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Translational Science

Cellular and Genetic Determinants of theSensitivity of Cancer to a-Particle IrradiationBrian D. Yard1, Priyanka Gopal1, Kristina Bannik2, Gerhard Siemeister2,Urs B. Hagemann2, and Mohamed E. Abazeed1,3

Abstract

Targeteda-particle–emitting radionuclideshave greatpoten-tial for the treatment of a broad range of cancers at differentstages of progression. A platform that accurately measurescancer cellular sensitivity to a-particle irradiation could guideand accelerate clinical translation. Here, we performed high-content profiling of cellular survival following exposure toa-particles emitted from radium-223 (223Ra) using 28 geneti-cally diverse human tumor cell lines. Significant variation incellular sensitivity across tumor cells was observed. 223Ra wassignificantly more potent than sparsely ionizing irradiation,with a median relative biological effectiveness of 10.4 (IQR:8.4–14.3). Cells that are the most resistant to g radiation,such as Nrf2 gain-of-function mutant cells, were sensitive toa-particles. Combining these profiling results with genetic

features, we identified several somatic copy-number alterations,gene mutations, and the basal expression of gene sets thatcorrelated with radiation survival. Activating mutations inPIK3CA, a frequent event in cancer, decreased sensitivity to 223Ra.The identification of cellular and genetic determinants of sensi-tivity to 223Ra may guide the clinical incorporation of targeteda-particle emitters in the treatment of several cancer types.

Significance: These findings address limitations in thepreclinical guidance and prediction of radionuclide tumorsensitivity by identifying intrinsic cellular and geneticdeterminants of cancer cell survival following exposure toa-particle irradiation.

See related commentary by Sgouros, p. 5479

IntroductionThe linear accelerator, the most commonly used device in

clinical radiotherapy practices, generates sparsely ionizing radia-tion in the form of X-rays or electrons (1). The penetrant butdispersed ionization tracks are maneuvered to conform to theshape of the target tumors using multiple incident beams thatsuperpose to effect tumor cell death (2). However, ionizationtracks rely on a confluence of variables to confer death. Tumorcells sustain lethal damage only when two or more lesions takeplace within one or two helical turns of a DNA strand (3–5). Thefrequency of clusters of damage also varies based on the orien-tation of the DNA, its compactness, and the probabilistic trajec-tories of the ionization tracks (6). Contributing to the uncertainty,the genetic variation across and within distinct cancer types hasbeen shown tomodulate the risk of tumor cellular death by eithermitigating damage or facilitating its repair (7). The mere condi-tional ability of sparsely ionizing radiation to cause lethal DNAdamage is reflected in the overall incomplete clinical local controlacross several cancer types (8).

Shortly after the discovery of theX-ray in 1895,Marie andPierreCurie described the activity of radium and its more potent phys-iologic properties compared with X-rays (9). Short exposures ofradium to the skin produced inflammation that exhibited similareffects to thoseobtainedaftermuch longer exposure toX-rays. Thissuggested that radium's emitted particles are substantially morepotent than sparsely ionizing X-rays. It is now evident that short-range charged a-particles emitted by radium induce clusteredDNA damage along their tracks, resulting in significantly moreeffective cell death per unit of absorbed dose (10–12). Despitethe qualitatively observed potency, the exact relative biologicaleffectiveness of a-particles compared with sparsely ionizing radi-ation across a panel of cancer cells remains unknown (13, 14), andit is unclearwhether physical (e.g., cellular size, shape, andnuclearvolume) or cancer genetic variables can modulate the survival oftumors to this more potent form of radiation.

The long half-life (�1,600 years) of radium's most stableisotope, radium-226, precluded radium's medical use for over acentury. However, an artificially generated isotope with a muchshorter half-life, radium-223 (223Ra, half-life ¼ 11.4 days), hasbeen effectively incorporated into routine cancer therapeutic use.223Ra, the first a-emitter approved by the FDA, takes advantage ofradium's bone mimetic properties for the treatment of patientswith castrate-resistant prostate cancer with bone metasta-ses (15, 16). The established clinical efficacy of 223Ra has led tosignificant interest in expanding the use of a-particles to targetcancers other than those that are localized in bone. The treatmentof extraskeletal cancers canbe achieved through the use of targetedradionuclide therapy, which use molecular carriers with highaffinity to antigens on the surface of tumor cells (17–21). Theability to target a-emitting particles to visceral disease is poised toimprove response rates across a range of cancers, including appre-ciably more radiation resistant solid tumors.

1Department of Translational Hematology Oncology Research, Cleveland Clinic,Cleveland, Ohio. 2Research and Development, Pharmaceuticals, Bayer AG,Berlin, Germany. 3Department of Radiation Oncology, Cleveland Clinic, Cleve-land, Ohio.

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

Corresponding Author: Mohamed E. Abazeed, Cleveland Clinic, 2111 East 96thSt, NE6-315, Cleveland, OH 44195. Phone: 216-212-0599; Fax: 216-636-2498;E-mail: [email protected]

Cancer Res 2019;79:5640–51

doi: 10.1158/0008-5472.CAN-19-0859

�2019 American Association for Cancer Research.

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Despite the potential for the use of targeted a-emitting radio-nuclides in solid tumors, very little is known about the interplaybetween a-particles and tumor cell sensitivity. To date, there havenot been extensive analyses of the cellular vulnerability toa-particle radiation within or across cancer types. Inhibition ofDNA double-strand break repair has been shown to sensitizecancer cells to a-particle treatments (22, 23), suggestingthat the composition of the cancer genome could regulatesensitivity. However, a genetic basis for response to a-particlesacross distinct tumors has yet to be established. An improvedunderstanding of the relationship between the sensitivity oftumors to a-particles and their cellular and genetic character-istics can more appropriately inform their future application.These could include the tailoring of a-particle prescriptions andschedules, biomarker-guided patient selection, and/or usingmore precise drug/a-particle treatments.

