CANCER RESEARCH | RESOURCE REPORT

Organoids Reveal That Inherent Radiosensitivity of Smalland Large Intestinal Stem Cells Determines OrganSensitivityMaria Laura Martin1,2, Mohammad Adileh2, Kuo-Shun Hsu1,2, Guoqiang Hua3, Sang Gyu Lee1, Christy Li1,John D. Fuller1, Jimmy A. Rotolo1, Sahra Bodo4, Stefan Klingler1,2, Adriana Haimovitz-Friedman4,Joseph O. Deasy5, Zvi Fuks4, Philip B. Paty2, and Richard N. Kolesnick1

ABSTRACT◥

Tissue survival responses to ionizing radiation are nonlinear withdose, rather yielding tissue-specific descending curves that impedestraightforward analysis of biologic effects. Apoptotic cell death oftenoccurs at low doses, while at clinically relevant intermediate doses,double-strand break misrepair yields mitotic death that determinesoutcome. As researchers frequently use a single low dose for exper-imentation, such strategies may inaccurately depict inherent tissueresponses. Cutting edge radiobiology has adopted full dose survivalprofiling and devised mathematical algorithms to fit curves toobserved data to generate highly reproducible numerical data thataccurately define clinically relevant inherent radiosensitivities. Here,we established a protocol for irradiating organoids that delivers

radiation profiles simulating the organ of origin. This techniqueyielded highly similar dose–survival curves of small and large intes-tinal crypts in vivo and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherentradiation profile of their respective Lgr5þ stem cell populations. Asthis technological advance is quantitative, it will be useful for accurateevaluation of intestinal (patho)physiology and drug screening.

Significance: These findings establish standards for irradiatingorganoids that deliver radiation profiles that phenocopy the organ oforigin.

See related commentary by Muschel et al., p. 927

IntroductionAn extensive literature shows that organoids derived from small and

large intestines recapitulate structural and functional characteristics ofthe tissue of origin (1, 2). The purpose of the current set of investiga-tions was to define conditions for delivery of radiation to intestinalorganoids that would accurately reflect impact of ionizing radiation onthe organ of origin. In contrast to the many biologic events that can bedefined by straight lines that relate input to outcome, for instance,ligand–receptor binding, Michaelis–Menten enzyme kinetic analysis,and stimulus-secretion coupling of hormone, mitogen, and cytokinerelease to list a few, ionizing radiation–induced dose survival data ineukaryotic cells are best-fit by nonlinear descending curves, analyzedby complex mathematical algorithms. Whereas radiation is increas-ingly employed as a tool in biological and translational research,frequently conducted by scientists who may not have had formalradiobiologic research training, we highlight here straightforward

methods and data analytic techniques to facilitate high-quality radio-biologic data management using organoid culture. The specificapproach taken in the current studies was to exploit recently detailedin vivo small and large intestinal crypt radiation dose survival data toestablish organoid growth settings that yield comparable radiationdose–survival curves ex vivo (3).We provide a set of growth conditionsfor radiation dose survival colony assays and a simple analytic tool thatyields a single quantitative readout that defines the inherent radio-sensitivity of the organ of origin from which organoids were derived.Using these protocols, we provide evidence in organoid culture that theestablished radiation sensitivities of the small and large intestine reflectthe inherent sensitivity of their Lgr5þ stem cell populations.

Materials and MethodsMice

Lgr5-lacZ and Lgr5-EGFP-ires-CreERT mice were used as describedpreviously (4). C57/Bl6micewere purchased from JacksonLaboratories.Mouse protocols were approved by Memorial Sloan Kettering CancerCenter Institutional Animal Care and Use Committee.

Crypt microcolony assayWhole-body radiation was delivered with a Shepherd Mark-I Unit

(Model 68, SN643, J. L. Shepherd & Associates) operating a 137Cssource. The crypt microcolony assay was performed as describedpreviously (5). Briefly, at 3.5 days after irradiation small intestineswere excised, fixed in 10% formalin overnight, embedded in paraffin,cut into transverse cross-sections, and circumferences were stainedwith hematoxylin and eosin. Surviving (regenerating) crypts aredefined as containing 10 or more violaceous, thick-walled non-Paneth cells that are enclosed with a lumen. Number of survivingcrypts was counted in each circumference. At least 4 or 5 circumfer-ences were scored per mouse, and 4 mice were used to generate eachdata point.

1Laboratory of Signal Transduction, Memorial Sloan Kettering Cancer Center,New York, New York. 2Department of Surgery, Memorial Sloan Kettering CancerCenter, New York, New York. 3Institute of Radiation Medicine, Fudan University,Shanghai, China. 4Department of Radiation Oncology, Memorial Sloan KetteringCancer Center, New York, New York. 5Department of Medical Physics, MemorialSloan Kettering Cancer Center, New York, New York.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M.L. Martin and M. Adileh contributed equally to this article.

