effects of carbon ion beam on putative colon …...tumor and stem cell biology effects of carbon ion...

Tumor and Stem Cell Biology

Effects of Carbon Ion Beam on Putative Colon Cancer StemCells and Its Comparison with X-rays

Xing Cui1, Kazuhiko Oonishi2,4, Hirohiko Tsujii2, Takeshi Yasuda3, Yoshitaka Matsumoto1,Yoshiya Furusawa1, Makoto Akashi4, Tadashi Kamada2, and Ryuichi Okayasu1

AbstractAlthough carbon ion therapy facilities are expensive, the biological effects of carbon ion beam treatment may

be better against cancer (and cancer stem cells) than the effects of a photon beam. To investigate whether acarbon ion beam may have a biological advantage over X-rays by targeting cancer stem–like cells, human coloncancer cells were used in vitro and in vivo. The in vitro relative biological effectiveness (RBE) values of a carbonion beam relative to X-rays at the D10 values were from 1.63 to 1.74. Cancer stem–like CD133þ, CD44þ/ESAþ

cells had a greater ability for colony and spheroid formation, as well as in vivo tumorigenicity compared with theCD133�, CD44�/ESA� cells. FACS (fluorescence-activated cell sorting) data showed that cancer stem–like cellswere more highly enriched after irradiation with X-rays than carbon ion at doses that produced the same level ofbiological efficacy. A colony assay for cancer stem–like cells showed that RBE values calculated by the D10 levelswere from 2.05 to 2.28 for the carbon ion beam relative to X-rays. The in vivo xenotransplant assay showed anRBE of 3.05 to 3.25, calculated from the slope of the dose–response curve for tumor growth suppression. Carbonion irradiation with 15 Gy induced more severe xenograft tumor cell cavitation and fibrosis without significantenhancement of cells with putative cancer stem cell markers, CD133, ESA, and CD44, compared with 30 GyX-rays, and marker positive cells were significantly decreased following 30 Gy carbon ion irradiation. Takentogether, carbon ion beam therapy may have an advantage over photon beam therapy by improved targeting ofputative colon cancer stem–like cells. Cancer Res; 71(10); 3676–87. �2011 AACR.

Introduction

Colorectal cancer is currently the most common gastro-intestinal malignancy and remains the third most commoncancer and second leading cause of cancer-related deaths indeveloped countries (1). Although surgical resection has beenthe first choice for treatment of colorectal cancer, half ofpatients still suffer recurrence, presumably because of dis-seminated micrometastases present at the time of surgery (2).Radiation therapy is the most effective nonsurgical interven-tion for cancer treatment, but most cancers also invariablyrecur after radiation therapy. Therefore, determination of themechanisms of recurrence and radioresistance in thesetumors and development of powerful therapeutics could leadto advances in the treatment of cancer.

The Heavy-Ion Medical Accelerator in Chiba (HIMAC) isthe first heavy-ion accelerator specially dedicated to medi-cine in the world and has now become the world's leadingheavy-ion cancer treatment facility (3,4). Although heavy-ionbeam facilities are large scale and hugely expensive, severalnew heavy-ion therapy facilities, such as CNAO (Italy), PTCMarburg (Germany), SAGA HIMAT (Japan), ETOILE(France), Shanghai Particle Therapy Hospital (China), andMayo Clinic (USA), are under construction or in planningworldwide. A one-third smaller and cheaper carbon ionradiotherapy facility based on the highly advanced technol-ogy was designed and constructed in Gunma University inJapan, and clinical trial was successfully initiated inMarch 2010. High linear energy transfer (LET) particle ther-apy has various advantages because of the production ofspread out Bragg's peaks (SOBP), which cover tumors withbiologically equivalent dose distributions. Therefore, highLET heavy-ion therapy has several potential advantages overlow LET photon therapy such as increased relative biologicaleffect, reduced oxygen enhancement ratio, decreased cell-cycle–dependent radiosensitivity, and induced complex DNAdamage that is not easily repaired (5,6). Over the pastdecades, HIMAC has been successful in treating more than5,000 cases of various human cancers and achieved promis-ing clinical outcomes for many radioresistant tumor types,including recurrent colorectal cancer, hepatocellular carci-noma, chondroma, and sarcoma (7–10).

Authors' Affiliations: 1Heavy-Ion Radiobiology Research Group,2Research Center Hospital for Charged Particle Therapy, and 3Departmentof Radiation Emergency Medicine, Research Center for Radiation Emer-gency Medicine, National Institute of Radiological Sciences (NIRS);4Department of Medicine and Clinical Oncology, Graduate School ofMedicine, Chiba University, Chiba, Japan

Corresponding Author: Xing Cui, Heavy-Ion Radiobiology ResearchGroup, Research Center for Charged Particle Therapy, National Instituteof Radiological Sciences, 4-9-1 Anagawa Inage-ku, Chiba, Chiba 263-8555, Japan. Phone: 043-206-3231; Fax: 043-206-4149; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-10-2926

�2011 American Association for Cancer Research.

CancerResearch

Cancer Res; 71(10) May 15, 20113676

Research. on February 13, 2020. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 31, 2011; DOI: 10.1158/0008-5472.CAN-10-2926





http://cancerres.aacrjournals.org/



Recently, cancer stem cells have been identified in a grow-ing number of solid tumors, which are typically recognized byvirtue of the expression of cell surface markers; most of themare transmembrane glycoproteins, such as CD133, CD44, andEpCAM (ESA; refs. 11,12). Cancer stem cells have the ability togenerate tumors that recapitulate the original tumor whenxenotransplanted into animals, whereas the remaining non-cancer stem cell tumor bulk most often cannot (13–18). Thecancer stem cells that populated the original tumor may haveresistance to the treatments to repopulate the recurrenttumor even after the bulk of the tumor has been removedby resection or chemoradiation therapy (19–22). Therefore,the key point in curatively treating cancer is how to effectivelyeradicate those minor cancer stem cells in the bulk of thetumor (23–25). Because heavy-ion radiotherapy has potentialadvantages in treating many human radioresistant cancers,we hypothesize that heavy-ion irradiation may effectivelytarget these cancer stem–like cells. To the best of our knowl-edge, our study is the first to explore whether heavy-ionirradiation may have advantages over X-rays in targetinghuman colon cancer stem–like cells.

Materials and Methods

Cell linesThe colon adenocarcinoma cell lines HCT116 and SW480

were purchased from American Type Culture Collection. Thecells were cultured in Dulbecco's modified Eagle's medium(DMEM; Invitrogen) supplemented with 10% with heat-inac-tivated 5% (v/v) fetal calf serum (FCS; Beit-Ha’Emek), 100unit/mL penicillin, and 100 mg/mL streptomycin (Invitrogen)in a 37�C with 5% CO2 in air. The medium was changed everyother day.