Here, we develop an integrated imaging, microdosimetric,cellular, and genomic high-content platform that measures thesurvival across a diverse panel of tumor cells following exposureto a-particles and leverages cancer genetic data for biomarkeridentification.

Materials and MethodsCell culture and irradiation

Cell lines from the Cancer Cell Line Encyclopedia (CCLE)were authenticated per CCLE protocol (24) and grown inrecommended media supplemented with 10% fetal bovineserum (Thermo Fisher Scientific) and 100 U/mL penicillin,100 mg/mL of streptomycin, and 292 mg/mL L-glutamine (Corn-ing). Immortalized bronchial epithelial BEAS-2B cells werepurchased from ATCC and maintained in advanced DMEM/F12 media (Thermo Fisher Scientific) supplemented with 1%fetal bovine serum and 100 U/mL penicillin, 100 mg/mL ofstreptomycin, and 292 mg/mL L-glutamine. All cultures weremaintained at 37�C in a humidified 5% CO2 atmosphere andtested to ensure absence ofMycoplasma. Plates were treated withg-radiation delivered at 0.85 Gy/minute with a 137Cs sourceusing a GammaCell 40 Exactor (Best Theratronics) or radium-223 dichloride (223RaCl2). The specific activity of 223Ra is 1.9MBq/ng. The six-stage-decay of 223Ra to lead-207 (207Pb)occurs via short-lived daughters, and is accompanied by anumber of a, b, and g emissions with different energies andemission probabilities. The fraction of energy emitted from223Ra and its daughters as a-particles is 95.3% (energy range,5.0–7.5 MeV). The fraction emitted as b-particles is 3.6%(average energies are 0.445 MeV and 0.492 MeV), and thefraction emitted as g-radiation is 1.1% (energy range, 0.01–1.27 MeV).

High-throughput proliferation assayCells were plated using a Multidrop Combi liquid handler

(Thermo Fisher Scientific) in at least 6 replicates at a singlepreviously determined optimal cell density (range, 30–1600cells/well) in a white 96-well plate with opaque walls (Corning).Plates were irradiated with a single dose of 137Cs or a continuousdose of 223RaCl2 with treatments delivered 24 hours after plating.Where indicated, hydroxyapatite (Sigma, H0252) was diluted(1:20,000) and added to the 100 mL of cell culture media at thetime of seeding. At 9 to 11 days after irradiation, media wereremoved, and 50 mL of CellTiter-Glo reagent (50% solution in

PBS; Promega) was added to each well (25). Relative lumines-cence units (RLU) were measured using an Envision multilabelplate reader (PerkinElmer) with a measurement time of 0.1seconds. Luminescence signal is proportional to the amount ofATP present. The luminescence signal was plotted as a function ofcell density, and a cell density within the linear range for lumi-nescence (or growth) was selected to generate integral survivalvalues for each cell line.

Clonogenic survivalCells were plated at appropriate dilutions, irradiated,

and incubated for 7 to 21 days for colony formation. Colonieswere fixed in a solution of acetic acid and methanol 1:3 (v/v)and stained with 0.5% (w/v) crystal violet as previouslydescribed (26). A colony was defined to consist of 50 cells orgreater. Colonies were counted digitally using ImageJ software asdescribed (27).

Integral survival and relative biological effectivenessThe integral area under the curve (AUC) was estimated by

trapezoidal approximation. The survival values for each trapezoidwere multiplied by the dose interval, [f(X1)þ f(X2)/2] � DX, andsummed. To avoid differences in relative biological effectiveness(RBE) along the shape of the dose response curves (e.g., D50, D10,or Do), values were calculated as the ratio of (AUCg/AUCa) foreach cell line. To relate our estimates with RBE values reported byothers, we also calculated RBE using D37.

MicroscopyTumor cells were plated in a 96-well half area high-content

imaging glass bottom microplate (Corning) at a density of 500to 2,000 cells/well. Wells were pretreated using poly-D-lysine(Sigma) 2 hours before cellular plating. Cells were then fixedand permeabilized using the Image-iT Fixation/PermeabilizationKit (Thermo Fisher Scientific). After fixation, the cytosol andnuclei were stained using actin green probes (green; ThermoFisher Scientific) and DAPI (blue) or propidium iodide (red;Thermo Fisher Scientific), respectively. For each tumor cell line,at least 2 wells and 4 images per well (8 images in total) werecaptured at �4 magnification using a Cytation 1 cell imagingmultimode reader (BioTek). All images were processed manuallyusing the ImageJ software. Tomeasure the diameter of single cells(rather than clusters of cells), a thresholdwas set for theminimumandmaximumpixel area size to exclude clusters of cells. Estimatesof cytoplasmic and nuclear diameter were made by circular orellipsoid fitting, outlining the chosen pixel area based on fluo-rescence intensity. The average cytoplasmic and nuclear diameters(the average of the major and minor axes in the case of ellipsoidfitting) of at least 100 cells per tumor cell line were calculated andtheir radii were used as input for microdosimetric calculations.

Cellular microdosimetryIn the decay chain of 223Ra, a total of four high-energy daughter

a-particles (219Rn, 215Po, 211Bi, and 211Po) and two beta decays(211Pb and 207Tl) are generated, with 207Pb as the final stable endproduct. We used the microdosimetry schema proposed byRoeske and Stinchcomb for calculations of absorbed a-particledose, D, in Gy, for given a-particle energies, our source location,and individual target sizes (28). The values for the input quan-tities are calculated such that D ¼ n x z1, where n is the averagenumber of hits to the target and z1 is the average of the single-hitspecific energy (energy deposited per unit mass). z1 values were

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either explicitly tabulated or obtained by linear interpolation foreach cellular andnuclear radii pair. For the source-target geometrywhere the source is the medium outside the cell, its volumewas taken to be one cm3 with the target cell at the center.This choice for the source volume allows for using thecumulated activity per cm3 such that S is equal to the absorbeddose per unit of cumulated activity (Gy*cm3/Bq*s). We used

