The Blebbishield Emergency Program Overrides Chromosomal Instability and Phagocytosis ... · The Blebbishield Emergency Program Overrides Chromosomal Instability and Phagocytosis

Tumor and Stem Cell Biology

The Blebbishield Emergency Program OverridesChromosomal Instability and PhagocytosisCheckpoints in Cancer Stem CellsGoodwin G. Jinesh and Ashish M. Kamat

Abstract

Genomic instability and immune evasion are hallmarksof cancer. Apoptotic cancer stem cells can evade cell deathby undergoing cellular transformation by constructing"blebbishields" from apoptotic bodies. In this study, we reporta novel linkage between genomic instability and phagocytosisevasion that is coordinated by the blebbishield emergencyprogram. Blebbishield emergency program evaded genomicinstability checkpoint, expressed genomic instability–associated genes at distinct phases of cellular transformation,exhibited chromosomal instability, and promoted increase

in nuclear size. Blebbishields fused with immune cells toevade phagocytosis, and the resultant hybrid cells exhibitedincreased migration, tumorigenesis, metastasis, red bloodcell recruitment to tumors, and induced hepatosplenomegalywith signatures of genomic instability, blebbishield emergencyprogram, and phagocytosis evasion to offer poor prognosis.Overall, our data demonstrate that the blebbishield emer-gency program drives evasion of chromosomal instabilityand phagocytosis checkpoints by apoptotic cancer stem cells.Cancer Res; 77(22); 6144–56. �2017 AACR.

IntroductionGenomic instability, particularly chromosomal instability, and

immune evasion are hallmarks of almost all cancers (1). Trans-formed cells and multinucleated cells have been widely impli-cated in tumorigenesis for centuries (2–5). While multinucleatedcells with unstable genome were implicated in tumorigenesis(2, 4), recent studies have termed these tumor-initiating cells ascancer stem cells (6). Thus, the link between cancer stem cellsand tumorigenesis is genomic instability. Studies using multinu-cleated p53-deficient/null cells have further confirmed this con-cept (5, 7).

Cancer stem cells are capable of transformation in soft agar(3), and we have demonstrated that the blebbishield emergencyprogram is required for transformation of apoptotic cancer stemcells (8–16). We previously demonstrated that the blebbi-shield emergency program has two prominent phases. The firstphase is construction of blebbishields by assembling apoptoticbodies to facilitate membrane fusion (8, 9, 17): construction ofblebbishields is not sufficient to evade apoptosis because

blebbishields from bulk cells undergo secondary necrosis at thisstage, whereas only cancer stem cells enter the second transfor-mation phase. The VEGF/VEGFR2/K-Ras/PKC-z/p70S6K axisdrives the second transformation phase (sphere formation fromblebbishields) by overriding secondary necrosis and establishingVEGF autocrine loop (9–11, 13, 18). The transformation phase ischaracterized by fusion between blebbishields (8) or betweenblebbishields and mitotic cells (9) to generate spheroids. Wehypothesized that fusionof blebbishields could generate genomicinstability and multinucleation. Apoptotic cells/bodies are wellknown to be removed by immune cell–mediated phagocytosis(19); it is not known whether blebbishields can evade phagocy-tosis. Therefore, we also hypothesized that if genetically unstablecells can generate tumors in vivo, these cellsmust alsobe capable ofevading phagocytosis.

Here we demonstrate that the transformation phase of theblebbishield emergency program overrides p53-mediated geno-mic checkpoints and helps to pool multiple nuclei under onecommon plasma membrane by cell fusion. Furthermore, wedemonstrate that during subsequent exit phase of blebbishieldemergency program, the transformed spheres release daughtercells with regular sized nucleus or with giant nucleus with chro-mosomal instability. The cells that have undergone the bleb-bishield emergency program exhibit a pan-cancer genomic insta-bility signature. The blebbishield emergency program results inthe increase in the number of cells with ploidy-level DNAcontent with giant nuclei to increase tumorigenesis and aggres-siveness. Importantly, blebbishields fuse with immune cells toavoid phagocytosis by generating hybrid cells that exhibitenhanced tumorigenesis, migration, red blood cell (RBC) recruit-ment within tumors to evade phagocytosis, and metastasis tooffer poor prognosis of tumor-bearing mice. Blebbishieldscause hepatosplenomegaly in vivo with protein expression signa-tures characteristic of genomic instability, phagocytosis evasion,and blebbishield emergency program. Thus, the blebbishield

Department of Urology, The University of Texas MD Anderson Cancer Center,Houston, Texas.

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

Corresponding Authors: Goodwin G. Jinesh, Department of Urology, Unit 1373,The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard,Houston, TX 77030. Phone: 713-792-3250; Fax: 713-794-4824; E-mail:[email protected]; and Ashish M. Kamat, Department of Urology, Unit1373, The University of Texas MD Anderson Cancer Center, 1515 HolcombeBoulevard, Houston, TX 77030. Tel: 713-792-3250; Fax: 713-794-4824; Email:[email protected]

doi: 10.1158/0008-5472.CAN-17-0522

�2017 American Association for Cancer Research.

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emergency program drives the evasion of chromosomal instabil-ity and immune phagocytosis checkpoints by apoptotic cancerstem cells.

Materials and MethodsReagents

Nocodazole (M1404), colchicine (C9754), PKH67 (green;PKH67GL-1KT), PKH26 (red; PKH26GL-1KT), GGTI-298(G5169), LiCl2 (L-4408), and b-actin antibody AC-40 clone(A4700) were purchased from Sigma. Cisplatin (NDC 0015-3220-22; a chemotherapeutic used against bladder cancer cells;ref. 20) was purchased from Bristol Laboratories. Antibodies toFoxP3 (Sc-166212), HO-1 (Sc-10789), IGFBP5 (Sc-6006),VEGF-A (for Western blot: Sc-7269), PKC-z (Sc-17781),galectin-3 (Sc-20157), FBW7/cdc4 (Sc-33196), K-Ras (Sc-30), andVEGFR2 (for Western blot: Sc-504) were purchased from SantaCruz Biotechnology. Antibodies to p70S6K (2708), Ral-A (4799),p53 (2524), RalBP-1 (5739), PAK-1 (2602), and cdc42 (2466)were purchased from Cell Signaling Technology. Rap-1 antibody(MA1-147) was purchased from Thermo Fisher Scientific. Anti-bodies to CXCR4 (Western blot þ FACS; MAB172) and phyco-erythrin-conjugated VEGFR2 (FAB 357P, for FACS) were pur-chased from R&D Systems.

