore Lo Vullof,Tiziana Camerinif, Luigi Marianif, Domenico
Deliac, Elisa Calabro`g, Ugo Pastorinog, and Gabriella Sozzia,2
aMolecular Cytogenetics Unit, bPreclinical Chemotherapy and
Pharmacology Unit, cCell Cycle Control Unit, Department of
Experimental Oncology,dDepartment of Pathology, fUnit of Medical
Statistics and Biometry, and gUnit of Thoracic Surgery, Fondazione
Istituto di Ricovero e Cura a CarattereScientifico, Istituto
Nazionale dei Tumori, 20133 Milan, Italy; and ePathology Unit, Casa
di Cura La Maddalena, 90136 Palermo, Italy
Edited by Carlo M. Croce, The Ohio State University, Columbus,
Ohio, and approved August 10, 2009 (received for review May 29,
2009)
The identification of lung tumor-initiating cells and
associatedmarkers may be useful for optimization of therapeutic
approachesand for predictive and prognostic information in lung
cancerpatients. CD133, a surface glycoprotein linked to
organ-specificstem cells, was described as a marker of
cancer-initiating cells indifferent tumor types. Here, we report
that a CD133, epithelial-specific antigen-positive (CD133ESA)
population is increased inprimary nonsmall cell lung cancer (NSCLC)
compared with normallung tissue and has higher tumorigenic
potential in SCID mice andexpression of genes involved in stemness,
adhesion, motility, anddrug efflux than the CD133 counterpart.
Cisplatin treatment oflung cancer cells in vitro resulted in
enrichment of CD133 fractionboth after acute cytotoxic exposure and
in cells with stable cispla-tin-resistant phenotype. Subpopulations
of CD133ABCG2 andCD133CXCR4 cells were spared by in vivo cisplatin
treatment oflung tumor xenografts established from primary tumors.
A ten-dency toward shorter progression-free survival was observed
inCD133 NSCLC patients treated with platinum-containing regi-mens.
Our results indicate that chemoresistant populations withhighly
tumorigenic and stem-like features are present in lungtumors. The
molecular features of these cells may provide therationale for more
specific therapeutic targeting and the definitionof predictive
factors in clinical management of this lethal disease.
ABC transporters cancer stem cells chemoresistance CXCR4
xenografts
Lung cancer is the leading cause of cancer deaths
worldwidebecause of its high incidence and mortality, with
5-yearsurvival estimates10% for nonsmall cell lung cancer
(NSCLC)(1). Refined investigation on the mechanisms of
tumorigenesisand chemoresistance of lung cancer is needed to
improvesurvival rate.Recently, the cancer stem cell (CSC) theory
has been pro-
posed to explain the tumor heterogeneity and the
carcinogenesisprocess (2, 3). According to this model, tumor can be
viewed asa result of abnormal organogenesis driven by CSCs, defined
asself-renewing tumor cells able to initiate and maintain the
tumorand to produce the heterogeneous lineages of cancer cells
thatcompose the tumor (4, 5). The existence of CSCs was first
provedin acute myeloid leukemia (6), and more recently in
glioblastoma(79), melanoma (10, 11), and epithelial cancers
(1218).CSCs were identified by using flow cytometry-based cell
sorting of tissue-specific surface markers or
sphere-formingassay in selective serum-free medium. CD133
(prominin-1), afive-transmembrane glycoprotein, was initially
described as amarker specific for CD34 human hematopoietic
progenitorcells (19, 20), normal stem cells of the neural (21, 22),
epithelial(23, 24), and endothelial lineages (25), and their
tumoral coun-terparts (1416, 26, 27). However, it is still a matter
of debate
whether CD133 cells truly represent the ultimate
tumorigenicpopulation, particularly in colon (28) and brain (29,
30) cancer.Long-term cultures of sphere-growing cells derived
from
human lung tumors were shown to be highly enriched for
CD133expression, able to self-renew, and able to be the only
tumori-genic population in vivo (18). A recent report further
demon-strated that Oct-4 expression plays a critical role in
maintenanceof stem-like properties in lung cancer CD133 cells
(31).CSCs may be inherently resistant to the cytotoxic effect
of
chemotherapy because of their low proliferation rate and
resis-tance mechanisms, such as the expression of multidrug
trans-porters of the ATP-binding cassette (ABC) superfamily.
ABCB5was found to be expressed on a distinct subset of
chemoresistantCD133 melanoma cells (32), and its selective
targeting causedtumor growth inhibition in xenograft models (11).
Expression ofABCG2 (BCRP1), a transporter involved in resistance to
mul-tiple drugs (33), was reported in normal lung
undifferentiatedcells (34) and lung cancer cell lines (35).By using
both in vitro systems and implemented in vivo models
of direct xenografts of human primary lung cancers in mice,
weprovide evidence that lung tumor CD133 cells are
highlytumorigenic, are endowed with stem-like features, and,
impor-tantly, are spared by cisplatin treatment.
ResultsIdentification of CD133 Cells in Lung Cancer. In an
attempt toidentify lung cancer-initiating cells, we analyzed 60
primarytissue samples derived from a consecutive series of lung
cancerpatients (Table S1). By using the surface marker CD133
aloneor performing double staining with the epithelial-specific
anti-gen (ESA) marker to exclude potential contamination by
he-matopoietic and endothelial progenitors, we noted a very
similarfrequency of CD133 and CD133ESA populations in all
casesexcept one (LT23; Table S1), indicating that most CD133
cellswere of epithelial origin.We demonstrated the presence of a
rare(mean, 1%) CD133ESA population in normal lung tissuesof the
patients, whereas tumor samples consistently showed anoticeable
(mean, 5%; P 0.0004) CD133ESA population
Author contributions: G.B., L.R., P.P., L.G., G.P., F.A., U.P.,
and G.S. designed research; G.B.,M.T., E.F., L.G., A.F., S.T.,
E.R., R.C., S.L.V., T.C., L.M., and E.C. performed research; E.C.
andU.P. contributednewreagents/analytic tools; G.B., L.R., P.P.,
L.G., G.P., F.A., S.L.V., T.C., L.M.,D.D., and G.S. analyzed data;
and G.B., L.R., and G.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1G.B. and L.R. contributed equally to this work.