S ¼ 10�12pðz1Þ½Rar2 þ ð23Þfr3 þ ðs2 � r2Þ3=2 � s3g�, where Ra isthe range of the a-particle, and s and r are cellular and nuclearradii, respectively. In our system, all of our cells adhered to thebottom of the well so we applied a correction of 0.5 to the S-valueto take into account the exclusively hemispheric irradiation (onlyfrom the top). In the presence of hydroxyapatite, the correctionwas not required due to the spherical distribution of irradiation;the cells are fully immersed in the matrix. For cells that had anellipsoid shape, a 0.9 correction was applied. This correction isderived from the relationship between S and electron energy foran ellipsoid geometry and is based on an average particle range of�60 mm and its corresponding electron energy. We usedD ¼ A x S to calculate the absorbed dose for each a-particle.The cumulated activity for 223Ra at initial activity A0 was calcu-lated usingA ¼ A0e�lt , where l ¼ �0:693=Thalf-life, and integrat-ed for the duration of exposure. The cumulated activityfor each a-particle emitting daughter was calculated usingA2 ¼ ð l2

l2� l1ÞA0ðe�l1 t � e�l1tÞ, where l2 is the decay constant for

thedaughternuclideandl1 is thedecay constantof223Ra.Weset the

decay constant l1 to 223Ra for all daughters because the activity ofthe daughters is short-lived and dependent on the half-life of 223Ra,which is significantly greater than 219Rn, 215Po, and 211Bi (3.97 s,1.78 ms, and 2.14 min, respectively). In addition, although there issome initial activity attributed to the daughters, we assume that thedaughter nuclides have zero activity at zero time. Because the half-life of 223Ra >> 219Rn, 215Po, and 211Bi, the activity contributed bythe daughters immediately upon cellular irradiation is negligiblecompared with the overall accumulated activity.

Variant generation in lentiviral vectorsWe performed mutagenesis in three steps: PCR, in vitro recom-

bination and transformation. Briefly, the gene ORF was PCRamplified by using primers that contain incorporated mutatedsequence. Fragments were then transferred directly to thedestination vector (pLEX306 or pLEX307) by LR recombination(Invitrogen) and the constructs were transformed into competentcells. The discontinuity at the mutation site was repaired byendogenous bacterial repair mechanism. After virus infection(multiplicity > 1), BEAS-2B cells were selected in the presence of1 mg/mL puromycin.

Western blot analysisWhole-cell lysatesweremadeusingM-PER lysis buffer (Thermo

Fisher Scientific). Proteins were separated on 4% to 12% bis-trisSDS-PAGE gels with MOPs buffer and transferred onto 0.45mmol/L nitrocellulose (Thermo Fisher Scientific). Blots weredeveloped with ECL Prime Western blotting detection reagent(Amersham/GE Healthcare). Anti-PIK3CA (clone C73F8, #4249,1:2,000), anti-AKT (#9272, 1:2,000), antiphospho-S473-AKT(#9271, 1:1,000), anti-HER2 (clone D8F12, #4290, 1:2,000),antiphospho-Y1248-HER2 (#2247, 1:1,500), and anti-b-actin(clone 13E5, #4970, 1:5,000) were from Cell SignalingTechnology.

Information-based association scoreThe association between genomic alterations [e.g., mutations

or somatic copy-number alteration (SCNA)] or single-samplegene set enrichment analysis (ssGSEA) profiles for each gene setand the radiation response profile was determined using theinformation coefficient (IC; refs. 25, 29, 30).

Genetic dataCancer cell lines were profiled at the genomic level and pro-

cessed as described in detail (24). The processed data are avail-able for download at http://www.broadinstitute.org/ccle. Briefly,mutation information was obtained by using massively parallelsequencing of exomes.Genotypeswere transformed to categoricalvalues (mutation ¼ 1, no mutation ¼ 0) and were used as inputto compute the IC.

Genotyping/copy-number analysis was performed usingAffymetrix Genome-Wide Human SNP Array 6.0. Raw Affyme-trix CEL files were converted to a single value for each probe setrepresenting an SNP allele or a copy-number probe using aGenePattern pipeline (31) and hg18 Affymetrix probe annota-tions. Copy numbers were then inferred based upon estimatingprobe set specific linear calibration curves, followed by normal-ization by the most similar HapMap normal samples. Segmen-tation of normalized log2 ratios [specifically, log2(CN/2)] wasperformed using the circular binary segmentation algo-rithm (32), followed by median centering of the segmentvalues to a value of zero in each sample. Next, quality checkingof each array was performed, including visual inspection ofthe array pseudo-images, probe-to-probe noise variationbetween copy-number values, confidence levels of Birdseedgenotyping calls, and appropriate segmentation of the copy-number profiles (33). Finally, the Genomic Identification ofSignificant Targets in Cancer (GISTIC) algorithm was usedto identify focal regions of copy-number alterations in indi-vidual samples (34). A gene-level copy number was alsogenerated, defined as the maximum absolute segmented valuebetween the gene's coordinates, and calculated for allgenes using the hg18 coordinates provided by the refFlat andwgRna databases from the UCSC Genome Browser (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/). Sepa-rate binary variables representing amplifications (above 0.7)and deletions (below �0.7) were generated based on theGISTIC gene-level copy-number output described above. Thesebinary amplification/deletion variables for each gene were usedas input to compute the IC against the radiation sensitivityphenotype.

mRNA gene expression was measured by RNA-seq. RPKMvalues were used as input to calculate the ssGSEA enrichmentscores based on the weighted difference of the empirical cumu-lative distribution functions of the genes in the set relative to thegenes not included in an individual set (35). The result is a singlescore per cell line per gene set, transforming the original data setinto amore interpretable higher-level description. Gene sets wereobtained from the C2 subcollection of the Molecular SignaturesDatabase (MSigDB), an additional collection of oncogenic sig-natures, and other cancer-related gene sets curated from theliterature, resulting in a data set that has 5,826 pathway profilesfor each sample (36). ssGSEA values were used as input tocompute the IC.