Colony and spheroid formation assayA clonogenic survival assay was conducted as described

previously (6). In brief, the appropriate plating density wasdesigned to produce 20 to 40 surviving colonies in each 6-cmdish or T25 flask. After incubation for 14 days, the colonieswere fixed and stained with 0.3% methylene blue in ethanol,and colonies containing more than 50 cells were counted assurvivors. At least, 3 parallel samples were scored in 3 to 5repetitions conducted for each irradiation condition.For assays of clonogenicity and ability to grow as "tumor

spheres" in suspension, HCT116 and SW480 cells were sortedto obtain populations of CD133þ and CD133�, CD44þ/ESAþ

and CD44�/ESA�cells by BD FACSAria (Becton Dickinson).For the clonogenicity assay and spheroid assay, the sortedCD133þ and CD133�, CD44þ/ESAþ and CD44�/ESA� cellsubpopulations were then resuspended in a cell density of500 or 2,000 cells/mL and were plated triplicate in a 6-cmdish or 96-well plates precoated with a layer of 1% agaroseand left to grow for 1 to 2 weeks. To quantify the number ofcolonies and sphere formations as well as spheroid forma-tion rates, each positive or negative stem-like cell wasapplied to 12 wells, the rate of spheres per well calculated,and the data are presented as percentage of the wells thatcontained spheres.

In vivo tumorigenic assaysImmediately after sorting, aliquots of the particular cell

populations were counted and cell viability was determinedusing a conventional trypan blue test. The sorted cells werecentrifuged at 300 � g for 5 minutes at 4�C, suspended inserum-free medium (DMEM with 1% penicillin/streptomy-cin), then various numbers of CD133þ/CD133�, CD44þ/ESAþ,and CD44�/ESA� cells ranging from 1 � 103 to 2 � 105 cellsare injected subcutaneously into the hind legs of 6- to 8-week-old male NOD/SCID (severe combined immunodeficientmice)mice. The animals were sacrificed at the indicated timeintervals (4–10 weeks) when tumor nodules were identified ontheir body surfaces. Tissues were fixed in formaldehyde andexamined histologically. All experiments involving the use ofanimals were carried out in accordance with NIRS institu-tional animal welfare guidelines. NOD/SCID mice (CharlesRiver Laboratories) weremaintained under defined conditionsat the NIRS Animal Facility.

Tumor growth delay assaysBALB/cAJcl-nu/nu male mice (5-week-old) were purchased

from CLEA Japan, Inc. Mice were provided with water andfood ad libitum and were housed at 5 animals per cage. Allsurgical procedures and care administered to the animalswere in accordance with the NIRS Animal Care and UseCommittee. Tumors were established by subcutaneous inocu-lation of 8 � 105 HCT116 cells into the right leg of the mouse.Tumor growth was monitored every 3 days by measuring 2perpendicular diameters. Tumor volume was calculatedaccording to the formula: 0.52 � a � b2, where a and b arethe largest and smallest diameters, respectively. The tumorgrowth delay (TGD) of xenograft tumors after treatment withX-rays or carbon-ion was estimated at the tumor volume of500mm3. The relative biological effectiveness (RBE) of carbon-ions at the middle of a 6-cm SOBP relative to 200 keV X-rayswas calculated by KaleidaGraph software.

IrradiationCells or mice were irradiated with carbon-ion beams accel-

erated by the HIMAC at NIRS. The details concerning thebeam characteristics of the carbon-ion beams, biologicalirradiation procedures, and dosimetry have been describedelsewhere (3, 4). Briefly, the initial energy of the carbon-ionbeams was 290 MeV/n, 50KeV/mm, 6-cm, SOBP. The energy ofheavy-ion beams at the irradiation site was obtained bycomparing the calculated and measured depth–dose distribu-tion. As a reference, mice were also irradiated with conven-tional 200 kVp X-rays (Pantac HF-320S; Shimadzu Co.) at NIRS.Cells were irradiated with 2, 4, 6, or 8 Gy of X-rays or 1, 2, 4, or 6Gy carbon ions. Transplanted xenograft tumors were irra-diated with various doses of X-rays (15, 30, and 60 Gy) orcarbon-ions (5, 15, and 30 Gy).

FACS analysisFACS (fluorescence-activated cell sorting) analysis for the

cells or xenograft tumors irradiated with X-rays or carbon ionwas conducted with BD FACSAria according to the man-ufacturer's protocol (Becton Dickinson). For in vitro analysis,

Heavy-Ion Irradiation Disrupts Cancer Stem Cells

www.aacrjournals.org Cancer Res; 71(10) May 15, 2011 3677




the cells were prepared and labeled with conjugated anti-human CD133�PE (phycoerythrin; Miltenyi Biotec),CD44�FITC (Miltenyi Biotec), and ESA-PE (Miltenyi Biotec).Isotype-matched immunoglobulin served as control. Cellswere incubated for 20 minutes at each step and were washedwith 2% FCS/PBS between steps. The percentage of CD133þ,CD44þ, and ESAþ present was assessed after correction for thepercentage of cells reactive with an isotype control. For in vivoanalysis, tumors were minced with scissors under sterileconditions, rinsed with HBSS and incubated for 2 hours at37�C in serum-free DMEM supplemented with 200 units/mLcollagenase type II and type IV (Sigma-Aldrich), 120 mg/Lpenicillin, and 100 mg/mL streptomycin. Cells were furtherdisaggregated by pipetting and serial filtration through celldissociation sieves (size: 40 and 80 meshes; Sigma-Aldrich).Contaminating erythrocytes were lysed by incubation inammonium chloride. Single-cell suspensions were assessedwith BD FACSAria in the same way as in vitro cell analysis.

Gross morphology and histopathologyGross morphologic changes were followed up to 12 weeks

after a single fraction of X-ray or carbon-ion radiation. Atselected time points, tumors were excised and histopathologicexaminations were conducted. Xenograft tumors from differ-ent groups were fixed in 10% neutral formalin and processedin paraffin-embedded sections followed by sectioning (4 mm)onto slides. Sections were stained with hematoxylin and eosin(H&E) and assessed microscopically.