2To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at
www.pnas.org/cgi/content/full/0905653106/DCSupplemental.
www.pnas.orgcgidoi10.1073pnas.0905653106 PNAS September 22, 2009
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(Fig. S1). FACS analysis showed the presence of a
variablefraction of CD133ESA cell populations in 47 of 56
(83.9%)tumor samples, varying from 0.02% to a maximum of
35%.However, 60% of the cases showed small amounts (2%) ofCD133
cells. Further investigation of CD133 expression
byimmunohistochemistry (IHC) on paraffin-embedded tumor sec-tions
confirmed the existence of CD133 cells and identified 32of 58
(55.2%) positive cases (Fig. 1 and Table S1). Positivity oftumor
cells was defined as membranous staining or staining ofmembrane and
cytoplasm, and the intensity was always specificand strong. Because
the immunoreactivity was heterogeneous,no cutoff was applied, and
only cases with no CD133 immuno-reactivity were scored as negative.
IHC generally confirmed theresults obtained with FACS analysis;
however, FACS was moresensitive in detecting positive cases with a
very small percentageof CD133 cells. Indeed, by categorizing FACS
results into fourclasses (negative or positive, with 2% and 5%
positivity cutoffs),the frequency of positive samples at IHC
gradually increasedfrom 11% to 92% (test for trend P 0.0005). When
data wereanalyzed in terms of discrimination between negative and
pos-itive IHC results on the basis of FACS classes, the areas
underthe receiver-operating characteristic curve (nonparametric
esti-mate) were 0.764 (P 0.0001; or 0.761, P 0.0001, by usingFACS
score for computation as a continuous variable), valuebetween 1
(perfect discrimination) and 0.5 (lack of discrimina-tion).
Cross-tabulation of CD133 expression data using IHCstaining and
clinicopathological features indicated that low-grade (G1G2) tumors
(P 0.0356) and adenocarcinomahistology (P 0.0441) were more
frequently represented amongCD133-positive cases (Table S2).
Follow-up information was toolimited (median follow-up duration, 14
months; eight recordedtumor progressions and two tumor deaths) for
investigation ofCD133 expression prognostic value.
Establishment of Xenograft Models of Lung Primary Tumors.
Todevelop novel preclinical models, we successfully grew 10
xeno-
grafts in nude mice starting from 29 human lung primary
tumors.These xenografts were serially maintained in vivo as tumor
linesthat represented a continuous source of tumor tissue for
subse-quent functional studies. IHC for CD133 and for a panel of
lungcancer antigens showed high similarity between parental
tumorsand mouse xenografts (Fig. 2A and Fig. S2) and a similar
fractionof CD133ESA cells by FACS (Fig. 2B) that was maintainedover
time in serial transplantations. No association was observedbetween
the levels of CD133 expression in the original primarytumor and the
ability to grow and propagate xenografts in mice.
Tumorigenic in Vivo Potential of Isolated CD133 Cells. Because
thedefinition of cancer-initiating cells relies mainly on
functionalproperties, like the ability to sustain tumor growth
recapitulatingthe original cellular heterogeneity, we investigated
the tumori-genic ability of lung tumor-derived CD133 and CD133
cellpopulations.After depletion of mouse cells expressing the
H2K-MHCI
antigen by immunomagnetic separation (when necessary),CD133
cells were isolated by FACS or magnetic cell sorting(Miltenyi
Biotech) from four xenografts and one primary tumor.
Fig. 1. IHC analysis of CD133 expression in two representative
positive (LT74and LT60) and one negative (LT66) adenocarcinoma lung
tumor samples atlow and high magnifications. As controls (CTR) for
staining specificity, anormal lung sample negative for CD133
(Bottom Left) and a control antibody-stained tumor sample (Bottom
Right) are also shown.
Fig. 2. Tumor xenografts resemble the original primary tumor.
(A) IHC oflow-molecular weight cytokeratins (CK-LMW), cytokeratin 7
(CK7), surfactantprotein C (SP-C), transcription thyroid factor 1
(TTF-1), CD133, and MIB-1performed in LT45 parental tumor,
corresponding xenograft, and xenografttumor derived from injection
of CD133 cells. (B) FACS analysis ofCD133ESAcells in xenografts
compared with parental tumors.
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Considerable enrichment of CD133 cells was observed in
thepositive fraction (80% purity), as determined by FACS (Fig.S3A).
The same numbers of CD133, CD133, and unsortedcells mixed
withMatrigel were injected in the flank of SCIDmiceby using
limiting doses derived from preliminary experiments(104 down to
102). Palpable tumors were visible after a variabletime interval in
the majority of CD133-injected mice, whereasno tumor growth was
observed in CD133-injected mice (Fig.S3B and Table S3) except for
LT73 (see below). Tumorigenicityof the unsorted population was
variable for the different cases,and for the xenografts it appeared
to be related to the percentageof CD133 cells in the bulk
population; however, tumor forma-tion of enriched CD133 cells was
faster and resulted in in-creased tumor take compared with that
observed after injectionof CD133 or unsorted cells. Morphological
and IHC analysesrevealed that tumors derived from CD133 cells
faithfullyreproduced the original tumor (Fig. 2A). Similar results
wereobserved after s.c. injection of FACS-isolated CD133 cells
fromthe A549 lung adenocarcinoma cell line (Table S3). These
datasuggest that the CD133 cell population is enriched in
cellscapable of initiating lung cancer in SCID mice.To investigate
whether lung cancers originating from CD133
cells possess higher long-term tumorigenic potential
comparedwith the rarely observed tumors originating from the
CD133fraction, we performed serial transplantation assays in
SCIDmice of cells isolated from LT73 tumor xenografts
originallyderived from CD133 or CD133 cell injection (Fig. S3C).
Cellsderived from CD133 tumors were able to generate
tumorsmaintaining the original morphology and proportion of
CD133
cells in primary, secondary, and tertiary
transplantation,whereas cells from CD133 tumors lost tumorigenic
potentialduring serial transplantations (Fig. S3C). In principle,
the gen-eration of tumors from CD133 cells could be caused
bycontamination of the negative fraction by CD133 cells duringcell
sorting. However, it is likely that for this specific
highlyaggressive tumor the CD133 fraction also had some
tumor-initiating potential at low dose. Here, we demonstrated
thatthese CD133 cells could not maintain tumor growth in
serialtransplantations, indicating a lower and fading
tumorigenicability compared with CD133 cells.