Cells, special cell lines, and cell maintenanceRT4 human bladder cancer cells [ATCC; HTB-2, referred to as

RT4 parental (RT4P) in this study], RT4v6 cells (obtained byserially passaging RT4P cells in mice six times to enrich cancerstem cells; ref. 8), RT4P-BSE-2 cells (RT4P cells that underwentblebbishield emergency program serially twice; ref. 8), PBSHMScells [established in this study after fusion of RT4P bleb-bishields with peripheral blood mononuclear cells (PBMC);see below], and LNCaP prostate cancer cells were cultured inMEM (Sigma: 4655, with L-glutamine) with 10% FBS, pyruvate,nonessential amino acids, vitamins, penicillin, and streptomy-cin supplements. Cell lines were STR fingerprinted as perinstitutional standards, expanded, and frozen. Fresh vials wereused for every 6 months or approximately 25 passages from thefrozen stock. The cells in use were tested for mycoplasmaperiodically using MycoAlert Kit (Lonza). The cell lines usedin this manuscript were maintained from December 2010 toMay 2016 in strict accordance with institutional guidelines.

Isolation of blebbishields and mitotic cells for fusion, live 2D,time-lapse, and transmission electron microscopy

Blebbishields were isolated from RT4P cells as described pre-viously (8). Briefly, 1 � 107 RT4P cells were plated in T-75 flaskswith 10% FBS containing MEM (15 mL of medium per flask) toget complete monolayer in 24 hours. The cells were then exposedto Blebbishield Ejection Medium [BE medium: 10% MEM (Sig-ma: 4655) with 1 mmol/L cisplatin and 20 mmol/L freshlyprepared LiCl2, with essential amino acids and L-glutamine (com-ponents of MEM) but without additional nonessential aminoacids or additional vitamin supplements] for 24 hours. At24 hours after treatment, floating apoptotic blebbishields (butyet anchored to substratum by a stalk) were collected by gentlystreaming the existingmediumover themonolayer using a 10-mLwide-mouth pipette three times. Care was taken during this stepnot to touch the monolayer lawn with the pipette (to avoiddislodging the adherent live cells) and to flush the medium

gently. The medium was collected in flat-bottom tipped 50-mLtubes (which makes resuspending the pellet easier later), and theblebbishields were pelleted down at 1,200 rpm for 3 minutes,resuspended gently with wide-mouthed pipette tips (over-pipetting is avoided at this step), counted, and then subjected toPKH-26 labeling as described below.

Mitotic cells were isolated in parallel (by treating RT4P cellswith 400 ng/mL nocodazole in 15% FBS containing MEM for4 hours: longer durations may induce apoptosis) using thepreviously described mitotic shake-off method (21), pelleteddown at 1,200 rpm for 3 minutes, resuspended gently withwide-mouthed pipette tips (overpipetting is avoided at thisstep), counted, and then subjected to PKH-67 labeling asdescribed below.

PKH labeling was performed as described previously (9).Briefly, isolated blebbishields and mitotic cells were washed with10 mL of serum-free MEM at 1,200 rpm for 3 minutes andresuspended in diluent-C of PKH membrane linker kits. Bleb-bishields andmitotic cellswere linkedwithPKH26-redor PKH67-green, respectively, for 5 minutes while protected from light. Theremainder of the PKH-staining procedure was performed accord-ing to the manufacturer's instructions. The PKH-linked bleb-bishields and mitotic cells were cocultured in equal proportions(1: 1) in 10% FBS containing MEM in 6-well plates and imagedat 16–24 hours.

For DNA staining in spheres [PKH labeling and mitoticcomponents were omitted for propidium iodide (PI) staining],fixed spheres at 4 hours were used for PI staining, and Hoechst-33342 was used at 16 hours in live spheres.

Time-lapse imaging was done using phase-contrast microscopeas described previously with heating stage, humidity, andCO2 (8)using BE medium to image blebbishield formation (using lactateto adjust pH to 6.5 to enhance blebbishield formation) andnormal completeMEM to image the exit phase of the blebbishieldemergency program (cells exiting spheres).

Transmission electron microscopy of freshly isolated bleb-bishields from RT4P cells was described previously (8).

Evaluation of phagocytosis in vitro and generation of PBSHMScells

Blebbishields were isolated from RT4P bladder cancer cells asdescribed above and previously (8) using BE medium for24 hours, labeled with PKH26 red dye as described above andpreviously (9), were plated at equal density with freshly isolatedandPKH67green–labeledPBMCs (fromhumanbuffy coat; bloodbank, The University of Texas MD Anderson Cancer Center), andallowed to undergo phagocytosis or sphere formation for16 hours. A single fusion colony with both dyes was picked andallowed to expand and confirmed for the presence of yellowfluorescence in all cells until 1 week before expansion to establishthe PBSHMS cell line (see text for naming).

Microarray analysisRNAs from RT4P cells, RT4P-blebbishields (apoptotic), bleb-

bishield-mediated transformed spheres (at 4 hours: post-apoptotic but undergone fusion and attachment to substratum),RT4P-BSE-2 cells, PBSHMS cells, and RT4v6 cells were iso-lated using mirVana kit (Ambion) and subjected to whole-tran-scriptome microarray analysis (Illumina, HumanHT-12 V4.0expression beadchip, catalog # BD-103-0204); data were quantilenormalized. The microarray data discussed in this publication

Blebbishields Evade Genomic and Immune Checkpoints

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have been deposited in NCBI's Gene Expression Omnibus (22)and are accessible through GEO Series accession numberGSE98980 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE98980). The top 100 pan-cancer genomic instability–specific genes (significantly associated with genomic instabilitybased on 41 cancer types; ref. 23) were picked from our tran-scriptome data, log-transformed, median-centered, average-linked in Cluster v3.0, and represented as a heatmap using JavaTreeView, to generate the blebbishield emergency programphase-specific expression of genomic instability genes.

To generate the transformation-specific (TS) gene expressionsignature, quantile-normalized transcriptome data of RT4P cells,RT4P-blebbishields, and RT4P-blebbishield-mediated trans-formed spheres (at 4 hours: post-apoptotic) were subjected toSD (gene vector) filtering (cutoff: 2,000), log-transformed, medi-an-centered, and clustered using average linkage method to gen-erate a heatmap. On the basis of this heatmap, genes specificallyup- or downregulated in transformed sample were picked togenerate a heatmap for the TS gene expression signature.