ImmunohistochemistryImmunohistochemical staining was conducted with the

Elite ABC Kit (Vector Laboratories) according to the man-ufacturer's protocol (26, 27). In brief, sections cut fromformalin-fixed, paraffin-embedded tissue blocks were depar-affinized and rehydrated through a graded series of ethanol andincubated in 0.3% hydrogen peroxidase in methanol to blockendogenous peroxidase action. For antigen retrieval, sectionswere placed in boiling 10 mmol/L citrate buffer (pH 6.0). Theslides were preincubated with normal horse serum (1:50 dilu-tion; Vector Laboratories) to diminish nonspecific binding ofthe secondary antibody and then incubated overnight at 4�Cwith anti-CD133 (AC133, human monoclonal; Miltenyi Biotec;1:200 dilution), anti-CD44 (mouse monoclonal; BD Transduc-tion Labs; 1:100 dilution), and anti-ESA (human monoclonal;Miltenyi Biotec; 1:200 dilution). Slides were then rinsed andincubated with universal secondary antibody containing anti-mouse/anti-goat IgG (Vectastain ABC Elite kit; Vector Labora-tories) for 30 minutes, developed with diaminobenzadine(Vectastain ABC Elite kit; Vector Laboratories) for 10 minutes,and counterstained with hematoxylin for 2 minutes. Ten fieldswere selected and expression was evaluated in 1,000 tumorcells with high power (200�)microscopy. As a negative control,the sections were stained without primary antibodies tomonitor the background staining level. Cytoplasmic and/ormembrane staining were considered to indicate specificCD133, CD44, and ESA immunoreactivity. Each slide wasassessed for the intensity of immunostaining, background,and percentage of cells expressing the target protein.

Western blottingWestern blot analyses were conducted as previously

described (26). Cells or tumor tissueswere lysed using the PierceNuclear and Cytoplasmic Extraction Reagent Kit (Pierce). Sam-ples were normalized for protein concentration using the PierceBCA protein assay; 50 mg of each cytoplasmic or nuclear extractsample was analyzed by SDS/5% PAGE and transferred to apolyvinylidene difluoride (PVDF) membrane. Membranes wereblocked with 2% ECL Advance Blocking Agent in TBS Tween 20for 1 hour and probed overnight with monoclonal antibodiesspecific for CD133, ESA, andCD44.b-Actinwas used as a loadingcontrol. Subsequently, membranes were incubated with rabbitanti-mouse or anti-rabbit horseradish peroxidase–conjugatedsecondary antibody (Sigma-Aldrich). Blots were visualized byECL Advanced Kit (GE Healthcare Life Sciences) and quanti-tated using ImageQuant LAS 4000 Mini Biomolecular Imager.Densitometric analysis was obtained with Fujifilm Multi GaugeSoftware (version 3.0).

Statistical analysisOne-way ANOVA and Bonferroni multiple comparison tests

were used when mean differences between the groups wereevaluated by StatView software (SAS Institute, Inc.). For allcomparisons, values of P < 0.05 were defined as significant.

Results

Survival fraction by carbon-ion versus X-ray irradiationThe HCT116 and SW480 cells were irradiated with carbon-

ion or X-rays up to 6 Gy, and their survival fraction wasmeasured from the colony formation. Figure 1 shows dose–response curves for cell killing effects on the 2 human coloncancer cell lines irradiated with X-rays or 50 keV/mm SOBPcarbon ion beams. The surviving fractions for the HCT116 andSW480 irradiated with X-rays and carbon ions decreasedexponentially with increasing doses. On the basis of thesesurvival curves, the RBE values calculated by the D10, which isdetermined as the dose (Gy) required to reduce the survivingfraction to 10%, relative to X-rays, is about 1.63 to 1.74 forcarbon-ion beams.

Determination of cancer stem–like cell properties ofCD133þ and CD44þ/ESAþ cells

Having isolated the CD133þ, CD44þ/ESAþ cells from theHCT116 and SW480 cells, we next determined their cancerstem–like cell properties. CD133þ, CD44þ/ESAþ cells havehigher clonal and spheroid formation capacities in vitro androbust tumorigenicity in xenograft model. When equal num-bers of 500 cells were plated in a dish, CD133þ, CD44þ/ESAþ

colorectal cancer cells from HCT116 formed 64� 10 and 87�6 clones, whereas CD133� or CD44�/ESA� cancer cells formedonly 20 � 6 and 22 � 3 clones (P < 0.01; Fig. 2A). These datashowed that CD133þ or CD44þ/ESAþ colorectal cancer cellshad much greater clonal formation capacities than that ofCD133� or CD44�/ESA� cancer cells. To investigate the abilityto form spheroid bodies, isolated CD133þ, CD44þ/ESAþ, andCD133�, CD44�/ESA� cells were cultured in 96-well platesprecoated with a layer of 1% agarose. After being in culture for

Cui et al.

Cancer Res; 71(10) May 15, 2011 Cancer Research3678




1 week, CD133þ, CD44þ/ESAþ aggregated and formed spher-oid bodies (Fig. 2B). The ability to form spheroid bodies inCD44þ/ESAþ was significantly higher than that in CD133�,CD44�/ESA� (P < 0.01; Fig. 2B). Figure 2C shows representa-tive positive cells for CD133, CD44, and ESA markers.

To determine the efficacy of tumor initiation from cells withor without CD133, CD44, and ESA markers, we carried outlimiting dilution experiments. A variable number of humancells, ranging from 1,000 to 2,000,000, were injected into NOD/SCID mice to test their xenotumor abilities. As few as 1000

Figure 1. Survival curves ofHCT116 and SW480 cells platedimmediately after X-ray or carbonion (C ion) irradiation. The graphsshow the mean and standard errorcalculated from 3 independentexperiments.

HCT116

Radiation dose (Gy)

Sur

viva

l fra

ctio

n

SW480

0.0001

0.001

0.01

0.1

1

0 1 2 3 4 5 6 7

Radiation dose (Gy)

Sur

viva

l fra

ctio

n

0.0001

0.001

0.01

0.1

1

0 1 2 3 4 5 6 7

X-ray

C ions

X-ray

C ions

0

5

10

15

20

25

30

35

40

45

CD133+ CD133–

Col

ony

num

bers

/500

cel

ls

Sph

ere

num

bers

/wel

l

Sph

ere

num

bers

/wel

l

Col

ony

num

bers

/500

cel

ls

0

20

40

60

80

100

120

CD44+ESA+ CD44+ESA+CD44–ESA– CD44–ESA– CD44+ESA+ CD44–ESA– CD133– CD133+ CD133– CD133+

Sph

ere

form

atio

n ra

te (

%)

Sph

ere

form

atio

n ra

te (

%)

**

0

2

4

6

8

10

12

14

16

18

20

**

0

10

20

30

40

50

60

70

80

90

100

02468

10121416182022

CD133–

CD133+A

B

C

D

CD133+

CD133– CD133+

*0

10

20

30

40

50

60

70

*

CD44–/ESA–

CD44–/ESA–

CD44+/ESA+

ESA+CD44+/ESA+ CD44+

Figure 2. Colony, spheroid, and tumor formation of cancer stem—like and noncancer stem—like cells delivered from HCT116 cells. Colony (A) andspheroid formation (B) of CD133þ, CD133�, CD44þ/ESAþ, and CD44�/ESA� cells after being in culture for 1 to 2 weeks. C, representative photographsof positive cancer stem—like HCT116 cells. D, tumorigenicity of CD133þ, CD133�, CD44þ/ESAþ, and CD44�/ESA� cells after subcutaneous injectioninto the hind legs of NOD/SCID mice. *, P < 0.01, compared with colony or sphere formed from CD133þ or CD44þ/ESAþ cells.