Biological and Molecular Features of Human Lung
Cancer-InitiatingCells. Down-regulation of CD133 expression was
observed inshort-term adherent cultures (n 7) grown in
serum-supplemented medium compared with the original cell
suspen-sion (Fig. 3A).Normal and neoplastic stem-like cells from
neural and epi-
thelial organs can be expanded as sphere-like aggregates
inserum-free EGFbFGF-supplemented medium (9, 15, 36) thatfavors the
proliferation of undifferentiated cells. However, es-tablishment of
long-term sphere cultures from primary lungtumor samples was
unsuccessful. Nevertheless from adherentLT73 and A549 cell lines we
were able to consistently expandcells growing as floating spheres
(A549/s and LT73/s) thatallowed a variety of cellular and molecular
analyses.To verify the clonal origin of A549 and LT73 spheres,
we
performed a label-retaining assay using vital red and
greenfluorescent dyes (PKH26 and PKH67, as described in SI
Text).
Fig. 3. In vitro characterization of CD133 cells. (A) FACS
analysis for CD133 expression in freshly dissociated primary LT73
and corresponding adherent cellculture after four passages in
vitro. (B) PKH26 staining of LT73 and A549 spheres. (Top) Single
cells from dissociated spheres were separately labeledwith PKH26and
PKH67 red and green fluorescent dyes, and cultures were harvested
by mixing red and green labeled cells. (Middle and Bottom) A549
(Middle) and LT73(Bottom) spheres, derived frommixed cultures,
showed a separate green or red fluorescent staining, with a
decreasing gradient of fluorescence intensity withinthe spheres.
Microscopic images of spheres derived from labeled cells were
acquired at 20 magnification in bright field, with rhodamine filter
for PKH26fluorescence (Left) and with fluorescein filter for PKH67
fluorescence (Right). (C) Time-course analysis of CD133 cells
sorted from A549 spheres. FACS analysisof CD133 after sorting and
at the first and second population doubling (PD). (D) Real-time PCR
analysis of stemness gene expression in CD133 and CD133
fractions, sorted from LT28 xenograft (Left) and A549 spheres
(Right). Unsorted cells were used as calibrator for the relative
quantification of gene expression.(E) Real-time PCR analysis of
transporters of ABCC-B-G families in CD133 and CD133 cells sorted
from A549/s, and ABCC family in CD133 and CD133 cellssorted from
LT73/s and LT45 xenograft. Unsorted spheres or LT45 unsorted cells
were used as calibrators for the relative quantification of gene
expression.
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Spheres derived from mixed cultures of PKH26-labeled
andPKH67-labeled cells showed a distinct red or green
fluorescentstaining, indicating that A549/s and LT73/s did not
representstochastic cellular aggregates. Moreover, the decreasing
gradientof fluorescence intensity of labeled cells within the
spheresallowed us to evaluate a difference in the rate of cell
divisionsbecause proliferating cells easily dilute the dyes,
whereas slowlydividing quiescent cells retain most of the
fluorescence (37).A549/s and LT73/s spheres showed a decreasing
gradient of redor green fluorescence, indicating that cells within
spheres havea clonal origin and are endowed with a differential
proliferationrate (Fig. 3B). FACS analysis indicated that A549/s
and LT73/sdisplayed an enrichment of CD133 cell fraction to
1%,whereas it was only 0.2% in the standard adherent culture.
Atime-course FACS analysis of the sorted CD133 fractionshowed that
CD133 cells remained quiescent for 2 weeks,whereas the CD133
fraction and the unsorted A549/s popula-tion showed a constant and
sustained proliferative rate. Afterthis time CD133 cells started to
proliferate, and CD133 ex-pression decreased along with cell
divisions and returned to theoriginal fraction in 50 days (Fig.
3C). In contrast, the CD133population remained CD133 negative
during the observationperiod.The expression of genes involved in
stem cell pathways [i.e.,
Sonic hedgehog (SHH, Gli-1), Notch (Notch1, Notch2,
Hes-1),CD133, Oct4/3, and Nanog] was investigated by real-time
PCRin CD133 and CD133 populations sorted from A549/s andLT73/s and
lung tumor xenografts (LT56, LT28, LT45). Anincreased expression
level of genes involved in the maintenanceof stemness, markedly of
Oct4/3 and Nanog, and adhesion andmotility genes, like -6 integrin
and CXCR4, was consistentlyobserved in CD133 cells compared with
the CD133 counter-part (Fig. 3D; detailed procedures described in
SI Text).To better define the molecular features of
cancer-initiating
cells with respect to cellular defense mechanisms, we
analyzedthe mRNA levels of transporters of the ABC superfamily
inCD133 and CD133 cell populations sorted from A549/s,LT73/s, and
LT45 xenografts by using TaqMan Micro Fluidiccards (Applied
Biosystems). By using this approach, we foundthat 70% of the 50
human ABC transporters were expressedat detectable levels (Fig.
3E). Compared with the CD133fraction, CD133 cells exhibited an
increased expression ofdifferent components of the ABCC family that
are known to beinvolved in the multidrug-resistant phenotype (i.e.,
ABCC1).The level of several ABCB family members and ABCG2 mRNAwas
also enhanced. Overall, these results indicate that CD133cells are
slowly dividing cells, able to give rise to a CD133 cellpopulation,
thereby recapitulating the original cellular hetero-geneity, and
CD133 cells express high levels of embryonic stem
cell genes, motility genes, and ABC transporters, suggesting
thatCD133 cells possess the features of stem-like cancer cells.
In Vitro Resistance of CD133 Cells to Cisplatin. Because of
their lowproliferation rate and the activation of defense
mechanisms, thesmall population of CD133 lung cancer-initiating
cells may thusbe inherently resistant to the cytotoxic effect of
chemotherapy.We therefore assayed whether standard chemotherapy
treat-ments result in enrichment of cancer-initiating cells in
thesurviving cell fraction. In vitro studies were performed
inadherent A549 cells that show a low fraction (0.2%) of CD133cells
(detailed procedures described in SI Text). Exposure of cellsto a
cytotoxic concentration of cisplatin, corresponding to theIC80,
resulted in an 8-fold enrichment of CD133 cells (from0.2% 0.05% to
1.6% 0.5%; Fig. 4A).We then extended such an analysis to cells with
acquired
resistance to cisplatin. By using the A549/Pt cells, generated
bychronic exposure of A549 to increasing concentrations of
cis-platin and endowed with a stable drug-resistant phenotype
whengrown in the absence of selecting agent (degree of
resistance10), we found that in vitro development of drug
resistance wasassociated with increased expression of CD133.
Indeed, a 13-foldenrichment in CD133 population (from 0.2% 0.05%
to2.7% 0.87%) was consistently observed in A549/Pt cells grownas
adherent culture (Fig. 4B).