Corresponding Author: Richard N. Kolesnick, Memorial Sloan Kettering CancerCenter, 1275 York Avenue, NewYork, NY 10065. Phone: 646-888-2174; Fax: 646-422-0281; E-mail: r-kolesnick@ski.mskcc.org

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Crypt isolationCrypt isolation and the dissociation of cells for flow cytometry

were performed as described previously (6, 7) modified as follows.Small intestine (�10 cm) and distal colon were removed andflushed with cold PBS containing 100 U/mL penicillin and100 U/mL streptomycin. Colonic fragments were sliced into 1–3 mm pieces, then suspended in 10 mL of DMEM high glucosecontaining 1% FBS, 500 U/mL collagenase IV. The mixture wasincubated for 30 minutes at 37�C in a shaking water bath. There-after, tissue fragments were vigorously shaken using a 10-mLpipette to isolate crypts, allowed to settle under gravity for 1 minute,and the supernatant was removed for inspection by invertedmicroscopy. The resuspension/sedimentation procedure wasrepeated three times, liberated crypts in suspension were combined,passed through 100-mm cell strainer to remove muscle material, andthen centrifuged at 4�C at 300 � g for 5 minutes. Isolated cryptswere pelleted, washed with cold 1% FBS/DMEM, and centrifugedthree times at 60 � g for 3 minutes to separate crypts from singlecells. For small intestine, tissue was chopped into 3–5 mm piecesand incubated in 5 mmol/L EDTA/PBS for 20–30 minutes on ice.After removal of EDTA buffer, tissue was washed once with coldPBS and vigorously resuspended in cold buffer using a 10-mLpipette to isolate intestinal crypts. This step was repeated twice.Supernatant was collected through a 100-mm cell strainer andcentrifuged at 300 � g for 5 minutes. Small intestinal (SI) cryptswere washed in cold PBS three times centrifuging at 60 � g for3 minutes.

Single stem cell isolationSingle epithelial cell preparation and flow cytometry analysis were

performed as described previously (8) with the modifications below.For the large intestine, we used what we called the “sausage protocol.”The whole large intestine was tied on one end with surgical thread,filled with 30mmol/L EDTA/PBS with 1mmol/LDTT, and tied on theopposite end afterward. Five colons were pooled and incubated in thesame solution (30 mmol/L EDTA in PBS with 1 mmol/L DTT) for 90minutes in a rocking platform at 4�C. After this incubation, knots werecut out, large intestines were washed once, fat was discarded, tissueswere opened longitudinally, cut into 1–2mmpieces, and shaken in PBSfor 5 minutes to get single crypts. For small intestine, tissue fragments(3–5 mm) were incubated with 30 mmol/L EDTA/PBS with 1 mmol/LDTT for 20 minutes on ice, followed by washing with PBS, and shakenfor 3 minutes to get single crypts. For both tissues, after shaking, cryptsuspension was passed through a 100-mm cell strainer and spin downtwice at 300 � g for 5 minutes. Pelleted crypts were dissociated in2–3 mL of TryplE containing 200 U/mL DNAase, 0.5 mmol/LN-acetylcysteine (NAC), and 10 mmol/L Y-27632 for 3 minutes at37�C in a water bath, shaking every minute to make a single-cellsuspension. Dissociated cells were washed with 1% FBS/DMEM andpassed sequentially through 70-, 40-, and 20-mm cell strainer. Singlestem cells were either obtained from Lgr5-GFPmice, sorting for GFPþ

cells or fromB6mice inwhich case cells were stainedwith the followingantibodies: Brilliant Violet 421 anti-mouse CD45, anti-mouse CD117(c-Kit) Alexa Fluor 700, and anti-mouse PE/Cy7–conjugated anti-CD44 for 30 minutes on ice, washed once with 1% FBS/DMEM,and analyzed in a BD FACSAria, as described previously (8, 9). DAPI(1mg/mL)was used to exclude dead cells in all sortings. Flow cytometrydata was analyzed using FCS Express Flow Cytometry Software(De Novo Software). The working concentrations of the antibodieswere determined by titration of each one of them (SupplementaryTable S1).

Organoid and colony cultureAfter centrifugation, pure crypts were counted and resus-