The nominal P values for the information-based associationmetric scores between the genetic parameters (alterations or

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Cancer Res; 79(21) November 1, 2019 Cancer Research5642



http://www.broadinstitute.org/ccle

http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/




ssGSEA scores) and radiation response scores were estimatedusing an empirical permutation test.

ResultsDevelopment and validation of a high-content a-particleirradiation survival platform

We profiled 28 cancer cell lines comprising 10 cancer types bymodifying an established high-throughput profiling platform forcellular survival after g irradiation (7, 25). To profile the responseof cancer cells to a-particles in a format amenable to high-throughput profiling, we first determined the proliferating frac-tion (mean RLU at dose x/mean RLU of control) as a function ofdose for all cell lines using optimized growth measurements in a96-well plate (Fig. 1A). We calculated the integral survival valuefor each cell line and compared it with the corresponding valueafter single fraction exposure to sparsely ionizing g (137Cs) irra-diation (Supplementary Fig. S1). We showed that although celllines that weremore resistant to g irradiation were generally morelikely to be less sensitive toa-particles, 223Rawasmore effective ininducing cellular growth delay and/or death in all 28 cell lines(Fig. 1B and C). There were notable exceptions to the overallcorrelation above and below the trendline. These, in part, includ-ed less sensitive cells, including two classes of small cell lungcarcinomawith neuroepithelial andmesenchymal differentiation(DMS53 and DMS114) and a breast carcinoma cell line withERBB2 amplification,AR overexpression, and a canonical PIK3CAmutation (HCC202). Interestingly, these and other relativelymore resistant cells had a longer cellular doubling time (Pearsonr ¼ 0.60, R2 ¼ 0.35, and P ¼ 0.03; Supplementary Fig. S2). Wetested the impact of the duration of exposure to a-particle on therelative sensitivity of cells with short (HSC4, RERFLCAI, andHCT15) or longer doubling times (LNCaP and BT474). Reduc-tion of the exposure time to 24 hours, with time to readout at 8 to10 days after washout, did not significantly alter the relativesensitivity of the five cell lines (Supplementary Fig. S3). Thesedata indicate that the relationship between doubling time andradiation sensitivity is not merely related to the experimentaldesign and that cellular repopulation is not a major determinantof decreased sensitivity up to 10 days after radiation treatments.

We next examined whether the high-throughput platformcorrelated with clonogenic survival following exposure to 223Ra.Mean integral survival values for 12 cell lines (for each cell line,n � 2) was calculated and compared with values from theclonogenic assay (Fig. 1D). Proliferation and colony integralsurvival values were significantly correlated, with Pearson r ¼0.78, R2 ¼ 0.61, and P ¼ 0.003. Therefore, the high-throughputplatform accurately profiles cancer cell lines for response toradium and demonstrated significant variation in survival acrossand within several cancer types.

a-Particle microdosimetry and cellular morphologyIn our experimental system, our targets (nuclei and cyto-

plasms) receive dose frommedium outside of the cell from 223Raand its daughters over 9 to 11 days of continuous exposure. In thissetting of cells irradiated in a uniform solution of a-particles, theactivity of the medium alone is insufficient to estimate theabsorbed dose for each tumor cell line. First, the average absorbeddose across a panel of cells with distinct cellular and nucleardimensions, and therefore target size, is likely to vary. Second,because the range of the a-particle is on the same order of

magnitude as the diameter of the target (e.g., the nucleus), notall cells of a particular cell line will receive the same dose.Therefore, a dosimetric framework that can account for intercel-lular and intracellular variation in absorbed dose estimates isessential to interpret biological results.

To accurately estimate the average absorbed dose, we firstcalculated the average specific energy deposited to each tumorcell based on the individual nuclear and cytoplasmic dimensions(i.e., radii). We used fluorescence capture of nuclear and cyto-plasmic staining to calculate the dimensions of each tumor cellline (Fig. 2A). Although we found some variation in the radialdimension and ploidy of tumor cell nuclei, the variation wassignificantly greater for cytoplasms compared with nuclei, s2 ¼3.2 vs. 0.7, respectively (F test, P � 0.001; Fig. 2B). With theseparameters as input, we calculated the average absorbed dose ineach cell line from all a-particle emissions (223Ra, 219Rn, 215Po,and 211Bi; Fig. 2C). We showed that the activity (in kBq) is highlycorrelated with the absorbed dose (in Gy) across the 28 cell lines(R2¼ 0.97, P� 0.001; Fig. 2D). Linear regression showed a slopeof 2.4 0.1, reflecting the lower areas under the curve afteradjusting for absorbed dose.

We also correlated integral survival values using the calculatedabsorbed dose with cellular parameters across the 28 cell lines.Cells with larger cytoplasmic, but not nuclear, radii appearedto correlate with integral survival (Supplementary Fig. S4a andS4b). Specifically, cells with larger cytoplasmic radii were morelikely to be sensitive to a-particles. Correspondingly, nuclear-to-cytoplasmic ratios were also associated with radiation sensitivity(R2 ¼ 0.5, P < 0.0001; Supplementary Fig. S4c). These resultssuggested that cellular shape and morphology may affect cancersurvival after a-particle irradiation.