Increased ABCG2 and CXCR4 Expression of Human Lung
Cancer-Initiating Cells After in Vivo Cisplatin Treatment.To
demonstrate thein vivo relevance of these findings, mice carrying
six differentlung cancer xenografts were treated i.v. with
cisplatin, accordingto a weekly schedule.Cisplatin treatment
resulted in a variable tumor volume
inhibition (TVI) among the xenografts, ranging from 35% to83%,
overall confirming the poor responsiveness to chemother-apy
observed in the clinical setting of lung cancer (Table S4).
Toverify whether cisplatin treatment was able to enrich the
fractionof cancer-initiating cells in the residual tumors, at 7
days after thelast treatment and at the time of tumor regrowth,
mice werekilled. FACS analysis of cells isolated from the resected
tumorsshowed an enrichment of 7 and 35 times in the CD133
fractionin A549 and LT66 tumors, respectively, shortly after
chemother-apy (day 7 after last treatment), which reverted to the
originalvalues at the time of tumor regrowth (Table S4). In tumors
withan original large content of CD133 cells, such as LT45 andLT56
(50% and 15%, respectively), the fraction of CD133 cellstaken as a
whole did not change after cisplatin treatment, but asubpopulation
of CD133ABCG2 cells showed a remarkableenrichment (Fig. 4C). A
similar change in the CD133ABCG2fraction was also noticed in A549
and LT66 treated tumors
Fig. 4. CD133 cells survive cisplatin treatment. (A) FACS
analysis of CD133 expression in A549 parental cell line and A549
treated for 1 h with cisplatin (IC80).CD133 expression increased in
the A549 cell line 72 h after treatment. (B) FACS analysis of CD133
expression in stable cisplatin-resistant A549/Pt cells comparedwith
the A549 parental cell line. (C) Enrichment of CD133ABCG2 (Upper)
and CD133CXCR4 (Lower) populations in xenografts LT45 and LT56
after in vivocisplatin treatment. (D) CD133 expression in
advancedNSCLCpatients treatedwith platinum-containing regimens.
Progression-free survival curvewas calculatedwith the KaplanMeier
method and compared by using the log-rank test.
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(Table S4). Overall, our findings indicate that
chemotherapy,even when reducing tumor burden, may spare CD133
tumorcells endowed with drug-resistance properties.Double staining
with CD133/CXCR4 antibodies also revealed
a subpopulation of positive cells that increased after
chemother-apy in 2 out of 3 tumors analyzed, and a persistent
increase incells expressing CXCR4 was noticed in most treated
tumors andin the A549-treated cell line (Fig. 4C and Table S4).
Such resultssuggest the survival of CD133 chemoresistant clones
alsoendowed with high mobility and metastatic
features.Cisplatin-treated tumors also consistently expressed
higher
mRNA levels of Nanog, Oct4/3, Notch, -6 integrin, and CXCR4genes
than their corresponding untreated controls (Fig. S4A).The same was
observed for ABC transporters (Fig. S4B). Thesefindings indicate
that the CD133-chemoresistant fraction pref-erentially expresses
genes important for the proliferation, self-renewal, homing, and
drug resistance of stem cells.
CD133 Expression as a Marker of Chemotherapeutic Response
inPatients. Finally, to investigate the possible relationship
betweenCD133 status and response to platinum-containing regimens
inthe clinical setting, we retrospectively analyzed CD133
proteinexpression by IHC in formalin-fixed biopsies obtained
beforetreatment from 42 stage IIIB/IV NSCLC patients
receivingcarboplatin and gemcitabine (Table S5). CD133 expression
onthese archival biopsies was found in 10 (23.8%) of 42
patients.Partial response to platinum-based chemotherapy was
ob-
served in 12 of 32 (37.5%) CD133 patients and 4 of 10 (40%)CD133
patients. Patients with CD133 tumors had a tendencytoward a shorter
median progression-free survival than patientswith CD133 tumors
(Fig. 4D); however, the difference did notreach statistical
significance. Interestingly, 9 of 10 CD133patients relapsed within
9 months (the last patient at 20 months)compared with 22 of 32
(68.7%) relapses in the group of CD133patients, suggesting that
NSCLC tumors carrying higher levels ofCD133 cells might be more
likely to develop early tumorrecurrence after chemotherapy.
DiscussionThe identification of distinct phenotypic and
functional markersassociated with stem-like properties of lung
cancer tumor-initiating cells would be instrumental for developing
therapeutictargeting strategies in lung cancer patients.Here, we
report that a CD133/ESA population is increased
in primary NSCLC samples compared with normal lung tissue ina
prospectively collected series of lung cancer patients.
Flowcytometry and IHC analyses showed a quite good concordancefor
the detection of the CD133 population in surgical tumorsamples,
supporting the use of the latter technique for
screeningparaffin-embedded samples and also small archival
biopsies.CD133 cells isolated from primary lung tumors showed
higher tumorigenic potential than their CD133 counterpartsand
were able to reproduce the original tumor heterogeneity inSCID
mice. This phenotype was associated with an increasedexpression
level of genes involved in stemness, adhesion, andmotility. An
interesting finding was the enrichment for severalABC transporters
in CD133 cells compared with the CD133fraction. Thus, CD133 cells
possess features of cancer-initiatingstem-like cells, including
ABCG2 expression and, moreover,they expressed additional ABC
transporters that may contributeto determine a drug-resistant
phenotype.Previous studies on the putative chemoresistant features
of
lung cancer-initiating cells have approached this issue by
usingonly a limited number of in vitro models of sphere-growing
cellsisolated from tumors or established cell lines (18, 31). Our
invitro experiments with cisplatin treatment also resulted in
en-richment of CD133 cancer-initiating cells in the
survivingfraction both after acute cytotoxic exposure and in an
estab-
lished lung cancer cell line model of acquired stable
cisplatinresistance. However, in vitro models may not perfectly
mirrorwhat is observed in primary cancer cells, whereas in vivo
modelsare more conducive to the evaluation of the functional
propertiesof tumor-initiating cells, particularly in the assessment
of treat-ment responses, because they best reflect the tumor
heteroge-neity observed in patients and the interaction with the
micro-environment. In novel preclinical models, i.e., xenografts
fromprimary lung tumors of patients, serially maintained in vivo
astumor lines, we show that cisplatin treatment resulted in
remark-able enrichment of the CD133 cell fraction in the
residualtumors, which then reverted to the original values at time
oftumor regrowth. Interestingly, in tumors with a high content
ofCD133 cells, only subpopulations of CD133ABCG2 andCD133CXCR4
cells displayed a clear enrichment after treat-ment, therefore