pended in Matrigel covered with advanced DMEM/F12 mediacontaining 1 mmol/L HEPES, 1 mmol/L glutamax, and 100 U/mLantibiotics (ADF), 10% R-spondin conditioned media and 50%Wnt conditioned media (when indicated); supplemented with 1�B27, 1� N2, and 1 mmol/L NAC containing 50 ng/mL murinerecombinant EGF and 100 ng/mL mouse recombinant Noggin.Wnt3a conditioned media was produced using a cell line kindlyprovided by Robert Vries (L-Wnt3a cell line, Hubrecht Institute,Utrecht, the Netherlands) and R-spondin conditioned media wasprepared as described previously (10). Intestinal stem cell colonieswere cultured from sorted single Lgr5-GFP cells based uponthe method reported by Yin and colleagues (11). Approximately3,000 intestinal stem cells were plated in 30 mL Matrigel andcultured in ENR-VC media with 1.5 mmol/L valproic acid (V)and CHIR99021 (C), containing 10 mmol/L Rho-kinase/ROCKinhibitor Y-27632 and 1 mmol/L Jagged-1 only for the first48 hours. Under these culture conditions, intestinal stem cellsdivide symmetrically without differentiating, growing into homo-geneous stem cell colonies. Intestinal stem cell colonies werepassaged once per week as single cells following a 3-minuteincubation with TrypLE containing 2 kU/mL DNase1, NAC, andY-27632.

Organoid irradiationIrradiation was delivered with a Shepherd Mark-I Unit (Model

68, SN643, J.L. Shepherd & Associates) operating a 137Cs source.Organoids were passaged one day prior to the experiment, plated ata density of 100–150 organoids/well in at least three wells for eachcorresponding dose. Organoids were exposed to single-fractionradiation (range, 0–16 Gy). Manual counting under an invertedbrightfield microscope of surviving organoids at day 4 to 10postradiation was used to generate classic dose–response curves.Brightfield imaging of organoids after radiation was performed withCitation 5 Cell Imaging Multi-mode Reader (BioTek). Survivalfraction was calculated as number of surviving organoids/numberof organoids in the unirradiated sample.

Radiation dose–survival curvesRadiation dose–survival curves are characterized on a log-

linear plot by an initial shoulder, or the quasi-threshold region,and an exponential region, where the curve approximates astraight line (12–17). In this study, we adapted the single-hitmulti-target (SHMT) model to characterize radiosensitivity pro-files of small and large intestinal organoids. The SHMT modelassumes that each cell contains [n] identical targets for prolethalradiation damage, and while inactivation of a single target issublethal, creating the shoulder region of the radiation dose–survival curve, inactivation of [n] targets is required for celllethality, expressed in the exponential portion of thecurve (12, 18, 19). Whereas intestinal organoids are multicell“mini-guts” dependent on a subset of Lgr5þ stem cells that driveorganoid growth and radiation dose survival in vitro, we modifiedthe baseline SHMT paradigm, introducing the concept that eachorganoid contains [n] identical stem cell targets. Inactivation ofone stem cell is sublethal, creating a shoulder region of theorganoid radiation dose–survival curve, and dose-dependentinactivation of [n] stem cells is required for organoid lethality,expressed in the exponential portion of the dose–survival curve.GraphPad Prism 6 Software (GraphPad) was used to generate the

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dose–survival curves applying the following equation:

S ¼ 1� 1� e�DD0

� �n

logen ¼ Dq

D0;

where S is the survival rate of the cell population,D is the dose,D0 isthe reciprocal slope value of the exponential portion of the curve, nis the extrapolation number, and Dq is the quasi-threshold doserange of the shoulder region. D0 that defines inherent radiosensi-tivity of the irradiated organoids is the dose required to reduce thefraction of surviving organoids to 37%, and systematically predictsorganoid-type specific 10% survival (D10) as the product of 2.3 �D0, where 2.3 is the natural log of 10. Dq defines the low dose rangewhere organoid reparable sublethal radiation damage prevails,resulting in disproportionate organoid survival per dose unitrelative to the inherent D0. The algorithm fits the dose–survivalcurve to the measured end point at each tested dose by iterativeweighted least square regression analysis of all data points, esti-mating covariance of survival curve parameters and correspondingconfidence limits, and reports the computed D0, Dq, and the nnumber. D0 values are used in this study to compare inherentradiosensitivities between tissues, organoids, and stem cellcolonies.

Whole-mount staining of organoidsb-galactosidase (LacZ) staining protocol was described by Barker

and colleagues (4) for tissue staining and here we adapted that protocolfor whole-mount organoids. Matrigel-embedded SI organoids grownfor 1 week in a 24-well plate were washed once with PBS and incubatedfor 30 minutes in ice-cold fixative (1% formaldehyde, 0.2% glutaral-dehyde, and 0.02% NP40 in PBS 1X) at 4�C on a rolling platform. Thefixative was removed and the organoids washed two times in PBSfor 20 minutes at room temperature on a rolling platform. Theb-galactosidase substrate [5 mmol/L K3FE(CN)6, 5 mmol/L K4Fe(CN)6 *3H2O, 2 mmol/L MgCl2, 0.02% NP40, 0.1% sodium deox-ycholate, and 1mg/mLX-gal (5-Bromo-4-chloro-3-indolyl b-D-galac-topyranoside, Sigma] in PBSwas then added and incubated in the darkovernight at room temperature. The substrate was removed and theorganoids washed two times in PBS for 20 minutes at room temper-ature on a rolling platform. The tissues were then fixed for 30 minuteswith 4% PFA/PBS at room temperature in the dark on a rollingplatform. PFA was removed and the organoids washed two times inPBS for 20 minutes at room temperature on a rolling platform.Originally, we used the LacZ Tissue Staining Kit from Invivogen, butthe company stopped producing it, and now all the reagents can beobtained separately (Table S1). Imaging of LacZ-stained organoidswasperformed under a Zeiss Lumar v.12 Stereoscope at Molecular Cytol-ogy Core Facility (MCCF) fromMSKCC (New York, NY). To confirmorganoid death, Matrigel-embedded intestinal organoids were stainedwith 1mg/mLHoechst 33342 and 1.5mmol/L propidium iodide 1 hourbefore taking images. Hoechst 33342 was also used to determine thenumber of cells at day 3. For these experiments, imaging was per-formed with inverted confocal Zeiss LSM 5 Live microscope at MCCF.