The osteomimetic hydroxyapatite and the cellular response toa-particles

Hydroxyapatite is a calcium phosphate similar to the humanbone in morphology and composition insofar as it has a hexag-onal structure and a similar stoichiometric calcium-to-phosphateratio (37). Hydroxyapatite has previously been shown to induceosteogenic differentiation from cellular precursors (38) and alterthe transcriptome and proteome of cocultured cells (39). Because223Ra exerts its effect by incorporation into the bone, we sought toassess the impact of hydroxyapatite on cellular sensitivity toa-particles using a coincubation method (Fig. 3A). First, wemeasured the cellular survival as a function of the initial activityof 223Ra in themediumwithout or with hydroxyapatite (Fig. 3B).Both the GI50 (compare Fig. 1A and Fig. 3B) and the integralsurvival values were significantly reduced by the addition ofhydroxyapatite (Fig. 3C). This suggested that 223Ra effectivelybound to the hydroxyapatitematrix andwasmore effective in thissource geometry per unit of activity than as an element insuspension.We then calculated the absorbeddose and consideredthe source geometry after exchange between calciumand 223Ra. Inthis system, irradiation occurs from both the top and bottom ofthe cell (no hemispheric correction; see Materials and Methods).After adjusting for the source geometry, there was no difference inthe overall sensitivity of cells to a-particles without or withhydroxyapatite (Fig. 3D).

Relative biological effectiveness of a-particlesWealso compared the efficacyofa-particle irradiation to that of

sparsely ionizing radiation (137Cs). For each cell line, we used






RBE¼AUCaAUCg

to calculate the relative biological effectiveness of a-par-ticles relative to g irradiation. The median RBE, calculated usingAUC estimates, was 10.4 (IQR: 8.4–14.3). The median RBE,calculated usingD37, was 9.7 (IQR: 4.5–12). These results indicatethat a-particles are significantly more effective in tumor cellkilling than g-rays.

Unlike a-particle emissions, sparsely ionizing radiation causesDNA damage mostly through intermediary reactive oxygen spe-cies. NRF2 and its binding partner KEAP1 are key regulators ofoxidative stress response (40, 41). We previously identifiedmuta-tions in the NRF2 pathway as some of the most highly correlatedwith resistance to g irradiation (7, 25). Accordingly, we sought toassess the relative importance of the oxidative response pathwayin regulating survival after a-particle irradiation. Cell lines withNRF2 (NFE2L2) or KEAP1 mutations were significantly moresensitive to a-particle treatments than g irradiation and wereamong some of the most sensitive cells to these treatments(Fig. 4A; Supplementary Table S1).

To study the effects of NRF2 directly, we expressed NRF2wild-type and T80K in an immortalized human bronchialepithelial cell line (BEAS-2B). Mutation T80K has previouslybeen shown to abrogate binding to KEAP1, resulting in acti-

vation of the NRF2 pathway (42). We showed that overexpres-sion T80K led to an increase in NRF2 protein level, followed bywild-type and then vector control cells (Fig. 4B). Correspond-ingly, NRF2 T80K proteins, and to a lesser extent wild-type,significantly increased the resistance of BEAS-2B cells to girradiation from a 137Cs source (Fig. 4C). In contrast, BEAS-2B cells expressing either T80K or wild-type NRF2 did notdemonstrate significantly different survival measurementscompared with vector alone cells after a-particle irradiation.We note that T80K mutations, and to a lesser extent wild-typeNRF2, contributed to marginally improved survival after a-par-ticle irradiation compared with vector control despite notachieving statistical significance (Supplementary Fig. S5).

Altogether, these results quantify the relative potency ofa-particles and demonstrate that they can effectively lethallydamage cells that are the most resistant to sparsely ionizingradiation at much lower absorbed radiation values.

Integrative genetic profiling of the cellular response toa-particles

We observed significant variation in survival across celllines, on the order of a 32-fold difference between the most

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High-throughput profiling of cellular survival after a-particle irradiation.A, The surviving fraction after treatment with 223Ra for all 28 cell lines. Dose representsthe initial activity of 223Ra. Data are expressed as the means SEM. B, Integral survival of dose (in kBq) was calculated for each cell line profiled by the high-throughput profiling platform after treatment with 223Ra and was compared with integral survival values of the corresponding cell lines after treatment withg-irradiation (137Cs). Data points represent the mean of at least two experiments. The diagonal represents the iso-sensitivity delimiter. C, Integral survival of dose(in kBq) after 223Ra and 137Cs treatments is plotted as box and rotated kernel density plots. , P < 0.001, Wilcoxon test for paired values. D, Integral survival ofdose (in Gy) after 223Ra treatment was measured by the high-throughput profiling (HTP) platform and by colony formation assay (CFA), was analyzed by linearregression, and the R2 value was calculated. Data points represent the mean of at least two experiments.

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sensitive and resistant cells (Fig. 5A). The variation in a-particleresponse juxtaposed with the genetic heterogeneity of theprofiled cell lines suggested that genetic parameters may reg-ulate response to a-particle irradiation. We sought to identifythese putative associations. We used ssGSEA projections as agene set identification tool to find genetic pathways that aredifferentially correlated with radiation response (see Materialsand Methods; Fig. 5B; refs. 25, 35). We also identified genemutations and SCNA that correlated with radiation sensitivityacross all cell lines (Fig. 5C and D). The recent availability ofwhole-exome sequencing data from the CCLE significantlybroadened our association analysis from �1,600 genes to�19,000 (43).

We compared the profiles of each gene set with the a-particleradiation response scores (integral survival). The ssGSEA scoresare displayed in a heat map with the top gene sets that correlatewith radiation survival organized by biological annotation(Fig. 5B; Supplementary Table S2). The top gene sets that corre-lated with reduced radiation sensitivity revealed pathways asso-ciated with breast cancer, cellular signaling and hypoxia. Withinthe breast cancer category, estrogen signaling, the luminal Bsubtype and ERBB2 signaling were associated with reduced sen-sitivity. Interestingly, several individual gene setswithin the breastcategory demonstrate that subtypes with a propensity to metas-tasize to the bone are less likely to be sensitive to a-particles (e.g.,SMID_RELAPSE_IN_BONE_UP).

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D

0.0 0.5 1.0 1.5AUC[Gy]

A

Figure 2.