suggesting that lung cancer CD133 cells com-prise populations of
cells with a similar phenotype but differentpotential. It is
noteworthy that if the subpopulation of tumor-initiating cells has
chemoresistant features, conventional mea-sures of treatment
efficacy (such as TVI) might only reflect howthe bulk tumor
responds to chemotherapeutic treatment, failingto provide relevant
information on long-lasting tumor-eradicating potential. In this
view, additional and differentendpoints should be considered to
evaluate innovative thera-peutic strategies.These chemoresistant
cell fractions preferentially expressed
genes relevant for proliferation, self-renewal,
differentiation,drug resistance, and homing of stem cells. Such
results indicatethat chemotherapy is effective in eliminating
drug-sensitiveCD133 differentiated tumor cells, whereas
cancer-initiatingcells with the above described phenotypes are
spared by thesetreatments and could be responsible for tumor
restoration afterchemotherapy cessation. Accordingly, by using
archived biopsieswe observed that CD133 protein expression tended
to correlatewith early recurrence in a series of advanced-stage
NSCLCpatients treated with platinum-containing regimens,
althoughmore data are needed to draw definitive conclusions.Our in
vivo findings showing coexpression of CD133/ABCG2
markers in the cisplatin-spared cell population are in
agreementwith the increase in the side population fraction observed
indrug-surviving cells of a H460 lung cancer cell line that can
bespecifically depleted by using the ABCG2 inhibitor fumitremor-gin
C (35). Our results suggest the interest of CD133/ABCG2expression
in relation to treatment effect in patients with lungcancer and a
rationale for a new generation of ABCG2 inhibitorsto be used in
combination therapy. Indeed, the use of a specificantibody against
ABCB5 transporter significantly inhibited tu-mor growth in treated
melanoma xenografts (11).An interesting finding was the elevated
expression of CXCR4
in CD133 cells compared with the CD133 fraction and aconsistent
enrichment of CD133CXCR4 population aftercisplatin treatment of
tumor xenografts. Our data are in line withthose reported in
pancreatic adenocarcinoma (38), suggestingthe existence of a
chemoresistant CD133CXCR4 subpopula-tion with highly tumorigenic
and metastatic properties also inlung cancer. Considering that
SDF-1, the specific ligand ofCXCR4, is strongly expressed in the
lung, targeting with specificinhibitors of the SDF1CXCR4 axis could
represent a thera-peutic option to eradicate the chemoresistant CSC
population.The demonstration of the presence in lung tumors of
highly
tumorigenic cells with stem-like properties and exhibiting
fea-tures of chemoresistant cells may be useful, together with the
useof carefully selected in vivo models, to evaluate novel
pathwaysto be targeted to increase the therapeutic response in the
clinicalsetting of this lethal disease.
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Materials and MethodsTumor and Normal Lung Tissue Dissociation
and Cell Cultures. Clinical specimenswere obtained from a
consecutive series of consenting patients according to theInternal
Review and the Ethics Boards of the Istituto Nazionale Tumori
ofMilan.
Single-cell suspensionswere obtained from lung tumor and normal
tissues,collected from clinical surgical specimens, and mouse tumor
xenografts aftermechanical and enzymatical disaggregation, as
described in SI Text. Foradherent cell cultures, cells were plated
in conventional medium, RPMI me-dium 1640 supplemented with 10%
heat-inactivated FCS (all from Lonza). Toobtain sphere cultures,
cells were plated at a density of 104 cells per milliliterin
serum-free medium DMEM/F12 (Lonza), supplemented with
commercialhormone mix B27 (Gibco Invitrogen), EGF (20 ng/mL;
PeproTech), bFGF (10ng/mL; PeproTech), and heparin (2 g/mL).
Floating sphere cultures wereexpanded by mechanical dissociation,
followed by replating of single cells incomplete fresh medium every
3 days.
For experiments using tumor xenografts, mouse cells were
depleted by usingthe Dynal cell-collection Biotin Binder kit
(Invitrogen) as detailed in SI Text.
Flow Cytometry Analysis. Single-cell suspensions were washed and
incubatedin staining solution containing 1% BSA and 2 mM EDTA with
the specificantibodies at appropriate dilutions. For CD133
staining, 106 cells were incu-batedwith 10L of
CD133/1-phycoerythrin antibody (50g/mL; AC133 clone;Miltenyi
Biotech) diluted in 80 L of staining solution and 20 L of
FcRblocking reagent (Miltenyi Biotech) for 10 min at 4 C.
Antibodies used werephycoerythrin (PE)-conjugated anti-CD133/1,
PE-conjugated anti-CD133/2,and FITC-conjugated anti CD326 (EpCAM),
all from Miltenyi Biotech; PE-Cy5-conjugated anti-CXCR4 (Becton
Dickinson); and FITC-conjugated anti-BCRP1(Chemicon).
Samples were acquired and analyzed by using a FACSCalibur
andCELLQuest Pro software (Becton Dickinson). For time-course
experiments,CD133 and CD133 fractions were plated in serum-free
medium after sort-ing, and the percentage of CD133 cells was
determined at each cell divisionby flow cytometry.
For the protocol of magnetic and cytofluorimetric CD133 cell
separation,refer to SI Text.
IHC. IHC was performed on formalin-fixed, paraffin embedded
samples.CD133 immunostaining was assessed on whole-tissue sections,
and CD133immunoreactivity was evaluated inside the neoplastic
epithelial component.All specimens were evaluated independently by
two observers (E.R. and G.S.),and interobserver agreement was
reached in all cases. Technical details ofimmunostaining are
reported in SI Text.
In Vivo Studies of Tumorigenicity.All experimentswere
carriedoutwith femaleCD-1 nude mice or SCID mice, 710 weeks old
(Charles River Laboratories).Miceweremaintained in laminar flow
rooms,with temperature and humidityconstant.Mice had free access to
food andwater. Experimentswere approvedby the Ethics Committee for
Animal Experimentation of the FondazioneIstituto di Ricovero e Cura
a Carattere Scientifico Istituto Nazionale Tumori,according to
institutional guidelines.
For establishment of xenograft models and tumor lines, regular
smallfragments were obtained by patient surgical specimens as
described (39).Fragments were implanted s.c. by trocar gauge in one
or both flanks of nudemice. Tumor lines were achieved by serial
s.c. passages of fragments fromgrowing tumors into healthy mice
(39).
To assess the tumorigenic potential of different cell
populations, aftersorting, viable 103 and 104 CD133, CD133, and
unsorted cells were sus-pended in Matrigel (BD Biosciences) at a
ratio of 1:1, and 200 L of cells wass.c. injected into the right
flank of SCIDmice. For serial transplantation assays,103 CD133 and
CD133 cells were injected s.c. in SCID mice, and derivedtumorswere
dissociated to single cells and serially reinjected inmice at 102
cellnumbers, generating secondary and tertiary tumors.