CD133þ, or CD44þ/ESAþ cells were sufficient to generate atumor within 6 to 10 weeks after implantation, whereas thenumber of CD133�, CD44�/ESA� cells that had a similarcapacity was more than 20,000 (Table 1). These data showedthat the tumorigenicity of CD133þ, CD44þ/ESAþ colorectalcancer cells was much higher than CD133� or CD44�/ESA�

cells (Fig. 2D and Table 1). Combining these results, our datasuggested that CD133þ, CD44þ/ESAþ cells isolated fromHCT116 and SW480 cells present the characteristics of cancerstem–like cells.

Changes in proportion of cancer stem–like cells in vitroby carbon-ion versus X-ray irradiation

In vitro FACS analyses showed that the percentage of cancerstem–like cells which were positive for CD133, CD44, and ESAwere more significantly increased after 48 or 72 hours X-raythan carbon ion irradiation. The percentage of CD133þ cells inunirradiated HCT116 cells was about 3%, and it was increasedmore than 3-fold after irradiation with 2 Gy X-rays and furtherincreased 5-fold after 4 Gy X-ray irradiation. In comparison,CD133þ cells were increased only 2-fold by 1 Gy carbon ionand 3-fold by 2 Gy carbon ion irradiation for which the dosesinduced equivalent effects by X-ray. The percentage of CD44þ

cells was increased by more than 1.5-fold after irradiation with2 Gy X-rays and further increased 2.5-fold after 4 Gy X-rayirradiation. In contrast, CD44þ cells were unchanged by 1Gycarbon ion and decreased 0.5-fold by 2 Gy carbon ion irradia-tion. Interestingly, the percentage of ESAþ cells did not changeby either 2 or 4 Gy but increased 2-fold by 6 Gy X-raysirradiation. In contrast, ESAþ cells were unchanged aftercarbon ion irradiation even by the dose up to 4 Gy(Fig. 3A). The proportion of CD133þ/CD44þ and ESAþ/CD44þ cells were enriched 2- to 3-fold by 2 or 4 Gy X-rays,whereas those of double positive cells were decreased orunchanged by 1 or 2 Gy carbon ion irradiation (Fig. 3A).

Clonogenic assays of cancer stem–like cells in vitro bycarbon-ion versus X-ray irradiation

Clonogenic assays were conducted to determine the differ-ent radiosensitivity of cancer stem–like cells between carbonion and X-ray irradiation. Figure 3B shows dose–responsecurves for cell killing effects on cancer stem–like CD133þ,

CD44þ/ESAþ cells and noncancer stem–like cells sorted fromHCT116 or SW480 cells. The results showed that the survivingfractions for cancer stem–like CD133þ, CD44þ/ESAþ cells aresignificantly higher than noncancer stem–like CD133�,CD44�/ESA� cells after exposure to either X-rays or carbonion beams, suggesting that cancer stem–like cells show resis-tance to both X-rays and carbon ionsWe have also determinedthe resistance of CD133þ, CD44þ/ESAþ cells to chemotherapy(data not shown). The surviving fractions for the cancer stem–like cells sorted from the 2 cell lines after irradiation with X-rays and carbon ions decreased exponentially with increasingdoses. On the basis of these survival curves, the RBE valuescalculated at the D10 level for cancer stem–like cells werecalculated to be about 2.05 to 2.28, whereas RBE values fornoncancer stem–like cells were about 1.22 to 1.44.

TGD and relative biological effects of carbon-ionrelative to X-ray irradiation

Transplanted xenograft tumors grow fast without anytreatment and the tumor volume became more than 400mm3 after being subcutaneously implanted into mice for 3weeks. Treatment with X-rays (30 Gy) effectively suppressedtumor growth and reduced the tumor size and volume about10%, but the tumor rapidly regrew after 4 weeks and todouble in volume after another 4 weeks (Fig. 4A). In contrast,treatment with carbon-ion (30 Gy) radiation increasedtumor size at the first week and then gradually decreased.The tumor was reduced to the same size as prior to radiationafter 4 weeks and actually became less than half in volumeafter 8 weeks, and finally disappeared after 12 weeks withoutany regrowth and relapse (Fig. 4A). To determine the tumorgrowth control possibility by carbon-ion radiation, the xeno-graft tumors were also treated with various doses. Carbon-ion irradiation with 15 Gy can suppress tumor growth with-out significant initial increase in tumor size and volume butregrew after 7 to 8 weeks. As expected, treatment with 5 Gycarbon-ion or 15 Gy X-ray failed to control tumor growthanymore; however, the tumor was completely regressedwithout regrowth with 60 Gy X-ray irradiation in the 12-week follow-up (data not shown). Xenotransplanted tumorcontrol possibility by carbon-ion and X-ray radiation atvarious doses is summarized in Table 2.

Table 1. Tumor formation ability of sorted HCT116 colorectal cancer cells using surface markers (numberof tumors formed/number of injections)

Groups 2 � 105 2 � 104 1 � 104 5 � 103 1 � 103

Unsorted 6/6 3/6 1/6 0/6 0/0CD133þ 0/0 6/6 5/6 3/5 3/5CD133� 0/0 2/6 0/6 0/5 0/5P 0.001 0.001 0.001 0.001CD44þ/ESAþ 0/0 5/5 5/5 4/5 3/5CD44�/ESA� 0/0 1/5 0/5 0/5 0/0P 0.001 0.001 0.001 0.001

NOTE: P < 0.01 compared with results from marker-negative cells.

Cui et al.