High-content cellular microdosimetery for 223Ra treatments.A, The cytosol and nuclei for each cell line were stained green and blue, respectively. Magnification,�20. Scale bars, 100 mm. B, Histogram and probability density function of measured radii for all 28 cell lines. C, Schematic depiction of cells exposed to 223Ra anda-particle products along its decay chain. The range for the path lengths of the four particles produced is shown. t¼ 10 days, the average number of days ofa-particle incubation with cells. D, Integral survival calculated from 223Ra initial radioactivity measurements (kBq) was plotted against values calculated from223Ra absorbed dose (Gy).






To assess the association of individual SCNA with radiationresponse, we correlated alterations with radiation survivalusing the IC (Fig. 5C; Supplementary Table S3). Consistentwith the association in ERBB2 cell signaling by ssGSEA, ERBB2amplification was also associated with decreased sensitivity toa-particle treatments. In fact, ERBB2 amplification scored sec-ond out of 46,637 potential gene-level SCNA associations (IC¼ 0.592, P � 0.001). Consistent with these results, overexpres-sion of ERBB2 has previously been shown to confer therapeuticresistance, and trastuzumab, a monoclonal antibody that inter-feres with ErbB2, sensitizes ErbB2-expressing cells to sparselyionizing radiation (44).

PIK3CA variants reduced cellular sensitivity to a-particleirradiation

To assess the association of individual mutations with radia-tion response, we correlated mutations with radiation survivalusing the IC (Fig. 5D; Supplementary Table S4). We associatedwhole-exome data, which included sequencing data from18,750 genes, with cellular survival to 223Ra. We identified muta-

tions in PIK3CA as one of the top genes associated with decreasedsensitivity to irradiation (IC ¼ 0.402, P ¼ 0.03), which ranked29th overall. We mapped the individual PIK3CA mutations on alinear protein coordinate and delineated its domains (Fig. 6A).E545K, E542K, andH1047Rwere well represented in the profiledcell lines, implicating functionally relevant and frequent muta-tions in PIK3CA (45).

We sought to examine the impact of activating mutations inPIK3CA on cellular survival after a-particle irradiation withoutthe confounding effects of varied background genetic altera-tions in the profiled human cancer derived cell lines. To achievethis, we expressed PIK3CA E545K and H1047R in the samegenetically defined, immortalized human bronchial epithelialcell line, BEAS-2B. PIK3CA variants and not wild-type led toconstitutive activation of the PI3K–AKT signaling pathway(Fig. 6b). PIK3CA variant expressing cells had a distinct cellularmorphology compared with wild-type expressing and vectorcontrol cells (Fig. 6C). Importantly, although the nuclear-to-cytoplasmic ratios did not vary substantially based on thegenotype, we accounted for the larger nuclear and cytoplasmic

32100

1

2

3

+Hydoxyapatite (kBq)

−Hyd

oxya

patit

e (k

Bq)

R2 = 0.87m = 1.77

CalciumOxygenPhosphorous

Cell

+ Hydoxyapatite (Gy)

−Hyd

oxya

patit

e (G

y)

R2 = 0.87m = 1.04

1.51.00.50.00.0

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1010.10.010.0010.0

0.5

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Ra223 (kBq/mL)P

rolif

erat

ing

fract

ion

C

Figure 3.

Hydroxyapatite and cellular survival after a-particle irradiation. A, Schematic depiction of cells cocultured with hydroxyapatite. B, The surviving fraction of cellscocultured with hydroxyapatite after 223Ra treatment. Dose represents the initial activity of 223Ra. Data are expressed as the means SEM. C, Integral survivalvalues calculated from 223Ra initial radioactivity measurements (kBq) were plotted with and without hydroxyapatite for each cell line. D, Integral survival valuescalculated from 223Ra absorbed dose (Gy) measurements were plotted with and without hydroxyapatite for each cell line.

A B

0

1

2

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137Cs 223Ra

NFE2L2 del (16-34)KEAP1 E258*

0 2 4 6 80.01

0.1

1C

Pro

lifer

atin

g fra

ctio

n

NRF2 wild-type NRF2 T80K φ

137Cs223Ra

**

KEAP1 G364C

AU

C[G

y]

Gy

**NRF2 alelles

NRF2

Actin

WT_1WT_2

T80K_1T80K_2φ

75 kDa

37 kDa

Figure 4.

Nrf2-mutant cells are effectively treated with a-particle irradiation.A, Integral survival values of cell lines with Nrf2 (NFE2L2) or KEAP1mutations after 223Ra and137Cs treatments are shown. Black bars, median values. B, BEAS-2B cells stably infected with vector alone (f) or vector expressing Nrf2 alleles were profiled forNrf2 protein level by immunoblot. C, BEAS-2B cells from Bwere irradiated with either 223Ra or 137Cs. Data points represent mean SE and are representative ofat least three experiments. P < 0.05 and q value (false discovery rate calculated using the two-stage step-upmethod) <0.01 were considered statisticallysignificant and are denoted by an asterisk.

Yard et al.





radii in PIK3CA mutant and, to a lesser extent, wild-type cellscompared with cells with vector alone in our calculation of theabsorbed dose of 223Ra. Both PIK3CA E545K and H1047R, butnot wild-type, enhanced cellular survival to a-particle irradia-tion (Fig. 6D). We evaluated H1047R using both proliferationprofiling (Fig. 6E) and colony formation assays to confirmthese findings (Fig. 6F and G). These results indicated that

activating mutations in PIK3CA decrease the sensitivity of cellsto a-particle irradiation.