The in vivo chemotherapy studies to evaluate response to
cisplatin wereperformed as described in SI Text.
ACKNOWLEDGMENTS. We thank Dr. Giacomo Cossa for technical
assistanceand Prof.MalcolmAlison for helpful discussions. F.A. is a
fellowof FondazioneErmenegildo Zegna. This work was supported by
the Associazione ItalianaRicercaCancro (toG.S. andU.P.),
ItalianMinistry ofHealth (Ricerca Finalizzata)(to G.S.), Compagnia
di San Paolo di Torino, and European Community Inte-grated Project
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Supporting InformationBertolini et al. 10.1073/pnas.0905653106SI
TextLung Tumor Tissue Disaggregation. Solid tissues were finely
mincedby razorblade, washed in DMEM/F12 (Lonza), and then
incubatedwith Accumax 1 (Innovative Cell Technologies) for 1 h at
37 C.Single-cell suspension was obtained by filtering digested
tissuethrough a 70-m cell strainer and then gently loaded onto a
layerofHistopaque-1077 gradient (SigmaAldrich).After
centrifugationat 400 g for 30 min at room temperature, red blood
cells, deadcells, and debris were removed from the bottomof the
tube, and livenucleated cells were collected at the interface.
Magnetic and Cytofluorimetric Cell Separation. CD133
separation.CD133 and CD133 cells were isolated from tumor
cellsuspension or sphere cultures by FACS or magnetic bead
sortingusing the MACS system (Miltenyi Biotech). Magnetic
beadseparation was used for the cases with a high content of
CD133cells (10%).For magnetic separation, cells were incubated with
the mono-
clonal CD133/1 antibody labeled with MicroBeads
(MiltenyiBiotech) for 30 min at 4 C, and CD133 cells were selected
byusing MS columns (Miltenyi Biotech), which retained positivecells
linked by beads. For FACS separation, cells were stainedwith
CD133/1-PE antibody (AC133 clone; Miltenyi Biotech)diluted 1:10 in
blocking buffer solution for 10 min at 4 C andwere sorted with FACS
Vantage-SE cell sorter (Becton Dick-inson). In both instances, the
purity of the CD133 and CD133cell populations was evaluated by
standard flow cytometryanalysis using a PE-labeled antibody against
human CD133/2(clone 293C3; Miltenyi Biotech).Mouse cell depletion.
Xenograft tumor cell mixture was incubatedwith anti-mouse
biotinylated H-2Kd antibody (clone SF1-1.1; BDBioscience) at 1 g
per 106 cells for 10 min at 4 C and thenincubated with 25 g per l06
cells of anti-biotin Dynabeads for20 min at 4 C. Bound human cells
were collected by using amagnet (Dynal MPB; Invitrogen).
IHC. Paraffin sections (2 m thick) were dewaxed in xylene
andrehydrated with distilled water. CD133 antigen retrieval
wasperformed in citrate buffer solution (5 mM/L, pH 6).
Afterperoxidase inhibition with a solution of 0.3% H2O2 in
methylalcohol for 30 min, the slides were incubated with the
followingantibodies: CD133/1 (AC133; Miltenyi Biotech); PE-10,
TTF-1,and MIB-1 (Dako); CK-7 (NeoMarkers); and a pool for
low-molecular weight cytokeratins (35h11 from Dako and CAM5.2from
Becton Dickinson) and high-molecular weight cytokeratins(34e12 from
Dako and KS8.12 from Sigma). The primaryantibody detection was
performed by using Ultra Vision detec-tion system-HPR polymer
(Thermo Fisher Scientific) and dia-minobenzidine substrate
chromogen (Dako), followed by coun-terstaining with
hematoxylin.
Real-Time PCR. Total RNA extraction from cells and
DNasedigestion were carried out with the RNAeasy kit (Qiagen).cDNA
synthesis was performed with the High-Capacity cDNAReverse
Transcription Kit (Applied Biosystems) using 1 g oftotal RNA in
final volume of 20 L.The relative quantification of the
stemness-associated genes
mRNA (Notch1, Notch2, Hes1, SHH, Gli-1, CD133, Oct4/3,CXCR4,
ITGA6, Nanog) was performed by TaqMan technologyusing the ABI PRISM
7900 HT Sequence Detection System(Applied Biosystems) and
ready-to-use Assay-on-Demand (Ap-plied Biosystems).
Human HPRT was used as endogenous control for thenormalization
of different samples and relative quantization ofgene expression;
the data were analyzed by comparative Ctmethod (Ct). For analysis
of xenograft mRNA, we previouslyvalidated the specificity for human
transcripts of all used Taq-Man assays (Applied Biosystems) to
exclude a possible cross-reactivity with mouse transcripts.Analysis
of ABC transporter mRNA levels was performed
with TaqMan Low-Density Arrays: 2 L of cDNA was mixedwith 48 L
of nuclease-free water and 50 L of TaqManUniversal PCR Master Mix
and then loaded into a sample portof the Micro Fluidic Cards (Human
ABC Transporters Panel;Applied Biosystems). The thermal cycling
conditions of real-time PCR were 2 min at 50 C and 10 min at 95 C,
followed by40 cycles of 30 s at 97 C and 1 min at 59.7 C. Data of
RQ assayswere analyzed with RQ Manager 1.2 and Sequence
DetectionSystem (SDS) 2.2.2 software (Applied Biosystems).
PKH26 and PKH67 Labeling. A549 and LT73 lung cancer sphereswere
labeled with the PKH26 red fluorescent and PKH67 greenfluorescent
dyes (Sigma). Briefly, harvested cells were dissoci-ated to single
cells, washed in PBS, and resuspended in 1 mL ofthe dilution
buffer. The cell suspension was mixed with an equalvolume of the
labeling solution containing 4 106 M PKH26or PKH67 in the dilution
buffer and was incubated for 5 min atroom temperature. A total of 2
mL of FBS was added to labelingsolution to stop the reaction, and
cells were washed three timesin serum-free medium. The labeling
ratio was determined byflow cytometry.Red- and green-labeled single
cells were mixed and plated at
clonal density of 1 104 cells per mL in six-well culture
plates(Corning). A distinct red or green fluorescent staining
ofgrowing spheres was monitored through fluorescence micros-copy,
indicating a clonal origin from one PKH26- or PKH67-labeled
cell.