DNAdouble-strand break repair focus staining and quantitationSingle Lgr5-GFPþ cells obtained from SI and large intestine (LI)

colonies were seeded onto 8-well Lab-Tek chamber (NUNC) pre-coatedwith 20mLof 100%Matrigel (�150–200 cells/well) and overlaidwith growthmedium. Lgr5-GFPþ cells were irradiatedwith 8Gy eitheron day 1 or day 4 postplating. At the indicated times, immunostaining

was carried out using a standard protocol. Briefly, colonies werewashed two times with PBS, fixed with 4% paraformaldehyde for30minutes, washed, and quenchedwith 50mmol/LNH4Cl for 15min-utes. After blocking for 2 hours (PBS containing 0.5% BSA, 0.2mg/mLsodium azide, 0.3mmol/L DAPI, and 0.25% Triton X-100), colonieswere incubatedwith primary antibodies in blocking buffer overnight at4�C. After three washes with PBS, colonies were incubated withsecondary antibodies in blocking solution for 2 hours at room tem-perature. gH2AX mouse mAB against gH2AX Ser139 [Millipore(clone JBW 301), #05-636] was used at a dilution 1:1,000 v/v, andsecondary antibody Alexa Fluor 488 F(ab’)2 fragment of goat anti-mouse IgG (HþL; 2 mg/mL) was used at a dilution of 1:400. Sampleswere mounted with ProLong Diamond Antifade Reagent (Life Tech-nologies). Fluorescent images were acquired with a 63�/1.4NA objec-tive oil lens on a Zeiss widefield microscope (Axio Observer.Z1)equipped with an AxioCam HRc camera. Ten consecutive 0.2-mmthin sections were imaged, deconvoluted with a theoretical PSF usingAutoQuant X software, and max-projected into a single image usingImage J (FIJI).

On average 100 nuclei/experiment were randomly selected fromeach sample for quantitation of focus numbers. Fiji software was usedfor image analysis (20). Briefly, foci were scoredwithin a nucleuswhoseboundary was defined automatically from a DAPI image. Focusthreshold was determined manually as a mean ¼ 8.28 pixels, basedon discreet gH2AX foci generated at a low radiation dose (2 Gy at 30minutes), consistent with published results (21). Because of extensiveoverlap of foci at 30minutes after high-dose irradiation, focus numberwas estimated at this time by measuring total gH2AX fluorescence pernucleus normalized to 8.28 mean pixels/focus.

ResultsThemicrocolony assay (also termed clonogenic assay) developed by

Withers and Elkind (5) directly quantifies radiation dose–dependentlethality of the crypt compartment, is predictive of eventual animaldeath from radiation gastrointestinal (GI) syndrome, and is considereda “gold standard” in the radiation field for evaluating normal tissueresponse to ionizing radiation (22, 23). This assay, which measuresnumber of regenerating crypts per SI circumference at 3.5 dayspostirradiation (typical histologic sections are shown in Fig. 1A), isregarded as a surrogate for stem cell survival, as a single surviving stemcell is considered sufficient for regeneration of an entire crypt. Numer-ous studies performed in mice reveal that 10–15 regeneratingcrypts per circumference, or approximately 10% crypt survival, isthe minimum required to fully recover the GI mucosapostirradiation (24). Figure 1B shows that crypt survival in C57BL/6 mice generates a descending exponential curve typical of radiationresponses in mammalian tissues (25) with 113 � 1 crypts/SI circum-ference after 7 Gy (a value not different from the 114 � 3 crypts/circumference in unirradiated mice), reduced to 12� 1 crypts after 13Gy (P � 0.001) and further to 1 crypt/circumference at 15 Gy (P �0.001 vs. 13 Gy). These data are consistent with a large body ofliterature on this topic (26, 27). Whereas cell death at low radiationdoses often occurs by mechanisms that are independent of DNAmisrepair (28–30), the clinically relevant mechanism of radiationkilling, algorithms have been developed to quantify inherent radio-sensitivity in the intermediate radiation dose range (12–17). Hence, tofurther characterize inherent radiosensitivity from our data, we trans-formed crypt survival data by nonlinear regression fitting according tothe well-established SHMT algorithm, yielding a single D0 value thatserves as a numerical estimate of the efficiency of double-strand break

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Figure 1.