Transformation-specific signature genes and pan-cancer top100 genomic instability–associated genes were independentlycompared among live RT4P, RT4P-BSE-2, PBSHMS, and RT4v6cells to examine the increase in expression with respect to tumor-igenicity. Apart from this, genes specifically up- or downregulatedamong highly tumorigenic cells (RT4P-BSE-2, PBSHMS, andRT4v6 compared with RT4P) were also identified using heatmapsgenerated using parameters similar to those used to generate TSgene expression signature.

To pick top differentially expressed genes between RT4P andPBSHMS cells, we subtracted RT4P gene values from PBSHMSgene values to get the difference in gene expression, sorted on thebasis of values high to low, and picked differentially expressedgenes with relevant functions to generate a heatmap.

Rap-1 and K-Ras activation assaysDH5a bacteria were transformedwith pGEX constructs [pGEX-

RalGDS-RBD [C-terminal 97 residues (24); a gift from Dr. Lawr-ence A. Quilliam, Indiana University School of Medicine, India-napolis, IN] and pGEX-4T control (a gift from Dr. SantoshChauhan,University ofNewMexico, Albuquerque,NM); inducedwith 1 mmol/L IPTG (I6758; Sigma) for 4 hours at 37�C; lysedusing sonication (output: 8; 15-second bursts, twice) in R1Mbuffer [25 mmol/L Tris-Cl (pH ¼ 7.2), 150 mmol/L NaCl, 1%(v/v) NP-40, 5% glycerol, 20 mmol/L MgCl2, 1�-protease inhib-itor cocktail, and 1 mmol/L PMSF]; clarified at 13,000 rpm for10 minutes; bound to glutathione sepharose-CL4B for 1 hour at4�C; washed 6 times using R1M buffer; and then incubated with200 mg/400 mL human cell/blebbishield lysates prepared in R1Mbuffer for 1hour at 4�C.Pulldowncomplexeswerewashed4 timeswith R1M buffer before being subjected to SDS-PAGE andimmunoblotting.

K-Ras activation assay was performed as described previously(11), and the interaction of PKC-zwithGST-Raf1-RBD [aa 1–149;a gift fromDr. ChanningDer (TheUniversity of North Carolina atChapel Hill, Chapel Hill, NC); Addgene-13338; described previ-ously in ref. 25] was considered as assay precision control.

Blebbishield-mediated transformation assay to test Rap-1requirement for transformation

Blebbishields were isolated from RT4P cells using BE mediumas described previously (9) and were allowed to form spheres in

6-well plates for 16 hours with or without 10 mmol/L Rap-1inhibitor GGTI-298. The floating cells were washed off, and theattached spheres were counted.

Tumorigenesis assays, overall survival, and immune/phagocytosis evasion of blebbishields in vivo

RT4P and PBSHMS cells (2 � 105 cells in 100 mL HBSS/mice;n ¼ 8/group) were subcutaneously inoculated in flanks of 3- to5-week-oldmale nudemice (athymic, NCr nu/nu, NCI, Bethesda,MD) to examine tumorigenesis, tumor growth, and overall sur-vival. Overall survival data were plotted using GraphPad Prism,and significance was calculated using log-rank test. Of note, thedeath events in the RT4P group were due to hernia and prolapsedrectum.

To examine immune/phagocytosis evasion of blebbishieldsin vivo, RT4P nonapoptotic cells, RT4P blebbishields (freshlyisolated apoptotic blebbishields as described above), andRT4P-BSE-2 cells (nonapoptotic cells that had undergonetwo rounds of blebbishield emergency program to enrichblebbishield-forming cells; described previously in ref. 8;2 � 105/100 mL HBSS) were subcutaneously inoculated inflanks of 3- to 5-week-old male nude mice (athymic, NCr nu/nu). Mice were euthanized as per Institutional Animal Careand Use Committee guidelines, and selected organs werecollected for further studies.

Immunofluorescence-FACS analysis of IGFBP5, surfaceVEGFR2, and surface CXCR4 and IGFBP5 immunofluorescence

For immunofluorescence-FACS (IF-FACS), cells were brieflytrypsinized, immediately washed with complete MEM, pelletedat 1,200 rpm for 3 minutes, and fixed in ice-cold methanol formore than 24hours at�20�C. The cells werewashed in 1%BSA inPBS thrice at 1,200 rpm for 3 minutes, blocked with 1% BSA inPBS for 30 minutes at 4�C, and incubated in primary antibodies(in blocking buffer: 1:50 for PE-VEGFR2; 1:100 for IGFBP5; 1:120for CXCR4: for IGFBP5 the blocking buffer was supplementedwith 0.3% Triton-X100 to permeabilize cells) for 30 minutes at4�C for VEGFR2; 1 hour at room temperature for IGFBP5; and 40minutes at 4�C for CXCR4. Cells were washed with PBS thrice andincubated with secondary antibodies conjugated with Alexa-555/Cy3 for 1 hour at room temperature. For VEGFR2, no secondaryantibodies were added as the primary antibody is conjugatedwithphycoerythrin. Cells were washed with PBS and then subjected toFACS in FL3 channel (Beckman-Coulter FC500) and analyzedusing Flowjo software.

For IGFBP5 immunofluorescence, the same protocol wasadapted in adherent cells skipping trypsin and centrifugationsteps, but supplementing blocking buffer with 0.3% Triton-X100 to permeabilize cells.

Western blotting (in vivo and in vitro samples) and secretedVEGF-A analysis

Snap-frozen (dry ice) liver and spleen samples (for hepatos-plenomegaly experiment) or subcutaneous tumors (for bloodytumor experiment) were ground using a Teflon Eppendorfhomogenizer in whole cell lysis buffer [50 mmol/L Tris-HCl (pH7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 25 mmol/L NaF, 1%Triton-X 100, 1% NP-40, 0.1 mmol/L Na3VO4, 12.5 mmol/Lb-glycerophosphate, 1 mmol/L PMSF, and 1x-complete proteaseinhibitor cocktail] and vortexed every 10 minutes for a total of40 minutes. Cells/blebbishields were lysed using whole cell lysis

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buffer for 40minutes with vortexing every 10minutes. The lysateswere clarified at 13,000 rpm for 10minutes, quantified using BCAassay reagent, and subjected to SDS-PAGE and transfer to nitro-cellulose membrane before probing with antibodies. The signalswere developed using enhanced chemiluminescence.

For secreted VEGF-A conditioned media and Western blottingwere performed as described previously (8), but used normalMEM instead of BE medium.