Cell Lines and Cell Sensitivity to Cisplatin. A549 and A549/Pt
cellswere maintained in RPMI medium 1640 plus 10% FCS.
Thecisplatin-resistant A549/Pt subline was generated from
parentalcells by continuous exposure to increasing concentrations
of cis-platin. Resistance was stable up to 6 months when cells were
grownin the absence of drug. Cellular sensitivity to drug was
evaluated byusing growth inhibition assays. For assessment of
modulation ofCD133 levels by cisplatin, exponentially growing A549
cells wereseeded in T75 flasks, and 24 h later they were exposed
for 1 h to acisplatin concentration corresponding to IC80. Cells
were harvested72 h after treatment for flow cytometric
analysis.
In Vivo Evaluation of Response to Cisplatin. Tumor fragments
wereimplanted at day 0, and tumor growth was followed by
biweeklymeasurement of tumor diameters with a Vernier caliper.
Tumorvolume (TV) was calculated according to the formula: TV(mm3)
d2D/2, where d andD are the shortest and the longestdiameters,
respectively. Cisplatin (Teva Pharma) was adminis-tered i.v. (10
mL/kg) at its optimal dose according to a scheduleof every seventh
day for three times (5 mg/kg per injection),starting when tumors
were palpable (50 mm3). The efficacy ofdrug treatment was assessed
as TV inhibition percentage(TVI%) in treated over control mice,
calculated as: 100 (meanTV-treated mice/mean TV control mice 100).
For tumorremoval, mice were killed by cervical dislocation under
lightanesthesia.
Bertolini et al. www.pnas.org/cgi/content/short/0905653106 1 of
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0%1%
0.1%1%
0.4% 1%
12%
0.2%
6%
1.5%2% 0.2%
0.5%
30%
3%
0.2%1%
0.5%0%
27%
0%
10%
20%
30%
17 la
mro
N
17 r
om
uT
27 la
mro
N
27 r
om
uT
37 la
mro
N
37 r
om
uT
57 la
mro
N
57 r
om
uT
67 la
mro
N
67 r
om
uT
77 la
mro
N
77 r
om
uT
87 la
mro
N87
ro
muT
08 la
mro
N
08 r
om
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18 la
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N
18 r
om
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38 la
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38 r
om
uT
/331DC
%+
ASEsllec
331DC
-EP
Primary tumor
30%
6%
ESA-FITC
0.5%
0.2%
Normal lungB
LT76
LT80
A
Fig. S1. Identification of CD133 cells in lung tumors and normal
lung tissues. (A) Percentage of CD133ESA cells in 10 primary tumors
and correspondingnormal lung tissues. (B) FACS analysis of CD133ESA
cells in two representative freshly dissociated lung tumors and
corresponding normal tissue.
Bertolini et al. www.pnas.org/cgi/content/short/0905653106 2 of
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Parental Tumor Xenograft Negative Ctr
H&E
20X 20X
CK7
TTF-1
SP-C
MIB-1
CD133
20X 20X 20X
20X 20X 20X
20X 20X 60X
20X 20X 20X
60X 20X 60X
CK-LMW
60X 60X 20X
Fig. S2. IHC for low-molecular weight CKs (CK-LMW), cytokeratin
7 (CK7), surfactant protein C (SP-C), transcription thyroid factor
1 (TTF-1), CD133, andMIB-1performed in a large cell carcinoma
parental tumor (LT56) and corresponding xenograft. Control
antibody-stained tumor sample is also shown (Negative Ctr).
Bertolini et al. www.pnas.org/cgi/content/short/0905653106 3 of
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Fig. S3. (A) FACS analysis of CD133 expression before and after
sorting of CD133 and CD133 fractions. CD133 cells were isolated
from freshly dissociatedlung tumors by FACS (LT28;Upper) or
bymagnetic beads (LT45; Lower). (B) In vivo tumorigenicity of CD133
and CD133 cells. Tumorigenic potential of CD133,CD133, and unsorted
cells, purified from two xenografts (LT56 and LT28) and one primary
tumor (LT45), after s.c. injection in immunocompromised mice.
Thetumor take for each subpopulation of injected cells is
indicated. (C) In vivo serial transplantation assay. A total of 103
CD133 and CD133 cells, purified fromLT73 xenograft, were injected
s.c. into SCID mice. Derived tumor xenografts were dissociated to
single-cell suspension and then serially reinjected in mice
(102
cells), generating secondary and then tertiary tumors. Tumor
growth curves of primary and tertiary tumors are shown.
Bertolini et al. www.pnas.org/cgi/content/short/0905653106 4 of
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Fig. S4. (A) Real-time PCR analysis of stemness genes expression
in cisplatin-treated A549, LT66, LT73, and LT56 xenografts. Control
untreated xenografts wereused as calibrator for the relative
quantification of gene expression. (B) Real-time PCR analysis of
ABC transporters in cisplatin-treated LT73 and LT56 xenograftsusing
TaqMan Micro Fluidic cards. Control untreated xenografts were used
as calibrator for the relative quantification of gene
expression.