SI organoids recapitulate in vivo response to ionizing radiation. A, Representative full-transverse section of proximal jejunum from C57BL/6 mice at 3.5 daysposttreatment with 15 Gy whole-body radiation compared with unirradiated mice. B, GI damage assessed by the crypt microcolony assay of Withers andElkind (5). Triangles, number of crypts in individual circumferences. C, Transformation of data in B using the SHMT algorithm. D, Representative live images ofSI organoids grown for 6 days from isolated crypts that had been irradiated 24 hours after plating with 10 Gy compared with unirradiated organoids. Shownare brightfield (left), Hoechst (middle), and propidium iodide (right) images. E, Dose–survival curve of SI organoids derived from irradiated SI crypts handledas in D. F, Representative images of SI organoids grown from crypts isolated from Lgr5-lacZ mice stained for lacZ expression 6 days postirradiation.G, Quantification of LacZþ area (blue) per organoid area (white þ blue). H, Dose–survival curve of organoids derived from single Lgr5-GFPþ SI stem cells thathad been irradiated at 24 hours after plating and counted at 6 days postirradiation. Data (mean � SD) are from 4 mice/dose analyzing four circumferences/mouse (B and C), or from triplicate determinations collated from seven independent experiments in E, three independent experiments in H, or at least twoexperiments in G. Scale bars, 200 mm in all images (A, D, and F).

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(DSB) repair (31). Note, the higher the D0 value the greater theradioresistance. Figure 1C shows that SHMT transformation of ourcrypt survival data yields a D0¼ 1.3� 0.1 Gy, virtually identical to D0

values previously calculated by ourselves and others in this strain(Fig. 1C; refs. 22, 32). In this context, we previously reported highlysimilarD0 values for SI crypt survival in vivo inmultiple mouse strains,as follows: 1.3� 0.2Gy for B6.129� 1/Jmice; 1.5� 0.1Gy forC57BL/6mice; 2.0� 0.2Gy for sv129/BL6mice, and 1.9� 0.1Gy forC3HeB/FeJmice (32).

Using organoid technology established byClevers and colleagues (1)that allows growth of intestinal organoids from crypts and from singleintestinal stem cells in a definedmedia containing EGF,Noggin, andR-spondin (ENR), we adapted principles of the in vivo clonogenic assayto profile radiation dose survival of SI organoids ex vivo. For initialstudies, SI crypts were isolated from Lgr5-lacZ mice. Crypts containLgr5þ stem cells at their base intermingled with Paneth cells overlaidby progenitor cells (4). Under these media conditions, once the upperopening seals, crypts undergo continuous budding events, creatingorganoids that consist of a lumen filled with apoptotic cells lined by avillus-like epithelium surrounded by multiple crypt domains (1). Forour studies, isolated SI crypts were irradiated 24 hours after plating toallow for recovery from isolation stress (which interferes with reliableradiation dose profiling). While unirradiated organoids grow loga-

rithmically and exclude propidium iodide (PI), an indication ofmembrane integrity, Fig. 1D shows that a dose of 10 Gy is lethal forSI crypts (shown at day 7) as the structure completely disintegrates,manifesting as a cloud of cell debris that readily incorporates PI.Radiation dose survival analysis of SI organoids irradiated withescalating doses (2–10 Gy) yields a curve with a D0 ¼ 2.1 � 0.1 Gyat 6 days postradiation (Fig. 1E), well within the range of ourD0 valuesfound for the in vivo microcolony assay of Withers and Elkind asdescribed above. These results indicate that SI organoids maintain theradiosensitivity profile of the intact organ.

Furthermore, organoids derived from Lgr5-lacZ mice when stainedfor b-galactosidase (LacZ) to evaluate stem cell response at day 6postradiation (2–10 Gy) show dose-dependent reduction in the stemcell compartment (LacZþ area/organoid) with escalating radiationdoses (whole-mount staining shown in Fig. 1F, quantified in Fig. 1G).To provide evidence supporting intestinal stem cells as determinant oforganoid and organ radiosensitivity, Lgr5-GFPþ stem cells isolatedfrom Lgr5-EGFP-ires-CreERT mice by FACS were irradiated with thesame dose range as for intact organoids at 24 hours after plating andallowed to grow into intact organoids (Fig. 1H). At day 6 postradia-tion, number of organoids was counted and SHMT transformation ofdose survival data generated a D0 ¼ 1.6 � 0.1 Gy, indicating thatindeed, for the small intestine, intrinsic radiosensitivity of Lgr5þ stem

Figure 2.