CIMICS cytogenetics, comparative chromosome radar scan,and average chromosome number

A total of 1 � 107 cells were subjected to mitotic arrest using20 ng/mL colchicine for 16 hours in 15mLof 10%FBS containingMEM. Themitotic cells were then collected by "mitotic shake-off",pelleted at 1,000 rpm, resuspended with 7mL of 0.067mol/L KClsolution, swelled for 2 minutes, fixed (two step: 7 drops and then7mL of fresh fixative 3:1methanol: glacial acetic acid as describedpreviously; ref. 26), incubated at �20�C overnight, prepared aschromosome spreads in fresh fixative, air-dried, aged at 70�C for1 hour, and stained with modified Giemsa stain using CTGbanding procedure with modifications (PBS, 70% ethanol,95%ethanol, 3dips each, air-dried, 2%Wright'smodifiedGiemsastaining for 5 minutes, 3 distilled water dips and air-dried). Fiftycountable chromosome spreads (there were overlaps in chromo-somes for ploidy with chromosomes >200/cell) were taken intoaccount per cell line for average chromosome number calculationand comparative chromosome radar scan (CCRS). For CCRS, thechromosome numbers of 50 spreads were sorted on the basis ofchromosome number and plotted as a radar plot in MicrosoftExcel 2010. For CIMIC-IS/CS counts, 50� 2 chromosome spreadswere taken into account and paired on the basis of morphologyand length of chromosomes. When all chromosomes had mor-phologically identical chromosomes, the chromosome spreadwas grouped to CIMIC-CS, and were grouped to CIMIC-IS whenthere were odd chromosomes (see text for naming).

Nuclear size and DNA content (nuclear radar scan), andmultinucleation analysis

Cell lines were plated at 5 � 104 cells/mL and 4 mL/well in6-well plates for 24 hours, medium was removed, and cells werefixed with freezing-cold (�20�C) methanol for more than24 hours to block any dye efflux activities of ABC-cassette pumps.Methanol was washed off using PBS, and cells were incubatedwith PI and nonyl-acridine orange (NAO) solution (10 mg/mL PIwith 0.2% sodium citrate, 0.2% Triton-X100 and 100 nmol/LNAO in PBS; 3 mL/well of 6-well plate) for 40 minutes at roomtemperature to allow saturation of DNA staining. Cells wereimaged at constant exposure (200ms/f) for PI staining, and NAOwas imaged to assess multinucleation status as a tracker ofcytoplasmic mitochondria. Nuclear area (calculated by compar-ing the area of a box created using the scale bar and represented inmm2) and PI intensity (DNA content) were quantified usingImageJ software (n¼ 1,000). PI intensities were normalized withrespect to nuclear size (so as to reflect actual low-intensity or high-intensity nuclei within the same frame, below or above the valuesof nuclear size, respectively) andplotted as a radar plot using Excel2010. The red lines represent DNA content, and black linesrepresent nuclear size in mm2. If the red line is visible toward thecenter/hub, it denotes the nuclei with low DNA content (nuclearhypertrophy), and if the red line is visible towards the outer ring,it denotes the nuclei with high DNA content. We fixed 250 mm2

as a cut-off point that matches the nuclear size increase totumorigenicity.

Statistical analysesStatistical analyses were performed usingMicrosoft Excel 2010.

Statistical significance was determined using Student t test withtwo-tailed distribution and two-sample unequal variance, andP values below 0.05 were considered significant. Error bars rep-resent SEM. For statistical details on survival curves, please see thetumorigenesis assay section.

ResultsDynamics of DNA, plasma membrane, and nuclei duringblebbishield emergency program–mediated cellulartransformation

Cancer stem cells undergo transformation after apoptosisthrough the blebbishield emergency program (8, 9, 11). Thestatus of the nucleus during the blebbishield emergency programis not yet understood. We used microscopy to study the status ofthe nucleus during the blebbishield emergency program. Duringthe first blebbishield construction phase, (Fig. 1A; SupplementaryMovie S1), multiple nuclei with nuclear membrane (Fig. 1B), ornuclear area without nuclear membrane (Fig. 1C) were foundwithin blebbishields using transmission electron microscopy.During the second transformation/sphere formation phase, [byfusion of blebbishields either with other blebbishields (8) or withmitotic cells (9)], the outer plasma membrane of the sphere wasderived fromboth fusionpartners, that is, fromblebbishields, andmitotic cells (Fig. 1D). At early stages of transformation (4 hours:from the timeof freshly isolated blebbishields allowed toundergotransformation), the DNA was distributed as a nucleoid in trans-formed spheres (Supplementary Fig. S1A). However, time-lapsemicroscopy revealed that beyond 12–16 hours, the transformedspheres harbored a nucleoid at the center and multiple nuclei atthe periphery, from which individual polarized cancer cellswere released at the polarization front (Fig. 1E; SupplementaryFig. S1B; Supplementary Movie S2). We called this third phase ofthe blebbishield emergency program as the "exit phase." A similartransformation from blebbishields and exit phase was alsoobserved in LNCaP prostate cancer cells (SupplementaryFig. S1C; Supplementary Movie S3).

Together, these results demonstrated that in the blebbishieldemergency program, transformed spheres harbor multiple gen-omes under one limiting membrane and release polarized nucle-ated cancer cells during the exit phase.

Genetic signatures of blebbishield emergency program: p53suppression overrides genomic instability checkpoint

As blebbishields and transformed spheres contained multiplenuclei, the blebbishield emergency programmay lead to genomicinstability. Therefore, we generated transcriptome profiles ofblebbishields (including sphere-forming and nonsphere-formingblebbishields), transformed spheres (sphere-forming bleb-bishields collected at 4 hours of transformation), and nonapop-totic control RT4P bladder cancer cells and examined the tran-scriptomes for expression of the top 100 genomic instabilitysignature transcripts (based on 41 human cancer types; ref. 23)during the blebbishield emergency program. We found that thesetranscripts were regulated in a stage-specific manner in nonapop-totic cells, blebbishields, and transformed spheres (Fig. 2A).






We further generated a blebbishield-mediated TS gene expres-sion signature to understand the mechanisms by which theblebbishield emergency program evades genomic instabilitycheckpoints. This TS gene expression signature included upregu-lation of transcripts related to stemness, cell fusion, apoptosis(both pro- and antiapoptotic), cell adhesion (VEGFR2 activationand focal adhesion), autophagy, malignancy, and xenobioticmetabolism (Fig. 2B; Supplementary Table S1). Only two proa-poptotic transcripts (DDIT and ATF4) were downregulated in theTS gene expression signature (Fig. 2B).