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Table S1. Clinicopathological characteristics and CD133/ESA
expression in NSCLC patients
ID Histology/grading Stage FACS analysis CD133, % FACS analysis
ESA, % FACS analysis CD133/ESA, % IHC analysis CD133
LT22 ADC G3 IB 23 80 20 PositiveLT23 LC G3 IIB 31 57 15
PositiveLT24 SCC G2 IIB 1.2 12 1 PositiveLT25 SCC G3 IIIA 0 10 0
NegativeLT26 ADC G3 IIB 0.3 10 0 NegativeLT27 ADC G2 IA 0 80 0
NegativeLT28 ADC G3 IIB 2 5 / NegativeLT29 ADC G3 IB 0 70 0
NegativeLT30 ADC G2 IB 33 89 29 PositiveLT32 SCC G2 IB 1 1.5 0.3
NegativeLT33 SCC G2 IA / / / NegativeLT34 SCC G2 IB 8 26 7
PositiveLT35 SCC G3 IA 0 42 0 PositiveLT36 ADC G3 IA 3 80 3
NegativeLT37 LC G3 IB / / / PositiveLT38 ADC G2 IA 2 13 0.7
PositiveLT39 ADC G3 IA 0.7 33 0.7 PositiveLT40 ADC G2 IIIB 0.9 70 /
PositiveLT42 ADC G3 IB 0 8 0 NegativeLT43 ADC G3 IV 2 12 2
PositiveLT44 SCC G3 IA 2 13 / NegativeLT45 ADC G3 IIIA 36 56 35
PositiveLT46 ADC G3 IIIA 0.5 88 0.5 /LT47 ADC G3 IIIA 2 50 2
NegativeLT48 ADC G3 IB 0.3 63 0.3 NegativeLT49 ADC G3 IIIA 3 48 3
PositiveLT50 ADC G3 IIA 2.5 35 2.5 PositiveLT51 ADC G2 IB 0 90 0
NegativeLT52 SCC G3 IA 6 43 5 PositiveLT53 SCC G3 IA 0.7 50 0.7
NegativeLT54 ADC G3 IIIB 1 78 0.7 PositiveLT55 ADC G1 IB 1.5 75 1.5
NegativeLT56 LC G3 IB 15 87 15 PositiveLT57 ADC G2 IIIA 2 57 0.7
PositiveLT58 ADC G3 IA 1 62 1 PositiveLT59 LC G3 IIIA 2 13 2
PositiveLT60 ADC G3 IA / / / PositiveLT62 SCC G3 IV 1.2 14 0.5
/LT63 ADC G2 IA / / / PositiveLT64 ADC G3 IA 5 50 5 NegativeLT65
ADCG3 IIIB 1 46 0.5 NegativeLT66 ADC G3 IIIA 0.02 72 0.02
NegativeLT67 ADC G3 IIIA 0 70 0 NegativeLT68 SCC G2 IB 3 5 0.6
PositiveLT69 ADC G3 IIIA 1.5 84 / NegativeLT70 SCC G3 IA 0 3 0
NegativeLT71 SCC G2 IA 1.3 6 1 NegativeLT72 ADC G2 IIIA 1 60 1
PositiveLT73 ADC G2 IB 27 93 27 PositiveLT74 ADC G2 IA 2 12 2
PositiveLT75 ADC G2 IA 12 40 12 PositiveLT76 ADC G2 IA 7.5 45 6
PositiveLT77 SCC G3 IIB 2 40 2 NegativeLT78 ADC G2 IA 0.2 10 0.2
PositiveLT79 SCC G3 IIA 3 55 3 NegativeLT80 SCC G3 IB 30 58 30
PositiveLT81 ADC G2 IV 3 20 3 PositiveLT82 ADC G2 IA 5 70 5
PositiveLT83 SCC G3 IB 1 26 1 NegativeLT84 SCC G3 IB 1.8 30 1.6
Negative
ADC, adenocarcinoma; SCC, squamous cell carcinoma; LC, large
cell carcinoma. Slash (/) indicates material not available.
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Table S2. Correlation of CD133 expression (by IHC) and tumor
histology, stage, and grading in NSCLC patients
IHC score
Overall Negative Positive
n % n % n % P
Histology 0.0441ADC 37 63.8 15 57.7 22 68.8SCC 17 29.3 11 42.3 6
18.8LC 4 6.9 4 12.5
Tumor stage 0.7091Ia 20 34.5 8 30.8 12 37.5Ib 16 27.6 8 30.8 8
25.0II 7 12.1 4 15.4 3 9.4IIIa 10 17.2 5 19.2 5 15.6IIIbIV 5 8.6 1
3.8 4 12.5
Grading 0.0356G1G2 22 37.9 6 23.1 16 50.0G3 36 62.1 20 76.9 16
50.0
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Table S3. In vivo tumorigenicity of CD133 and CD133 cells
Case Cell dose Stage Histology CD133, % Source (separation
technique)
Tumor incidence
CD133 CD133 Unsorted
LT28 1 103 IIb ADC 0.2 Xenograft (FC) 2/2 0/4 0/4LT45 1 104 IIIa
ADC 35 Primary tumor (MB) 2/3 0/2 0/2LT56 1 103 Ib LC 18 Xenograft
(MB) 3/4 0/4 2/4LT66 1 103 IIIa ADC 0.02 Xenograft (FC) 4/4 1/4
2/4LT73 Ib ADC 30 Xenograft (FC)Primary 1 103 3/4 3/4 3/4Secondary
1 103 4/4 4/4 /
1 102 4/4 2/4 /Tertiary 1 103 2/2 1/2 /
1 102 4/4 1/4 /A549/s 1 103 0.8 Cell line (FC) 3/4 2/4 4/4
1 102 5/6 1/6 4/6A549 WT 1 103 0.1 Cell line / / 2/4
1 102 / / 0/6
FC, flow cytometry separation; MB, magnetic bead separation.
CD133 and CD133 cells were sorted by FACS or magnetic beads from
xenograft, primarytumors, and A549 spheres. Cells were injected
s.c. into the flanks of immunocompromised mice at scalar doses. For
tumor incidence, / indicates not done.
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Table S4. Effect of chemotherapy [cisplatin (cisPt)] on tumor
cell subpopulations
Fraction of cells expressing, %
Xenograft cisPt* Max TVI, % CD133 CXCR4 ABCG2 CD133/ABCG2
CD133/CXCR4
A549 Control 0.3 5 36 0.3 NDcisPt 35 2.15 26 38 1.5 NDRelapse
0.7 30 ND ND ND
LT66 Control 0.02 1.3 33 0 0cisPt 70 0.7 7.8 48 0.7 0.7Relapse
0.15 1.3 50 0.15 0.15
LT28 Control 0.2 9.5 5.4cisPt 83 0.2 19.8 24 ND NDRelapse 0.15
19.5 20
LT45 Control 50 4.5 19 14.5 NDcisPt 64 40 9.5 56 25 ND
LT73 Control 5.3 8.5 13.5 5 5.2cisPt 55 5.7 8 22 5.5 5Relapse 5
9 18 4.5 4.5
LT56 Control 18 7 46 6.5 3.5cisPt 43 17 10 68 15 7.5Relapse 15 7
45 7.5 3.5
ND, not determined.*Regimen cisPt: i.v. 5 mg/kg q7d 3 weeks.
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Table S5. CD133 expression as a marker of chemotherapeutic
response in advanced-stage NSCLC patients treated
withcarboplatin/gemcitabine: Patient characteristics
Clinicopathologic features No. of cases (%); n 42 No. of CD133
cases (IHC); n 10 P
Sex 0.6966Male 29 (69) 6Female 13 (31) 4
Age 0.304961 y 19 (45) 361 y 23 (55) 7
Smoking behavior 0.4164Never/nonsmokers 9 (21.4) 1Smokers 33
(78.6) 9Packs per year, median (range) 18.2 (9.145.6)
Stage at diagnosis 0.4164IV 9 (21.4) 1IV 33 (78.6) 9
Histology 0.1189Adenocarcinoma 25 (59.5) 8Squamous cell
carcinoma 14 (33.3) 1
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