LI organoids are significantlymore resistant to ionizing radiation than SI organoids.A,Representative brightfield images of LI and SI organoids grown for 7 days fromfreshly isolated crypts irradiated 24hours after platingwith0Gy (control, left panels), 8Gy (middle panels), and 12Gy (right panels). LI organoids aremore resistant toionizing radiation than SI organoids, as live organoids can still be detected after 8 Gy and 12 Gy (white arrows), while SI organoids are dead, displaying only a debriscloud, at 8 Gy (black arrows). B, Dose–survival curves of murine LI and SI organoids grown from isolated crypts irradiated 24 hours after plating. Organoids werecounted 10 days postirradiation. C and D, Similar data were derived using passaged LI and SI organoids, handled as in A and B. Data (mean� SE) are collated fromthree independent experiments, each performed in triplicate in B and D. Scale bars, 100 mm (A and C).

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cells is highly similar to that of crypts in the intact organ in vivo and toorganoids derived thereof.

Employing Lgr5-lacZ transgenic mice, we recently published thatLgr5þ stem cells in the distal large intestine aremore radiation resistantthan their counterpart Lgr5þ stem cells in the small intestine in vivo(3). To extend these findings to the in vitro setting, initial studiesgenerated organoids from crypts isolated from small and distal largeintestines of Lgr5-lacZmice. In contrast to SI organoids, which containPaneth cells that provide Wnt LI organoids, require exogenous Wnt,because in vivo Wnt is provided by stromal cells (33) not included inour SI culture conditions. Hence, SI and LI organoids were grown inWENR (Wnt, EGF, Noggin, R-spondin) media for thesestudies. Figure 2A displays representative bright-field images oforganoids derived from LI and SI crypts irradiated at 24 hours afterplating and shows that LI organoids are more radioresistant than SIorganoids. While LI organoids are alive at 6 days post 8 Gy and 12 Gy,near-complete lethality is detected in SI organoids, manifested as acloud of cell debris in place of organoids. Radiation dose survivalanalysis of SI and LI organoids using 4–16 Gy (Fig. 2B) confirmsthat LI organoids aremarkedly radioresistant with aD0¼ 7.6� 1.3 Gyas compared with SI organoids that exhibit a D0 ¼ 2.2 � 0.3 Gy(P < 0.001, Fig. 2B). Furthermore, consistent with the well-describeddose-dependent cellular growth delay postradiation, all LI organoidstreated with 4–16 Gy are smaller than unirradiated organoids on day 7postirradiation, with growth recovery displaying a clear radiationdose dependence (Supplementary Fig. S1A and S1B). In addition,mechanically passaged LI and SI organoids retain the radiosensitivitiesobserved with organoids from fresh crypts, studied up to passage10. Figure 2C shows that passage 10 LI organoids retain radio-resistance compared with SI organoids, displaying live organoids at6 days post 8 Gy and 12 Gy, which appear capable of fasterpostradiation growth than freshly isolated organoids (compareSupplementary Fig. S1A–S1D). SHMT transformation of radiationdose–survival curves of SI and LI organoids grown from passagedorganoids confirm LI organoids retain radioresistance with a higherD0 ¼ 9.2 � 1.5 Gy than SI organoids D0 ¼ 2.2 � 0.2 Gy (Fig. 2D,P < 0.001). These results validate ex vivo organoids as legitimate

surrogates for in vivo radiation responses, a phenotype that is stableover time.

Unexpectedly, organoids derived from single LI stem cells sortedfrom Lgr5-GFP mice and irradiated 24 hours after plating display lossof radioresistance yielding a D0 ¼ 2.2 � 0.2 Gy as compared withorganoids derived from SI stem cells, which retain their originalradiosensitivity with D0 ¼ 1.8 � 0.1 Gy (Fig. 3A). This selective lossof LI stem cell radioresistance was confirmed using an alternativesorting strategy that allows obtaining a larger number of cells by FACSusing CD44 and cKit surface markers (see Supplementary Fig. S2 forsorting strategy; refs. 8, 9). For these studies, stem cell populationsmarked byCD44highcKit�were isolated from large and small intestinesof C57BL/6 mice and irradiated 24 hours after plating as above.Radiation dose–survival curves of these cell populations generatedD0 ¼ 2.2� 0.1 Gy for LI stem cells and D0 ¼ 1.9� 0.2 Gy for SI stemcells (Fig. 3B). However, by delaying delivery of radiation beyond24 hours postplating, we found that LI stem cells recover their inherentradioresistance by day 3 of culture (Fig. 3C), at a moment when singlecells have developed into mini-organoids containing an average of 30(with 95% confidence interval (CI) ¼ 12–40) cells per organoid,consistent with a doubling time of approximately 12 hours (1). Themechanism by which development of cell–cell contact interactions inLI organoids restores radioresistance remains unknown, althoughpreliminary data (see below) suggest these radiation phenotypes reflectdifferences in DNA repair, which will require further studies forelucidation.