Of the 39 transcripts in the TS gene expression signature,RAP1GAP transcript drew our attention for functional validationbecause RAP1GAP protein activates Rap-1, a molecule requiredfor reattachment of mitotic cells (27) and activation of VEGFR2(ref. 28; driver of transformation from blebbishields; ref. 9).Rap-1 was specifically activated in blebbishields (Fig. 2C), andits inhibition with GGTI-298 blocked transformation by 77.5%�0.19% (Fig. 2D), which validated the requirement of Rap-1activation for blebbishield-mediated transformation.

Further analysis of the TS gene expression signature led us tofocus on LGALS3 transcript (encodes galectin-3; mediates cellfusion and immune evasion), and associated p53 (genomic

instability checkpoint master switch, known to suppress LGALS3to induce apoptosis; ref. 29). In addition, p53 collaborates withFBW7 to eliminate cells with genomic instability (30). Hence, weexamined the protein-level expression of FBW7, p53, and galec-tin-3 in nonapoptotic RT4P cells, sphere-forming blebbishields(surviving blebbishields), and nonsphere-forming/floating bleb-bishields (dead blebbishields). FBW7 was expressed only in cellsthat underwent apoptosis (spheres and dead blebbishields),whereas p53 was selectively suppressed in sphere-forming bleb-bishields, resulting in the expression of galectin-3 (Fig. 2E).

Together, thesedata demonstrated that blebbishield emergencyprogramexhibits genomic instability signature andoverrides p53-mediated genomic checkpoint to retain galectin-3 expression, andutilize Rap-1/VEGF/VEGFR2 signaling for reattachment duringtransformation.

Blebbishields evade phagocytosis and acquire migratorybehavior by fusion with immune cells

Apoptotic cells/bodies are phagocytosed by immune cellsbecause apoptotic cells expose "eat-me" signals (31). Galectin-3 induces apoptosis in tumor-associated immune cells (32) andmediates cell fusion (33). These facts, together with our finding

Figure 1.

Nuclear amalgamation during transformation phase and release of mononucleated cells during exit phase of blebbishield emergency program. A, Time-lapsephase-contrast images of blebbishield formation (arrow) from RT4P cancer cells. B and C, Transmission electron micrographs showing multinucleatedblebbishields with nuclear membrane (B, white arrows) and nuclear envelope breakdown (C, black arrows). N, nucleus; C, cytoplasm. D, PKH67-labeledmitotic cells (green) and PKH26-labeled blebbishields (red) fuse to undergo transformation (sphere formation). The outer plasma membrane is contributedby both mitotic cells and blebbishields (arrows). E, Spheres in exit phase exhibit a central nucleoid with mixed DNA and peripheral nuclei at the polarizationfront (PF). PM, plasma membrane; DIC, bright field. Supplementary Movie S2 shows exit of polarized cells from transformed spheres. Blebbishield andmitotic cell isolation time was considered as 0 hour.

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that transformed spheres expressed galectin-3, raised the possi-bility that blebbishields may induce apoptosis in immune cellsand fuse with them to avert phagocytosis. We tested this conceptby coculturing PKH67-green–labeled human PBMCs (whichinclude a number of professional antigen-presenting cell typesand phagocytes) with PKH26-red–labeled blebbishields fromRT4P cancer cells. Interestingly, blebbishields fused with PBMCs,and the fused cells showed transformed phenotype with numer-ous filopodia protruding from spheres, demonstrating that bleb-bishields exposed "fuse-to-me" signals rather than "eat-me" sig-nals (Fig. 3A). We picked one of the transformed blebbishield–PBMC hybrid spheres to establish a hybrid cell line, which wenamed "RT4P-blebbishield to human peripheral blood mono-nuclear cell hybrid sphere-derived" (PBSHMS) cells (Fig. 3A).

Both PKH dyes were tracked up to 1 week from the time of colonypicking to ensure that all PBSHMS cells harbored membranecomponents from both blebbishields and immune cells (Fig.3A). We next examined the expression levels of the essentialcomponents of the blebbishield emergency program [secretedVEGF-A, VEGFR2, RalBP-1, Ral-A, K-Ras, cdc42, PAK-1, p70S6K(9), and PKC-z (13)] and galectin-3 in RT4P and PBSHMS cells.We found that increased expression of total VEGFR2 and reducedexpression of PKC-z isoform-2 were hallmarks of PBSHMS cellswithminor increase in surface VEGFR2-positive cell number (Fig.3B). Of note, blebbishield formation by itself does not result inreduction of PKC-z isoform-2 expression (13). Furthermore,PBSHMS cells had higher baseline activation of oncogenic K-Rasthan RT4P cells (Fig. 3C; Supplementary Fig. S2A and S2B).

Figure 2.

Blebbishield emergency program overrides genomic checkpoint and drives genomic instability. A, Expression of top 100 pan-cancer genomicinstability–associated transcripts in RT4P live cells, blebbishields (at 0 hours of isolation), and transformed spheres (at 4 hours) examined by microarray.The genes specifically upregulated or downregulated in each phase of the blebbishield emergency program are listed in the boxes at right. B, Generation ofTS gene expression signature and functional classification of TS signature genes. C, Rap-1 activation assay showing specific activation of Rap-1 inblebbishields BE medium, cells treated with BE medium (includes blebbishields and live cells). BD cells� , blebbishield-depleted cells; NS� , nonspecificGST background. D, GGTI-298, a Rap-1 inhibitor, blocks transformation from BE medium–generated blebbishields. E, Expression of genomic checkpointregulators (FBW7 and p53) and galectin-3 (p53-suppressed gene) in live RT4v6 cells, transformed spheres, and blebbishields (BS) that did notsurvive (floating BS�) by Western blotting. (Note: RT4v6 is capable of enhanced transformation from blebbishields compared with RT4P cells; ref. 8).






We further investigated the differences in gene expressionbetween RT4P and PBSHMS cells by microarray transcriptomeanalysis. We found that PBSHMS cells had upregulation oftranscripts implicated in genomic instability, cell survival/death,stemness, cell adhesion/migration, phagocytosis (includes don'teat me signals), metabolism, and proliferation (Fig. 3D; seeSupplementary Table S2 for complete list).

We chose to validate the top altered IGFBP5 transcript becauseboth IGFBP5 and VEGFR2 are linked to migration (34, 35). Wefound that 70.2% � 0.4% of PBSHMS cells but only 14.0% �0.9%of RT4P cells were IGFBP5 positive (Fig. 3E), suggesting thatblebbishield–immune cell fusion results in increased migration.Furthermore, IGFBP5 was expressed more in PBSHMS cells withmigratorymorphology (Fig. 3E) andPBSHMS cells acquiredmoredramatic migratory phenotype compared with RT4P cells

(Supplementary Movie S4). More interestingly, the cells withmigratory morphology had multiple nuclei (migratory cells fromSupplementary Movie S4 marked in Fig. 3E).