To calculate in vivo RBE values, the TGD of xenotrans-planted tumors after treatment with 15 or 30 Gy of X-rays and5 or 15 Gy carbon-ions were estimated. Due to the tumor bedeffect, the extent of TGD determined from tumor growthcurves is highly dependent on the end volume chosen. To

minimize the influence of the tumor bed effect on the growthdelay, we calculated by choosing a smaller size and essentiallyan earlier time for regrowth. The TGD was obtained accordingto the endpoint of the tumor regrown to a volume of about 500mm3. As shown in Table 3, TGD was 4 and 28 days for 15 and

CD133+

A

B

0 Gyisotypecontrol

isotypecontrol

isotypecontrol

3.2%

6.0%

6.9%

30

0.00010 1 2 3 4 5 6 7

0.001

0.01

0.1

HCT116-CD133

X ray cd133+Cion cd133+Xray cd133–Cion cd133–

1

0.00010 1 2 3 4 5 6 7

0.001

0.01

0.1

SW480-CD133

X ray cd133+Cion cd133+X ray cd133–Cion cd133–

1

0.00010 1 2 3 4 5 6 7

0.001

0.01

0.1

HCT116-CD44/ESA

X ray cd133+esa+Cion cd133+esa+Xray cd133–esa–Cion cd133–esa–

1

0.00010 1 2 3 4 5 6 7

0.001

0.01

0.1

SW480-CD44/ESA

Xray cd44+esa+Cion cd44+esa+Xray cd44–esa–Cion cd44–esa–

1

25

20

15

10

5

0non-IR 2 4

**

* *

**

* *

6

X-rays C-ion

8 1

P < 0.01

P < 0.01

P < 0.01

2 4 6 Gy Gy

CD

133+

cells

(%

)

Sur

viva

l fra

ctio

n

6

7

5

4

3

2

1

0non-IR 2 4

* *

**

* *

*

6

X-rays C-ion

8 1

P < 0.01

P < 0.01

P < 0.05

2 4 6 Gy

CD

133+

/CD

44+ce

lls (

%)

3

2.5

2

1.5

1

.5

0

non-IR 2 4

* *

**

* *

6

X-rays C-ion

8 1

P < 0.01

P < 0.01

2 4 6 Gy

ES

A+/C

D44

+ce

lls (

%)

30

35

40

25

20

15

10

5

0non-IR 2 4

*

* * *

**

6

X-rays C-ion

8 1

P < 0.01

P < 0.01

P < 0.01

2 4 6

CD

44+ce

lls (

%) 17.5

2022.5

1512.5

107.5

52.5

0non-IR 2 4

*

**

6

X-rays C-ion

8 1

P < 0.01

P < 0.01

2 4 6 Gy

ES

A+ce

lls (

%)

17.5%

23.0%

9.2% 6.0%

1.5%

0.6% 1.2% 0.7%

3.2% 0.4%

13.3%

6.1%

0 Gy

0 Gy

X-2 Gy

X-2 Gy

X-8 Gy

Cion-1 Gy

0 Gy

CD133-PE

ESA-PE

CD

44-F

ITC

X-2 Gy Cion-1 Gy

0 Gy X-2 Gy Cion-1 Gy

Cion-1 Gy

Cion-4 Gy

CD44+

ESA+

Non-IR

+ –

X-2 Gy Cion-1 Gy

+ – + –HCT116CD133

Non-IR

+ –

X-2 Gy Cion-1 Gy

+ – + –HCT116CD44/ESA

Radiation dose (Gy)

Figure 3. A, percentage changes of CD133þ, CD44þ, ESAþ, CD133þ/CD44þ, and CD44þ/ESAþ cells by FACS analysis 48 or 72 hours after irradiationwith X-rays or carbon ions in HCT116 cells. *, P < 0.01, compared with unirradiated cells. B, survival curves of cancer stem–like cells and noncancerstem–like cells sorted from HCT116 and SW480 cells after irradiation with X-rays or carbon ion. Representative photos of colony formation fromCD133þ, CD133�, CD44þ/ESAþ, and CD44�/ESA� cells after irradiation with X-ray or carbon ions are displayed. The graphs show the mean and standarderror calculated from 3 independent experiments.






Figure 4. A, HCT116 xenograft tumor growth control by 30 Gy X-ray or 15 and 30 Gy carbon-ion irradiation. B, HCT116 xenograft TGD after treatmentwith X-rays or carbon-ion irradiation. Gross morphologic changes (C) and histopathologic features (D) of HCT116 xenograft tumors at 4 weeks afterbeing treated with 30 Gy X-rays or 15 and 30 Gy carbon-ion irradiation. Arrows indicate microvessels in which red blood cells were seen. #, P < 0.05;*, P < 0.01 compared with that of control tumors.

Cui et al.





30 Gy of X-rays but 10 and 76 days for 5 and 15 Gy of carbon-ion beams, respectively (Fig. 4B). On the basis of this TGD, theRBE values of 50 keV/mm carbon ion at the middle of a 6-cmSOBP relative to X-ray were calculated as 3.05 to 3.25 accord-ing to the formula analyzed by KaleidaGraph software (Fig. 4Band Table 3).

Gross morphologic and histopathologic changes aftercarbon-ion versus X-ray irradiationFigure 4C illustrates gross tumor morphology before and

after treatment with carbon-ion (15, 30 Gy) or X-ray (30 Gy)irradiation. Tumor-supplying vessels were very clearly seen inthe unirradiated mice as well as in the X-ray irradiated mice,but markedly reduced in carbon-ion irradiated mice. It isfurther confirmed by microscopy observation that the micro-vessel counts were significantly reduced by carbon ion com-pared with X-ray irradiation (Fig. 4D). Histopathologicchanges of xenograft tumors after irradiation with X-rays orcarbon-ion for 4 weeks were examined by H&E staining.Histopathologic features showed that most of the tumor cellswere not disrupted by 15 Gy X-rays or 5 Gy carbon ionirradiation (data not shown). At the same level of biologicalefficacy, 15 Gy carbon-ion irradiation predominantly inducedcolon cancer cell cavitations, fibrosis and the duct-like archi-tecture was completely disrupted. In contrast, 30 Gy X-rayirradiation only partially destroyed colon cancer cells and theduct-like architecture still remained (Fig. 4D). It is clearlyshown that almost all of the tumor cells were destroyed afterirradiated with 30 Gy carbon-ions (Fig. 4D).

Expression of cancer stem–like cell marker CD133,CD44 and ESA after carbon-ion versus X-ray irradiation

The expression changes of cancer stem cell marker CD133,CD44, and ESA in the xenograft tumors at 4 weeks afterirradiation with 30 Gy X-rays or carbon-ion (15, 30 Gy) wereexamined by immunohistochemical staining. It was shownthat expression of both CD133 and CD44 was significantlysuppressed by 15 Gy carbon-ion irradiation compared withunirradiated tumors, except ESA protein, but all 3 putativecancer stem cell proteins were remarkably inhibited aftertreatment with 30 Gy carbon-ion irradiation. In contrast, 30Gy X-rays significantly increased expression of CD133, CD44,and ESA compared with those of unirradiated tumors(Fig. 5A). It was further confirmed by Western blotting ana-lyses that expression levels of all CD133, CD44, and ESAproteins was significantly enhanced by 30 Gy X-rays comparedwith 15 Gy carbon-ion irradiation and all 3 proteins werepredominantly reduced by 30 Gy carbon-ion irradiation(Fig. 5B).