To further evaluate the inherent radiosensitivity of SI and LI stemcells, we used a niche-independent high-purity Lgr5þ intestinal stemcell culture system. In this system, combination of a GSK3b inhibitorand histone deacetylase inhibitor enables culture of homogenoussymmetrically-dividing Lgr5þ stem cell colonies from single-purified stem cells. The potent activation of the Wnt and Notchpathways in this culture systemmaintains the high frequency of Lgr5þ

cells (60%–100%, see Supplementary Fig. S3), in contrast to SI and LIorganoids that display 14% � 11% and 4% � 3% Lgr5þ stem cells,respectively. For these studies, LI and SI colonies were grown fromsorted Lgr5-GFPþ cells [isolated from 5 (for large intestines) or 3 (for

Figure 3.

Transient loss of LI stem cell radiation resistance upon plating of single cells.A,Dose–survival curves of organoids derived from single Lgr5-GFPþ intestinal stem cellsthatwere obtained fromsymmetrically dividing stemcell colonies, irradiated at 24hours after plating, and counted at 6dayspostirradiation.B,Dose–survival curveoforganoids derived from single CD44highcKit� intestinal stem cells that had been sorted fromC57BL/6mice, irradiated at 24 hours after plating, and counted at 6 dayspostirradiation. C, LI stem cells recover resistance to radiation at 3 days after plating. Single CD44highcKit- intestinal cells were sorted from C57BL/6 mice, irradiatedwith 8 Gy on successive days after plating, and organoids were counted at 6–7 days postirradiation. Data (mean � SE) are collated from at least two independentexperiments (A–C).

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small intestines) mice], which allows for availability of an unlimitednumber of stem cells. It is worth pointing out that although this culturesystem has been reported (11), it has never been used before to ourknowledge to study biologic processes. For radiation dose survivalexperiments, these pure stem cell colonies were passaged as single cellsby trypsinization and irradiated at day 3 after plating, at the time whendeveloping LI organoids regain their radioresistance. Note that singleLI stem cells lose their radioresistance irrespective of the media inwhich they are resuspended, as this radiosensitive phenotype isobserved in single LI stem cells grown in either WENR (where theygrow as organoids) or ENR-VC (where they grow as stem cellcolonies), associatedwith reduced resolution of gH2AX foci, indicativeof diminished DNADSB repair capacity (Supplementary Fig. S4A andS4B). Figure 4A and B show that LI stem cell colonies are moreradioresistant than SI colonies, displaying live colonies at 7 days post 8Gy and 12 Gy. In contrast, a dose of 8 Gy eliminates most SI stem cellcolonies and a dose of 12 Gy is almost completely lethal, manifested asnear absence of colonies under brightfield andGFP imaging. Radiationdose survival SHMT analysis of SI and LI colonies, grown for 7 to10 days post 2–16 Gy shows that LI stem cell colonies, like LIorganoids, LI crypts, and stem cells in the intact organ in vivo, aremarkedly radioresistant (3) with a D0¼ 10.9� 2.0 Gy compared withSI colonies that manifest a D0 ¼ 1.6 � 0.1 Gy (P < 0.001, Fig. 4C).

DiscussionAltogether, our results indicate that we have generated an in vitro

radiation sensitivity assay validated against in vivo–published datausing classic radiobiology concepts. Using this technology, we providestrong evidence that in vivo responses reflect inherent radiosensitivityof the Lgr5þ stem cell compartment of these organs.Whether the Bmi-1 population of resting small intestinal stem cells at position þ4 fromthe crypt base, or an analogous population in the large intestine, mightalso play a role in this radiation response is currently unknown (34).

A large body of literature reports data on mammalian normal andtumor stem cell radiosensitivity, but correlation with organ/tumorradiosensitivity remains inconclusive (35–42). A potentially con-

founding factor in this analysis is that SI and LI stem cell radiosen-sitivity is often measured by employing methodologies that did notcomplywith the rigorous criteria developed and validated in thefield ofradiobiology over the past four decades (43–45). For example, while arecent study argues that a dose of 12 Gy renders colonic Lgr5þ stemcells more radiosensitive than colonic KRT19þ stem cells based onlineage-tracing experiments (43), we have shown that at this dose levelcolonic Lgr5þ cells are totally resistant to radiation (3), and hence thereshould be no stress-induced incentive for colonic stem cell lineagetracing. We thus posit that the reason lineage tracing is observed inKRT19þ cells is that unlike colonic Lgr5þ CBCs, KRT19 cells undergosignificant damage at 12 Gy, leading to KRT19 lineage tracing duringthe rapid division phase of population recovery. One recent studyutilized ionizing radiation to study whether Dll1high precursor cells ofthe small intestinal secretory lineage might revert to stem cells uponradiation damage using a dose of 6 Gy, repeatedly referring to this doseas one that induces “extensive” radiation damage (45). In fact, radi-ation dose–survival curves inC57BL/6mice uniformly show 6Gy to bewithin the Dq region of the dose–survival curve, which reflectspotentially lethal damage that is mostly reversible. Of note, theobservation that 6 Gy is a relatively low dose in no way contradictsthe interpretation of the data that Dll1high precursor cells can revert tostem cells upon genotoxic stress. These and other studies (46–48) pointto a need for standardized assays to achieve the goal of translatingin vitro data successfully into animal models and humans.