Together, these results demonstrated that blebbishields evadephagocytosis by fusion with immune cells and that the bleb-bishield-immune cell hybrids have increased expression ofVEGFR2 and IGFBP5, leading to increased migration of multi-nucleated cells, and decreased expression of PKC-z isoform-2.

Blebbishield emergency program drives novel CIMICS typegenomic instability

We next examined the numerical alterations in mitotic chro-mosomes of RT4P and PBSHMS cells by CCRS to test whether cellfusion leads to chromosomal instability. CCRS in mitotic cellsrevealed that nearly 34% of PBSHMS cells but only 4% of RT4P

Figure 3.

Blebbishields fuse with immune cells to evade phagocytosis: signatures of blebbishield-immune cell hybrid cells. A, Establishing PBSHMS cells duringan in vitro phagocytosis assay. RT4P blebbishields evaded phagocytosis by fusion with human PBMCs and showed transformed phenotype (top row) withnumerous protruding filopodia (arrows). One of the transformed blebbishield-PBMC hybrid spheres was picked to establish PBSHMS cell line (RT4P-blebbishieldto human PBMC hybrid sphere-derived cells; bottom); at 1 week, both PKH dyes were detected in all cells. Arrows, weaker green label. B, Expression of componentsof blebbishield emergency program in RT4P and PBSHMS cells by Western blotting (�sVEGF-A is secreted VEGF-A analyzed using conditioned media; left) andsurface VEGFR2 FACS showed the increase in VEGFR2–positive cells in PBSHMS (right). C, Densitometry of K-Ras activation assay to show increased basaloncogenic activation in PBSHMS cells. PKC-z, an interacting partner, was used as an assay precision control (see Supplementary Fig. S2A and S2B). D, Key genesdifferentially expressed between RT4P and PBSHMS cells. A complete list of differences in gene expression is available as Supplementary Table S2. The functionalclassifications of these genes are indicated in the boxes at right. E, Quantification of IGFBP5-positive RT4P and PBSHMS cells by immunofluorescence-FACS (left),IGFBP5 in migrating PBSHMS cells by total immunofluorescence microscopy; arrow, migratory cell; arrowhead, nonmigratory cells (middle); and migration ofmultinucleated cells marked by orange arrows and nuclei by asterisks (right, these panels were taken from Supplementary Movie S4).

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cells had more than 92 chromosomes per cell (>4n, where onechromosome includes both sister arms; Fig. 4A). Average chro-mosome number analysis in mitotic cells revealed that PBSHMScells had nearly double (111.7/cell) the chromosome numbercompared with RT4P cells (60.0/cell; Fig. 4A). We also found thatan in vivo serial passaged and highly tumorigenic version of RT4Pcells named RT4v6 cells (8) had larger genome size and higheraverage chromosome number than RT4P cells, which confirmedthat numerical chromosome gain is associated with cell fusion invivo (Fig. 4A).

We further examined surface CXCR4 expression becauseimpeding CXCR4 results in multinucleation (36). RT4P andPBSHMS cells had similar percentages of surface CXCR4-positivecells; however, 62.4% of PBSHMS cells but only 29.9% of RT4Pcells had high surface CXCR4 expression (Fig. 4B). Cytogeneticobservations revealed that RT4P and PBSHMS cells had novelchromosomal instabilitywithmorphologically identical chromo-some sets (CIMICS).We recognized two types of CIMICS: CIMIC-incomplete set with one or more odd/unpaired chromosomes(CIMIC-IS), and CIMIC-complete set without any odd/unpairedchromosomes (CIMIC-CS; Fig. 4C). CIMIC-IS were detected in25% of RT4P cells and 63% of PBSHMS cells, whereas CIMIC-CS

were detected in 30% of PBSHMS cells, but not in RT4P cells,demonstrating that the blebbishield emergency program drivesCIMIC-CS–type genomic instability (Fig. 4C).

We next examined whether increased CIMICS genomicinstability in PBSHMS cells had any effects in vivo. We foundthat PBSHMS cells generated substantially more tumors onday 9 compared with RT4P cells in nude mice (Supplemen-tary Fig. S3A); furthermore, the tumors from PBSHMS cellsgrew significantly larger and were associated with a signifi-cantly worse prognosis (Fig. 4D). The tumorigenic ability ofPBSHMS cells confirmed that the isolated PBSHMS colony(Fig. 3A) was indeed a result of blebbishield–immune cellfusion rather than blebbishields engulfed by immune cells byphagocytosis. The PBSHMS cells offered much worse prog-nosis than RT4P cells possibly because PBSHMS cells weremore efficient in metastasis than RT4P cells (SupplementaryFig. S3B).

Together, these results demonstrated that blebbishield emer-gency program drives CIMICS-type genomic instability andthat blebbishield–immune cell fusion leads to increasedtumor aggressiveness, and poor prognosis (able to evade immu-nity effectively).

Figure 4.

Blebbishield emergency program drives novel CIMICS type genomic instability, leading to increased tumor aggressiveness and poor prognosis. A, CCRS analysis ofchromosome number in RT4P, PBSHMS, and RT4v6 cells. Note: RT4P is the same in both CCRS as the experiment was done together. Blue font, chromosome spreadnumber. Average chromosome number in mitotic cells (n ¼ 50). B, Surface IF-FACS for CXCR4 in RT4P and PBSHMS cells. The gate segregates CXCR4 low versushigh populations. C, Cytogenetics of RT4P and PBSHMS chromosome spreads to show CIMIC-CS (top) and CIMIC-IS (bottom). Red arrow, odd chromosomes.Percentages of cellswith each type of CIMICS are shownat right (n¼ 50/replicate). Note: the photomicrographs are of differentmagnifications.D, Tumor growth andoverall survival after subcutaneous injection of RT4P and PBSHMS cells at flanks of nude mice (also see Supplementary Fig. S3A–S3C for tumor incidence andmetastasis).