Changes in proportion of cancer stem–like cells in vivoby carbon-ion versus X-ray irradiation

In vivo FACS analyses showed that the percentage of cancerstem–like cells which were positive for CD133, ESA wereincreased with 15 and/or 30 Gy but significantly decreasedwith 60 Gy X-rays irradiation after 1 month. In comparison,carbon-ion irradiation with 15 Gy did not change the percen-tage of these positive cancer stem–like cells, whereas irradia-tion with 30 Gy significantly reduced the percentage ofpositive cancer stem–like cells (Fig. 5C). It is not detectablein any of cancer stem–like cells by in vivo FACS analysis afterthe tumor irradiated with 30 Gy carbon ion or 60 Gy X-rays for2 to 3 months.

Discussion

We found that the in vitro RBE values, calculated by the D10relative to the X-rays, are about 1.63 to 1.74 for the 50-keV/mmSOBP carbon ion beam on HCT116 or SW480 cells in thisstudy. RBE values are known to be dependent on LET, and ourresults are almost consistent with previous reports usingHIMAC carbon-ion beams on various human cancer cell linessuch as lung, pancreas, and brain tumors, which reported 1.06to 1.33 for a 13-keV/mm-beam, 1.42 to 1.69 for a 50-keV/mm-beam, and 2.00 to 3.01 for a 77-keV/mm-beam (4, 28). Recentaccumulating experimental evidence suggests that cancerstem cell content may differ among tumors and that a higherproportion of cancer stem cells is correlated with higher

Table 2. Therapeutic efficacy of X-ray andcarbon-ion irradiation in HCT116 colon cancerxenograft (12-week follow-up)

Group Mice, n Completeresponse

Partialresponse

Unirradiated 5 – –

X-ray15Gy 5 0 530Gy 5 1 460Gy 5 5 0

Carbon-ion5Gy 5 0 515Gy 5 1 430Gy 5 5 0

Table 3. TGD and RBE values estimated in this study

X-ray Carbon ion RBE � SE (95% CI)

15 Gy 30 Gy 5 Gy 15 Gy

TGD � SE (95% CI), d4 � 1 (2–6) 28 � 3 (24–33) 8 � 2 (7–13) 76 � 4 (71–82) 3.15 � 0.16 (3.05–3.25)






Figure 5. A, immunohistochemistry for the expression changes of CD133, CD44, and ESA in the HCT116 xenograft tumors at 4 weeks aftertreated with carbon-ion or X-ray irradiation. Original magnification � 100. #, P < 0.05, *, P < 0.01, compared with unirradiated tumors. B, Westernblotting for the expression changes of CD133, CD44, and ESA in the HCT116 xenograft tumors at 4 weeks after being treated with carbon-ion orX-rays irradiation. b-Actin was used as loading control. C, percentage changes of CD133þ/CD44þ and CD44þ/ESAþ cancer stem–like cells by FACSanalysis at 4 weeks after X-ray or carbon ion irradiation in HCT116 xenograft tumors. #, P < 0.05; *, P < 0.01, compared with unirradiated tumors.

Cui et al.





radioresistance (22). To determine cancer stem properties ofCD133þ, CD44þ/ESAþ cells in HCT116 and SW480, we havesorted cancer stem–like positive and negative cells and con-firmed that CD133þ, CD44þ/ESAþ cells have a significantlyhigher possibility for colony and tumor sphere formation thanCD133�, CD44�/ESA� cells. In vivo tumorigenicity studyshowed that the tumorigenicity of CD133þ, CD44þ/ESAþ

colorectal cancer cells exactly much higher than CD133� orCD44�/ESA� cells.In the present study, FACS analyses showed that the

proportion of cancer stem–like cells which were positivefor CD133, ESA, and CD44 was more highly enriched afterX-ray compared with carbon ion irradiation, particularly, thepopulation of CD133þ, CD44þ cells was increased more than2-fold (2–4 Gy) by X-ray irradiation. In contrast, CD133þ andCD44þ cells were unchanged or decreased by carbon-ionirradiation (1–2 Gy). Interestingly, the proportion of ESAþ

cells was increased by 6 Gy X-ray irradiation but withoutchanges by carbon-ion irradiation even the dose was up to 4Gy. The proportion changes of double positive CD133þ/CD44þ and ESAþ/CD44þ cells by X-ray versus carbon-ionirradiation were showed same responses. These finding sug-gests that low LET X-ray irradiation may mainly kill the non–stem-like tumor cells, as a result the radioresistant cancerstem–like cell population was predominantly enriched. Incontrast, carbon-ion irradiation may concurrently kill bothnon–stem-like and stem-like tumor cells; consequently, thepopulation of cancer stem–like cells was only slightlyincreased or unchanged. To directly determine the radio-sensitivity of cancer stem–like cells between carbon ionand X-ray irradiation, a colony assay was conducted. Onthe basis of the dose–response curves for cell killing effecton cancer stem–like cells and noncancer stem–like cells afterirradiation with either X-rays or carbon ion beams, the cancerstem–like cells showed resistance to both X-rays and carbonions. The surviving fractions for the cancer stem–like cellsafter irradiation with X-rays or carbon ions decreased expo-nentially with increasing doses. The RBE values calculated atthe D10 level for cancer stem–like cells were about 2.05 to 2.28,suggesting that carbon ion beam has a promising potential todestroy cancer stem–like cells. In contrast, RBE values at theD10 level for noncancer stem–like cells were only 1.22 to 1.44,implying that there are no significant differences in the killingof noncancer stem–like cells between the carbon ion beamand X-ray irradiation. Altogether, these results can also par-tially explain why the proportion of cancer stem–like cellsafter irradiation with X-rays is more enriched than those ofcarbon-ion beams.In vivo study showed that 15 or 30 Gy carbon-ion irradia-

tion predominantly induced colon cancer cell cavitations,fibrosis, and completely disrupted the duct-like structure,whereas 30 Gy X-ray irradiation only partially disruptedcolon cancer cells and the duct-like structure still remainedwhen the xenograft tumors were histopathologically exam-ined after 4 weeks. The tumor-supplying vessels wereexactly reduced in carbon-ion irradiated mice comparedwith those of X-rays irradiated mice. This finding is inagreement with a previous report that heavy-ion irradiation

inhibits in vitro angiogenesis (29). Several previous studiesreported that the in vivo RBE values for high LET carbon-ionbeams ranged from 2.0 to 3.1 (30, 31). In the present study,the in vivo RBE was calculated as 3.05 to 3.25 from the slopeof the dose–response curve for tumor growth suppressionby carbon ions relative to X-rays, which is almost in linewith previous reports (30, 31). It is known that RBE is acomplex quantity, depending on many factors such asparticle type, dose per fraction, and LET, as well as onbiological factors like cell or tissue type and the selectedbiological endpoint. The extent of TGD determined fromtumor growth curves is highly dependent on the end volumechosen because of tumor bed effects (32). This may be thereason why the RBE values calculated in this study are alittle higher than other reports. In addition, because a lownumber of dose levels were applied in this study, furtherstudies are needed on in vivo RBE of carbon ions for localtumor control.