DiscussionWe developed and benchmarked a high-content platform

that measures 223Ra radiation survival across a diverse panel

ELVIDGE_HIF1A_AND_HIF2A_TARGETS_UP

VANTVEER_BREAST_CANCER_ESR1

ELVIDGE_HYPOXIA_DN

CHARAFE_BREAST_CANCER_LUMINAL_VS_BASALSMID_BREAST_CANCER_RELAPSE_IN_BONE_UP

ELVIDGE_HIF1A_AND_HIF2A_TARGETS

SMID_BREAST_CANCER_BASAL_DNSMID_BREAST_CANCER_ERBB2

DOANE_BREAST_CANCER_CLASSES_UPSMID_BREAST_CANCER_ERBB2_UP

SMID_BREAST_CANCER_LUMINAL_B_UPDOANE_BREAST_CANCER_CLASSES

YANG_BREAST_CANCER_ESR1_BULK

HAN_JNK_SINGALINGHALLMARK_PI3K_AKT_MTOR_SIGNALING

NIKOLSKY_BREAST_CANCER_19Q13.1_AMPLICON SMID_BREAST_CANCER_LUMINAL_B

REGULATION_OF_RHO_GTPASE_ACTIVITY

ELVIDGE_HYPOXIA_BY_DMOG_DN

SMID_BREAST_CANCER_RELAPSE_IN_LUNG_DN

REGULATION_OF_RAS_GTPASE_ACTIVITY

0.522

0.523

0.523

0.5240.53

0.536

0.5510.5550.5610.5630.5740.5790.583

0.5840.585

0.60.612

0.627

0.652

0.663 5.94e–05

5.94e–05

7.13e–05

0.673

Survival d (dose)

FDRIC P0.15

0.15

0.2830.0002020.40.000333

Bre

ast

Sig

nal

Hyp

oxia

0.4230.0005820.4230.000618

0.000749 0.4310.4610.001150.4610.00121

0.00133 0.4670.00151 0.5070.00266 0.539

0.5390.003040.00313 0.539

0.280.0001660.4230.000559

0.000559 0.423

0.5390.002380.5390.003140.5390.00311

0.15

SNORD124_AMPSOCS7_AMPERBB2_AMP

KAT7_AMP 0.4330.4580.463

0.592

DM

S114

HC

C20

2D

MS5

3BT

474

MD

AMB4

53VC

APN

CIH

1563

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1755

HSC

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1H

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NC

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66H

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18H

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1SQ

SFSQ

1SK

NM

CR

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0.4670.000798

0.6680.00970.0104 0.668

0.6680.0153

−0.395−0.346

−0.309−0.322

17q12

17q21

MALT1_DELSMAD4_DEL

CDH2_DELSNORD58C_DEL

18q12-2110.042510.032110.0210.00731

MAP3K11 0.486PIK3CA 0.402

0.9760.009580.9760.0382

HDAC9

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0.8920.0043−0.3930.8920.0166−0.339

B

C

D

Num

ber

1.20.80.40.00

2

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10

AUC[Gy]

A

Figure 5.

Gene-expression changes, SCNA, and mutations associated with cellular survival after a-particle irradiation. A, Histogram and probability density function ofcalculated integral survival values of 28 cell lines. B, ssGSEA identifies gene sets that correlate with radiation sensitivity and resistance. Heat map of ssGSEAscores (red, positive; blue, negative). Top gene sets, organized by biological processes, are shown. C,A subset of the top SCNAs that correlate with radiationresistance and sensitivity is organized by chromosomal positions. Red and blue bars represent an SCNA in the corresponding gene that is associated withresistance and sensitivity, respectively. D, A subset of the top genes that, when mutated, were associated with resistance or sensitivity. Red and blue barsrepresent a mutation in the corresponding gene that is associated with resistance and sensitivity, respectively. Heat map of integral survival (red, resistant; blue,sensitive) is parallel to the representations in B, C, and D.






5

0No.

of m

utat

ions

A

p85-BD RAS-BD PI3K C2 PIK

E545K

PI3-P14 kinase

H1047R

0 200 400 600 800 1000 1068 aa

PIK3CA alelles

100 kDa

50 kDa

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37 kDa

PIK3CA

AKT

P-AKT

(S473)

Actin

B

WT_1WT_2

E545K_1

E545K_2

H1047R_1

H1047R_2

φPIK3CA WT

PIK3CA H1047R

WT_1WT_2

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φ

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Gy

φ

φ

Ra223

PIK3CA WTPIK3CA H1047R

φ

PIK3CA WTPIK3CA H1047R

φ

φ

Figure 6.

PIK3CA activation mutations confer decreased sensitivity to a-particles. A, Amap of the PIK3CAmutations identified in the profiled cell lines displayed on theprotein and its domains. B, BEAS-2B cells stably infected with vector alone (f) or vector expressing PIK3CA alleles were profiled for PI3K/AKT pathway activityby immunoblot. C, The cytosol and nuclei for each cell line were stained green and red, respectively.D, Vector alone (f) or vector expressing PIK3CA alleles wereprofiled for survival from 223Ra irradiation. Data are expressed as the AUC and represent the mean SEM of at least three independent experiments. Vectoralone (f), vector expressing PIK3CAwild-type, or H1047R cells were profiled using either the high-content proliferation method (E) or by colony formation assay(F and G). Data points represent mean SE and are representative of at least three experiments. P < 0.05 and q value (false discovery rate calculated using thetwo-stage step-up method) <0.01 were considered statistically significant and are denoted by an asterisk. Representative irradiated colonies in G are from a doseof 0.076 Gy of 223Ra.

Yard et al.





of tumor cells. The platform integrates fluorescence microsco-py, individual cellular microdosimetry, and computationalapproaches that span the preclinical experimental continuumfrom measuring radiation sensitivity to associating cancergenetic data with treatment responses. We used this platformto profile 28 cancer cell lines for response to 223Ra and esti-mated the absorbed dose to each cell line. Critically, the doserange in which we observed a dose response to a-particletreatments across multiple cell lines (the steep aspect of thesurvival curves) significantly overlapped with clinical estimatesof the mean absorbed dose to metastatic tumors in boneafter Ra223 treatments (0.2–1.9 Gy; refs. 46–48). These findings,coupled with the expected significant stochastic variationsof energy deposited within small targets (29), suggest thatsurvival measurements from our platform are likely to beclinically relevant.