Blebbishield emergency programdrives increase in nuclear sizeto reflect genomic instability and tumorigenic potential ofcancer cells

Cytogenetics gave insight only into mitotic cells, and it wasdifficult to obtain proper chromosomal spread in cells withextremely complex genomes (e.g., cellswith>200 chromosomes).To examine chromosomal/genomic instability also in nonmitoticcells, we developed the nuclear radar scan, which displays thenuclear area (based on 2D imaging), and DNA content (PIfluorescence under constant exposure settings) of 1,000 nucleiper cell line to determine the frequency of cells with large nucleiand high DNA content, reflecting chromosomal instability. Thenuclear radar scan revealed that 28.6%ofPBSHMS cells (cellswitha history of in vitro immune encounter), 40.5% of RT4v6 cells(cells with a history of in vivo immune encounter) and 16.6% of

RT4P-BSE-2 cells (cells that had no immune cell exposure afterprocurement from ATCC but had undergone blebbishield emer-gency program twice; ref. 8) compared with only 6.5% of RT4Pcells had nuclear size above 250 mm2 (Fig. 5A and B). This lowerfrequency of giant nuclei in RT4P cells compared with RT4v6 cellsreflected the poor tumorigenic potential of RT4P cells observedpreviously (8). The presence of super-giant nuclei in RT4P-BSE-2cells demonstrated that the blebbishield emergency programdrives increase in nuclear size. Interestingly, we detected super-giant nuclei in metastatic cells established from mice subcutane-ously injectedwith PBSHMS cells, indicating that these cells couldalso metastasize (Supplementary Fig. S3C). Interestingly,PBSHMS cells, and blebbishields from RT4P cells could causesporadic mesenteric lymphadenopathy (Supplementary Fig.S3D). On the other hand, the proportion of multinucleated cells

Figure 5.

Nuclear size increase and transformation-specific gene expression signature reflects blebbishield-driven genomic instability. A, Nuclear radar scans (top)showing changes in nuclear size (black radial lines) and corresponding DNA content (red radial lines, PI) upon blebbishield-PBMC fusion (in vitro; PBSHMS), cancercell immune exposure (in vivo in mice; RT4v6), and two rounds of blebbishield emergency program (in vitro; RT4P-BSE-2) and corresponding representativephotomicrographs (bottom; note the super-giant nuclei in RT4P-BSE-2). B, Average nuclear size (left), nuclei with size >250 mm2 cutoff (middle), andmultinucleation percentages (right) for the cells in the first panel (n¼1,000). C, TS gene expression signature from Fig. 2B was examined in cDNA microarray dataof live RT4P, PBSHMS, RT4v6, and RT4P-BSE-2 cells, and only the genes upregulated in comparison with RT4P are shown (24/39). D, Expression analysis of top100 pan-cancer genomic instability–associated genes in cDNA microarray data of live RT4P, PBSHMS, RT4v6, and RT4P-BSE-2 cells. Only the altered genes incomparison to RT4P are shown.

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did not correlate with tumorigenicity; for example, RT4v6 was themost tumorigenic but ranked second to PBSHMS in proportion ofmultinucleated cells (Fig. 5B).

Given the correlation between nuclear size and tumorigeniccapacity, we hypothesized that giant nuclei could contribute to TSgene expression signature in live cells. Evaluation of the TS geneexpression signature in live cells revealed that 24 of 39 genes wereupregulated in anyone of the highly tumorigenic cells (RT4P-BSE-2, PBSHMS, and RT4v6) compared with RT4P cells, demonstrat-ing that the giant nuclei indeed contributed to TS gene expressionsignature in these cells (Fig. 5C). We also found that, among thetop 100 pan-cancer genomic instability–associated transcriptscorrelating with increased tumorigenicity, 11 were upregulatedand 18 downregulated in highly tumorigenic cells compared withRT4P cells (Fig. 5D).

Together, these results demonstrated that nuclear size increaseabove 250 mm2 reflects CIMICS/ploidy level genomic instabilityand tumor-initiating cells. This nuclear size cutoff also predicts thecells derived from blebbishield emergency program, whichexpress TS gene expression signature in live cells.

Blebbishields induce hepatosplenomegaly with signatures ofblebbishield-immune cell hybrid, evasion of phagocytosis, andgenomic instability

We further examined the capability of RT4P cells, blebbishieldsfrom RT4P cells (apoptotic), and blebbishield-derived but non-apoptotic cells (RT4P-BSE-2; described previously in ref. 8) toevade the immune systemor phagocytosis in vivo.RT4P andRT4P-BSE-2 cells, but not apoptotic blebbishields, formed subcutane-ous tumors innudemice; however, blebbishields andRT4P-BSE-2cells sporadically formed massive hepatosplenomegaly, suggest-ing that the blebbishields escaped phagocytosis, fused withimmune cells, migrated to lymphoid organs, and failed to under-go clonal deletion (Fig. 6A). Multiple genetic studies have linkedsplenomegaly/hepatosplenomegaly to defects in apoptosis (Sup-plementary Table S3). We thus examined the spleen and liverspecimens for expression of proteins associated with bleb-bishield-immune cell fusion signature (reduced PKC-z isoform-2, Fig. 3B), blebbishield emergency program [e.g., VEGFR2, K-Ras,RalBP-1, Ral-A, cdc42, PAK-1, and p70S6K/p45S6K (9)]; genomicinstability [FBW7 (30), p53 (30), and galectin-3 (23)], phagocy-tosis evasion [through HMOX1/HO-1-dependent hemolysis andinactivation of phagocytes (37, 38)], and hepatosplenomegaly[loss of FoxP3 (39) and CXCR4 (40)]. Increased expression of thecomponents of the blebbishield emergency program was clearlyevident in hepatomegaly specimens but obscured in spleen asRT4P-control spleens already expressed K-Ras, cdc42, and PAK-1(Fig. 6B). However, gain of RalBP-1, loss of PKC-z isoform-2, andgeneration of cleaved p70S6K (p45S6K) in both hepatomegalyand splenomegaly specimens confirmed the intrusion of humancells, blebbishield–immune cell fusion, and existence of theblebbishield emergency program, respectively, in these specimens(Fig. 6B). In support of this, we detected giant andmultinucleatedcells in hepatomegaly and splenomegaly formalin-fixed, paraffin-embedded sections (Supplementary Fig. S4). Furthermore, hepa-tosplenomegaly specimens exhibited reduced p53 expressionassociated with increased galectin-3, HO-1 (HMOX1 gene prod-uct), and FBW7 expression, indicating that the cells in hepatos-plenomegaly evaded genomic instability and phagocytosis check-points (Fig. 6C). Galectin-3 suppresses FoxP3þ Treg cells (41), andloss of FoxP3þ Treg cells leads to splenomegaly (39). In line with

this fact, we found that blebbishield and BSE-2–induced hepa-tosplenomegaly were associated with loss of FoxP3 and gain ofgalectin-3 (Fig. 6C).