Recently, accumulating evidence showed that expressionof cancer stem–like cell markers such as CD133, CD44 wereclosely related with patient's clinical outcome and prog-nosis as well as chemoresistance (33–36). In the presentstudy, we surprisingly found that 15 Gy carbon-ion irradia-tion induced more severe tumor cell disruption withoutsignificant increment of putative cancer stem cell markersCD133, CD44, and ESA, whereas X-rays irradiation remark-ably increased these protein levels. All these cancer stem–like cell markers were remarkably inhibited after treatmentwith 30 Gy carbon-ion irradiation. Furthermore, in vivoFACS analyses showed that the proportion of cancerstem–like cells which were positive for CD133, ESA wereenriched by 15 and/or 30 Gy but significantly decreased by60 Gy X-ray irradiation after 4 weeks. In comparison,carbon-ion irradiation with 15 Gy did not change thepercentage of these positive cancer stem–like cells, whereasirradiation with 30 Gy significantly reduced the percentageof positive cancer stem–like cells. However, none of cancerstem–like cells were detectable by FACS analysis after thetumors were irradiated with 30 Gy carbon ions or 60 GyX-rays for 2 to 3 months. Presumably, the doses to com-pletely destroy the cancer stem cells are dependent on thetumor cell type as well as cancer stem cell population in thetumor bulk.

Altogether, according to the cancer stem cell model, ther-apeutic approaches that are not capable of eradicating thecancer stem cell subset are unlikely to be successful becausethey might be able to kill the majority of tumor cells andinduce regression of tumor lesions but fail to prevent diseaserelapse andmetastatic dissemination (23–25, 37, 38). Based onthis understanding, although carbon ion beam facilities arevery expensive, to achieve better outcomes as well as a betterquality of life for patients with some types of cancer, such asunresectable sacral chondromas (39) or locally recurrentrectal cancer (40), carbon-ion beam therapy may be worththe cost. In conclusion, our findings presented here are thefirst to show that carbon ion beam therapy may have advan-tages over photon beam therapy in targeting putative coloncancer stem–like cells.






Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Mrs. Noda Y, Mr. Maeda T, Mr. Takano H, Mrs. Koike S,and Mrs. Uzawa A for their kind technical support. They also thank Drs. Ando Kand Niwa O for their helpful suggestions.

Grant Support

This work was a part of Research Project with Heavy-ion at NIRS-HIMAC.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 11, 2010; revised March 25, 2011; accepted March 28, 2011;published OnlineFirst March 31, 2011.

References1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer

statistics, 2006. CA Cancer J Clin 2006;56:106–30.2. Wolmark N, Rockette H, Fisher B, Wickerham DL, Redmond C, Fisher

ER, et al. The benefit of leucovorin modulated 5-fluorouracil as post-operative adjuvant therapy for primary colon cancer: results from theNational Surgery and Adjuvant Breast and Bowel Project protocol C-03. J Clin Oncol 1993;11:1879–87.

3. Kanai T, Endo M, Minohara S, Miyahara N, Koyama-ito H, Tomura H,et al. Biophysical characteristics of HIMAC clinical irradiation systemfor heavy-ion radiation therapy. Int J Radiat Oncol Biol Phys1999;44:201–10.

4. Suzuki M, Kase Y, Yamaguchi H, Kanai T, Ando K. Relative biologicaleffectiveness for cell-killing effect on various human cell lines irra-diated with heavy-ion medical accelerator in Chiba (HIMAC) carbon-ion beams. Int J Radiat Oncol Biol Phys 2000;48:241–50.

5. Griffin TW. Particle-beam radiation therapy. In:Perez CA, Brady LW,editors. Principles and Practice of Radiation Oncology. 2nd ed.Philadelphia, PA: J.B. Lippincott Co.; 1992. p. 368–87.

6. Okayasu R, Okada M, Okabe A, Noguchi M, Takakura K, Takahashi S,et al. Repair of DNA damage induced by accelerated heavy ions inmammalian cells proficient and deficient in the non-homologous end-joining pathway. Radiat Res 2006;165:59–67.

7. Kamada T, Tsujii H, Tsuji H, Yanagi T, Mizoe JE, Miyamoto T, et al.Efficacy and safety of carbon ion radiotherapy in bone and soft tissuesarcomas. J Clin Oncol 2002;20:4466–71.

8. Tsujii H, Mizoe JE, Kamada T, Baba M, Kato S, Kato H, et al. Overviewof clinical experiences on carbon ion radiotherapy at NIRS. RadiotherOncol 2004;73Suppl 2:S41–9.

9. Tsujii H, Mizoe J, Kamada T, Baba M, Tsuji H, Kato H, et al. Clinicalresults of carbon ion radiotherapy at NIRS. J Radiat Res 2007;48Suppl A:A1–13.

10. Schulz-Ertner D, Tsujii H. Particle radiation therapy using proton andheavier ion beams. J Clin Oncol 2007;25:953–64.

11. Zou GM. Cancer initiating cells or cancer stem cells in the gastro-intestinal tract and liver. J Cell Physiol 2008;217:598–604.

12. Marhaba R, Klingbeil P, Nuebel T, Nazarenko I, Buechler MW, ZoellerM, et al. CD44 and EpCAM: cancer-initiating cell markers. Curr MolMed 2008;8:784–804.

13. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al.Identification of human brain tumour initiating cells. Nature 2004;432:396–401.

14. Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, LearyAG, et al. AC133, a novel marker for human hematopoietic stem andprogenitor cells. Blood 1997;90:5002–12

15. Vander Griend DJ, Karthaus WL, Dalrymple S, Meeker A, DeMarzoAM, Isaacs JT, et al. The role of CD133 in normal human prostate stemcells and malignant cancer-initiating cells. Cancer Res 2008;68:9703–11.

16. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cellcapable of initiating tumour growth in immunodeficient mice. Nature2007;445:106–10.

17. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, PeschleC, et al. Identification and expansion of human colon-cancer-initiatingcells. Nature 2007;445:111–5.

18. Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K,Perez Alea M, et al. Single-cell cloning of colon cancer stem cells

reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U SA 2008;105:13427–32.

19. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al.Glioma stem cells promote radioresistance by preferential activationof the DNA damage response. Nature 2006;444:756–60.

20. Hambardzumyan D, Squatrito M, Holland EC. Radiation resistanceand stem-like cells in brain tumors. Cancer Cell 2006;10:454–6.

21. Rich JN. Cancer stem cells in radiation resistance. Cancer Res2007;67:8980–4.

22. Baumann M, Krause M, Hill R. Exploring the role of cancer stem cellsin radioresistance. Nat Rev Cancer 2008;8:545–54.

23. DiehnM, Clarke MF. Cancer stem cells and radiotherapy: new insightsinto tumor radioresistance. J Natl Cancer Inst 2006;98:1755–7.

24. Dingli D, Michor F. Successful therapy must eradicate cancer stemcells. Stem Cells 2006;24:2603–10.

25. Ischenko I, Seeliger H, Schaffer M, Jauch KW, Bruns CJ. Cancer stemcells: how can we target them? Curr Med Chem 2008;15:3171–84.

26. Cui X, Wakai T, Shirai Y, Yokoyama N, Hatakeyama K, Hirano S, et al.Arsenic trioxide inhibits DNA methyltransferase and restores methyla-tion-silenced genes in human liver cancer cells. Hum Pathol2006;37:298–311.

27. Cui X, Wakai T, Shirai Y, Hatakeyama K, Hirano S. Chronic oralexposure to inorganic arsenate interferes with methylation status ofp16INK4a and RASSF1A and induces lung cancer in A/J mice. ToxicolSci 2006;91:372–81.

28. Matsui Y, Asano T, Kenmochi T, Iwakawa M, Imai T, Ochiai T. Effectsof carbon-ion beams on human pancreatic cancer cell lines that differin genetic status. Am J Clin Oncol 2004;27:24–8.

29. Takahashi Y, Teshima T, Kawaguchi N, Hamada Y, Mori S, Madachi A,et al. Heavy ion irradiation inhibits in vitro angiogenesis even atsublethal dose. Cancer Res 2003;63:4253–7.

30. Koike S, Ando K, Oohira C, Fukawa T, Lee R, Takai N, et al. Relativebiological effectiveness of 290MeV/u carbon ions for the growth delayof a radioresistant murine fibrosarcoma. J Radiat Res 2002;43:247–55.

31. Peschke P, Karger CP, Scholz M, Debus J, Huber PE. Relativebiological effectiveness of carbon ions for local tumor control of aradioresistant prostate carcinoma in the rat. Int J Radiat Oncol BiolPhys 2011;79:239–46.

32. Beck-Bornholdt HP, W€urschmidt F, Vogler H. Net growth delay: anovel parameter derived from tumor growth curves. Int J Radiat OncolBiol Phys 1987;13:773–7.

33. DiehnM, Clarke MF. Cancer stem cells and radiotherapy: new insightsinto tumour radioresistance. J Natl Cancer Inst 2006;98:1755–7.

34. Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY. CD133þ HCC cancerstem cells confer chemoresistance by preferential expression of theAkt/PKB survival pathway. Oncogene 2008;27:1749–58.

35. Pallini R, Ricci-Vitiani L, Banna GL, Signore M, Lombardi D, Todaro M,et al. Cancer stem cell analysis and clinical outcome in patients withglioblastoma multiforme. Clin Cancer Res 2008;14:8205–12.

36. Horst D, Kriegl L, Engel J, Kirchner T, Jung A. CD133 expression is anindependent prognostic marker for low survival in colorectal cancer.Br J Cancer 2008;99:1285–9.

37. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours:accumulating evidence and unresolved questions. Nat Rev Cancer2008;8:755–68.

Cui et al.





38. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, et al.Cancer stem cells–perspectives on current status and future directions:AACR Workshop on cancer stem cells. Cancer Res 2006;66:9339–44.

39. Imai R, Kamada T, Tsuji H, Yanagi T, Baba M, Miyamoto T, et al.Working Group for Bone, Soft Tissue Sarcomas. Carbon ion radio-

therapy for unresectable sacral chordomas. Clin Cancer Res2004;10:5741–6.

40. Mobaraki A, Ohno T, Yamada S, Sakurai H, Nakano T. Cost-effec-tiveness of carbon ion radiation therapy for locally recurrent rectalcancer. Cancer Sci 2010;101:1834–9.






Correction

Correction: Effects of Carbon Ion Beam onPutative Colon Cancer Stem Cells and ItsComparison with X-rays

In this article (Cancer Res 2011;71:3676–87), which was published in the May 15, 2011,issue of Cancer Research (1), the affiliation for Makoto Akashi is incorrect. The correctaffiliation for Dr. Akashi is Department of Radiation Emergency Medicine, ResearchCenter for Radiation Emergency Medicine, National Institute of Radiological Sciences(NIRS), Chiba, Japan.

Reference1. Cui X, Oonishi K, Tsujii H, Yasuda T, Matsumoto Y, Furusawa Y, et al. Effects of carbon ion beam on

putative colon cancer stem cells and its comparison with x-rays. Cancer Res 2011;71:3676–81.

Published OnlineFirst July 19, 2011.�2011 American Association for Cancer Research.doi: 10.1158/0008-5472.CAN-11-1905

CancerResearch

Cancer Res; 71(15) August 1, 20115360

2011;71:3676-3687. Published OnlineFirst March 31, 2011.Cancer Res Xing Cui, Kazuhiko Oonishi, Hirohiko Tsujii, et al. Cells and Its Comparison with X-raysEffects of Carbon Ion Beam on Putative Colon Cancer Stem

Updated version

10.1158/0008-5472.CAN-10-2926doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2011/03/31/0008-5472.CAN-10-2926.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/71/10/3676.full#ref-list-1

This article cites 39 articles, 11 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/71/10/3676.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

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

SubscriptionsReprints and

[email protected] at

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

Permissions

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

.http://cancerres.aacrjournals.org/content/71/10/3676To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-10-2926

http://cancerres.aacrjournals.org/content/suppl/2011/03/31/0008-5472.CAN-10-2926.DC1

http://cancerres.aacrjournals.org/content/71/10/3676.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/10/3676.full#related-urls

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/71/10/3676


effects of carbon ion beam on putative colon …...tumor and stem cell biology effects of carbon ion...

Documents