We believe that this study will help to establish conditions fororganoid irradiation that accurately reflect in vivo responses, andprovide a simplified analytic tool, the D0, to allow for quantitativecomparison of impact of pharmacologic and genetic regulation ofgenotoxic stress on the small and large intestine. In this context, ourpreliminary data indicate that this assay can be translated into a clinicalsetting in colorectal cancer. Treatment with neoadjuvant chemoradia-tion (nCR) followed by total mesorectal excision has been the standardof care for locally advanced rectal cancers for more than two decades.At present, only approximately 30% of neoadjuvant patients willachieve clinical or pathologic complete responses to nCR therapy andare candidates for organ preservation. Moreover, these patients have

Figure 4.

Pure stem cell colonies recapitulate the radiosensitivity of small and large intestines.A, Representative brightfield (left) and GFP (right) images of LI colonies grownfor 7days from single Lgr5-GFPþ cells irradiated at 3days after platingwith 8Gy and 12Gy (bottompanels) comparedwith unirradiated colonies (control, toppanels).B, Representative brightfield (left) and GFP (right) images of SI colonies grown for 7 days from single Lgr5-GFPþ cells and irradiated at 3 days after plating with 8 Gyand 12 Gy (bottom panels) compared with unirradiated colonies (control, top panels). C, Dose–survival curve of SI and LI stem cell colonies irradiated at 3 days afterplating and counted at 6–10 days postirradiation. Data (mean � SE) are collated from triplicate determinations from three independent experiments. Scale bars,100 mm in all images (A and B).

Adapting GI Organoids to Phenocopy In Vivo Radiosensitivity

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better outcomes in terms of disease-free and overall survival (49).However, success of this approach is hindered by lack of molecularand/or functional assays that predict response to therapy, emphasizingthe need for development of personalized models that can predicttreatment response. On the basis of results established in this study, wehave applied the assays described here to test radiation responses ofpatient-derived organoids (PDO) and early evidence indicates thatPDO radiation responses predict treatment response to nCR (Adileh,Paty, and Kolesnick, manuscript in preparation).

Finally, we propose these data will be of benefit to the scientificcommunity interested in using organoids to investigate organ biologyother than radiation biology, as validation against a high-qualityin vivo assay, the Clonogenic Assay of Withers and Elkind, indicatesthat growth conditions identified here closely resemble those of intactorgans in live animals.

Disclosure of Potential Conflicts of InterestR.N. Kolesnick reports receiving commercial research grant from NCI, National

Institute of Allergy and Infectious Diseases, other commercial research support fromCeramedix Holding, LLC, and has ownership interest (including patents) in CeramedixHolding, LLC. Z. Fuks is a co-founder of Ceramedix Holding LLC and has patentsunrelated to this work (US10413533B2, US20170333413A1, and US20180015183A1).No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: M.L. Martin, M. Adileh, Z. Fuks, P.B. Paty, R.N. KolesnickDevelopment of methodology: M.L. Martin, M. Adileh, K.-S. Hsu, G. Hua,S. Klingler, A. Haimovitz-Friedman, J.O. Deasy, P.B. PatyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.L. Martin, K.-S. Hsu, C. Li, J.D. Fuller, S. Bodo,S. Klingler, P.B. PatyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.L. Martin, S.G. Lee, C. Li, J.D. Fuller, J. A. Rotolo,S. Bodo, A. Haimovitz-Friedman, J.O. Deasy, Z. Fuks, P.B. PatyWriting, review, and/or revision of the manuscript: M.L. Martin, M. Adileh,J. A. Rotolo, J.O. Deasy, Z. Fuks, P.B. Paty, R.N. KolesnickAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M.L. Martin, C. Li, J.D. Fuller, S. Bodo, P.B. PatyStudy supervision: M.L. Martin, A. Haimovitz-Friedman, Z. Fuks, R.N. Kolesnick

AcknowledgmentsThis work was supported by the generous donations of Corinne Berezuk, Mike

Stieber, Patrick Gerschel, and Edouard Gerschel. This research was funded, in part,through the NIH/NCI Cancer Center Support Core Grant P30 CA008748. We alsothank Jim Russell for his insightful comments about the manuscript.

Received January 28, 2019; revised September 3, 2019; accepted October 29, 2019;published first November 5, 2019.

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