We showed that a-particle treatments were significantly moreeffective than sparsely ionizing radiation with an RBE of �10,which is 2-fold higher than its previously estimated magni-tude (13, 14). These results are consistent with the proposeddirect action of the incidenta-particle onDNA and the attenuatedcross-resistance to g irradiation with a-particles compared withthe converse (49). Coupled with approaches to estimate the RBEfor normal tissues (50), more individualized estimates of RBEbased on genomic biomarkers could guide clinical dose schedulesfor a-emitting radionuclides.

Despite the increased sensitivity of cells to a-particles, thedistribution of integral survival measurement across the panelof cells suggested significant underlying cellular and geneticdiversity. Specifically, a trend in association was observedbetween nuclear-to-cytoplasmic ratios and responses to a-parti-cles. This associations appear to be mainly driven by cytoplasmicsize, with larger cells demonstrating higher sensitivity to treat-ments. There is evidence to support amodest contribution towardDNA damage from cytoplasmic irradiation (51, 52). However,the magnitude of the effect that we observed suggests a moresubstantial association between cytoplasmic irradiation andsurvival. It is unclear whether this association is regulated byenergy deposition by an a-particle produced outside of the cellor whether continuous exposure with 223Ra allows for intra-cellular sequestration via exchanges with similar elements (i.e.,calcium, magnesium, iron, and/or copper). For example, thereis some evidence for sequestration of 223Ra by intracellularferritin (53).

We studied the effects of bonymatrix on cellular sensitivity. Wefound that the radioactivity required to effect iso-sensitivity wassignificantly less when cells were grown in the presence of bone-like hydroxyapatite matrix in vitro, indicating that there wasexchange between the hydroxyapatite (calcium) and 223Ra. How-ever, adjustment for the source geometry resulted in a correctedabsorbed dose of irradiation that indicated no significant differ-ences in sensitivity. These results indicate that our measurementof cellular sensitivity is not altered when cells are placed in amatrix that mimics bone. The implication of these findings is thatcandidate biomarkers identified through our profiling effort canpotentially direct the use of a-particles to treat tumors that arelocated in the bone or the viscera.

We identified several gene-expression set determinants ofresponse to a-particles. The identification of breast cancer cellsoverall and mainly those with a propensity to travel to bone,the luminal B subtype (54), as markers of decreased sensitivity

may inform clinical studies of 223Ra in patients with metastaticbreast cancer. We also identified genetic alterations that canpotentially have predictive capacity by identifying the likeli-hood of response to treatments. A subset of these alterations(e.g., ERBB2 amplification) can potentially guide combinato-rial therapeutic strategies because these alterations both con-ferred decreased sensitivity and are targets of approved drugs.Lastly, the oxygen enhancement ratio (OER) of high linearenergy transfer (LET) particles is predicted to be substantiallyless than sparsely ionizing radiation. Nonetheless, the LET ofa-particles is in the range of 60 to 110 keV/mm, resulting in anOER that remains >1 (55). This, coupled with the biologicaleffects of hypoxia beyond oxygen fixation of DNA damage,suggests that hypoxic tumors may remain relatively moreresistant to a-particles than their nonhypoxic counterparts asindicated in our gene set associations.

Importantly, we demonstrated that cells that are among themost resistant to g radiation can be effectively treated witha-particles. The largest radiation cell line profiling effort con-ducted to date revealed that cells with alterations in oxidativestress response, namely, NRF2 and its binding partner KEAP1, arehighly associated with g radiation resistance (7). Our results, inboth cancer cell lines and immortalized human bronchial epi-thelial cells made to express an activating allele of NRF2, indicatethat a-particles can effectively kill these cells. These results canaffect future strategies of radiation dose escalation in tumors witha preponderance of NRF2/KEAP1 alterations. Those includetumors of the head and neck, esophagus, lung, and bladder (56).

We validatedour genetic biomarker platformbydemonstratingthat PIK3CA-activating mutations confer decreased sensitivity toa-particles. This finding is critical because it demonstrates thatsome cancer cells, despite continuous and uniform irradiation,show decreased sensitivity to high LET irradiation. Moreover,these results demonstrate that survival is guided by geneticalterations, indicating that biologically guided radiotherapy willalso be relevant for a-particle treatments. The high frequency ofPI3K pathway alterations across several cancer types presents anopportunity for the combinatorial targeting of tumors with botha-particle and PI3K inhibitors.

In summary, we have established a platform designed forbiomarker and target identification to a-particle treatments. Ourstrategies could guide appropriate patient selection for thesetreatments and result in the improved clinical outcomes forpatients with tumors that are putatively the most resistant tosparsely ionizing radiation treatments.

Disclosure of Potential Conflicts of InterestM.E. Abazeed reports receiving a commercial research grant from Bayer AG,

other commercial research support from Siemens Healthcare Solutions USA,and honoraria from the speakers bureau of Bayer AG. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: B.D. Yard, G. Siemeister, U.B. Hagemann,M.E. AbazeedDevelopment of methodology: B.D. Yard, K. Bannik, M.E. AbazeedAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.D. Yard, P. Gopal, M.E. AbazeedAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B.D. Yard, P. Gopal, G. Siemeister, M.E. AbazeedWriting, review, and/or revision of the manuscript: P. Gopal, G. Siemeister,U.B. Hagemann, M.E. Abazeed






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): U.B. Hagemann, M.E. AbazeedStudy supervision: M.E. Abazeed

AcknowledgmentsM.E. Abazeed was supported by NIH KL2TR0002547, NIH R37CA222294,

and VeloSano.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 17, 2019; revised June 12, 2019; accepted July 29, 2019;published first August 6, 2019.

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2019;79:5640-5651. Published OnlineFirst August 6, 2019.Cancer Res Brian D. Yard, Priyanka Gopal, Kristina Bannik, et al. -Particle Irradiation

αCellular and Genetic Determinants of the Sensitivity of Cancer to

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