Next, we examined whether HO-1 expression in blebbishield-derived cells is linked to RBC recruitment because HO-1 isupregulated in macrophages that engulf RBCs by phagocytosis(42) where heme generation by hemolysis inhibits phagocytosis(37, 38) and because RBCs within the bladder (hematuria) are anearly warning sign for urothelial cancers (Supplementary TableS4). We previously noted that RT4v6 cells can generate bloodybladder tumors (8); however, RBCs within the bladder can havemany different causes, so we chose subcutaneous bloody tumorsfor examining HO-1 expression. HO-1 was upregulated inPBSHMS and RT4P-BSE-2 tumors in vivo (Fig. 6D) and in RT4v6,PBSHMS, and RT4P-BSE-2 cells in vitro (Fig. 6E), confirming thatthese cells recruited RBCs to evade phagocytosis in vivo (Fig. 6F).

Together, these results confirmed that the blebbishield emer-gency program protects apoptotic cancer stem cells from phago-cytosis, prevents clonal deletion of blebbishield-immune cellhybrid cells, promoting genomic instability and phagocytosisevasion by reducing p53 to result in HO-1 expression and asso-ciated RBC recruitment within tumors; and galectin-3-mediatedsuppression of Treg cells to result in hepatosplenomegaly.

DiscussionHuman bladder cancer has an extremely unstable genomewith

nuclear size alterations (43, 44). Serial passaging of cancer cells innude mice enriches cancer stem cells (45) and generates super-giant nuclei (46). We for the first time demonstrate that cancercells acquire chromosomal instability and increase in nuclear sizethrough apoptosis, to provide mechanistic basis for the extremegenomic instability in cancers. In addition, LNCaP prostate cancercells (known to have extreme chromosomal instability; ref. 47)exhibit robust blebbishield emergency program (SupplementaryFig. S1C). However, we continued to use RT4P/RT4v6 cell systemfor further characterization because RT4P/RT4v6 spheres attach tosubstratum much stronger than LNCaP spheres aiding properisolation of sphere-forming and nonsphere-forming bleb-bishields. The weak attachment of LNCaP spheres to substratumis demonstrated in Supplementary Movie S3. We previouslydemonstrated that in vivo serial passaged RT4v6 cells are capableof enhanced tumorigenesis and superior transformation fromblebbishields compared with its original RT4P cells (8), and herein this study we demonstrated the drastic change in nuclear sizeand chromosomenumber between these cells (Fig. 5A andB). Thegeneration of super-giant nuclei in RT4P-BSE-2 cells confirms thedefinitive role of blebbishield emergency program in nuclear sizeincrease (Fig. 5A).

The stage-specific expression of pan-cancer genomic instabilitygenes during the blebbishield emergency program (Fig. 2A), andsuppression of p53 in sphere-forming blebbishields, (Fig. 2E)explain why cells undergoing the blebbishield emergency pro-gram permit extreme CIMICS genomic instability (Fig. 5A and B)and evade clonal deletionof blebbishield–immune cell hybrids toresult in hepatosplenomegaly (Fig. 6C). Galectin-3 suppressesFoxP3þ Treg cells (41), and loss of FoxP3þ Treg cells is associatedwith splenomegaly (39). Thus, p53 suppression-mediated galec-tin-3 upregulation is directly linked to hepatosplenomegaly.Cyclophosphamide induces bladder cancer (48) when p53 func-tion is compromised (49). In addition, defects in apoptosis






are linked to splenomegaly/hepatosplenomegaly before or aftertherapy (Supplementary Table S3). These facts explain whyblebbishields and blebbishield-derived cells (RT4P-BSE-2)induced hepatosplenomegaly. Loss of PKC-z isoform-2 in hepa-tosplenomegaly specimens is specific to blebbishield-immunecell hybrid cells because sphere-forming or nonsphere-formingblebbishields from cancer cells do not show reduction in PKC-zisoform-2 (13).

HO-1 expression and RBC recruitment within tumors in vivoexplain how blebbishields evade phagocytosis, because heme

released from hemolysis induces filopodia-formation and inhibitphagocytes (Fig. 6F; ref. 38) and we previously demonstrated thatblebbishield formation depends on serpentine filopodia (9).Filopodia drives cell fusion (50) and hence filopodia formationis the "fuse-to me" signal on blebbishields that overcomes the"eat-me" signals (Fig. 3A). Or perhaps eat-me signals aid fuse-to-me signals to bring immune cells much closer to apoptotic cancercells. Of note, nonapoptotic and nonmitotic RT4P cells do notexhibit fusion behavior in vitro but may undergo fusion in vivoafter undergoing apoptosis or mitosis. The clinical relevance of

Figure 6.

Blebbishields induce hepatosplenomegaly with protein expression signatures of blebbishield emergency program, phagocytosis evasion, and genomicinstability. A, Subcutaneously injected freshly isolated blebbishields (apoptotic) and RT4P-BSE-2 cells (nonapoptotic but had undergone blebbishield emergencyprogram twice) but not live RT4P cells (control), generated hepatosplenomegaly. Arrow, liver; arrowhead, spleen; note the color change. B, Western blotanalysis of components of blebbishield emergency program (p45S6K, cleaved p70S6K) and signature of immune cell-blebbishield hybrid (PKC-z isoform-2 loss) inliver and spleen specimens. C, Western blot analysis of signatures of genomic instability (FBW7, p53, galectin-3, CXCR4) and immune/phagocytosis evasion(galectin-3, HO-1) in liver and spleen specimens. Note: The RT4P control spleen and liver were from age-matched mice (matched to RT4P-BSE-2, which tookthe longest duration to develop hepatosplenomegaly; 216 days, whereas blebbishields took only 29 days). D, Western blot and photos showing RT4P-BSE-2and PBSHMS subcutaneous tumors form bloody subcutaneous tumors with HO-1 upregulation compared with RT4P controls. E, Western blot showing thatblebbishield emergency program in vitro (RT4P-BSE-2 and PBSHMS) or in vivo (RT4v6) led to increase in HO-1 expression. F, Schematic showing how HO-1expression couples cell fusion, RBC recruitment, hemolysis, and phagocytosis inhibition.

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RBC recruitment in patients before or after therapy is evident inSupplementary Table S4.

Selective suppression of p53 in the blebbishield emergencyprogram explains how the cancer stem cells might evade genomiccheckpoints. The presence of nuclear abnormalities with ploidy-level alterations in bladder cancer patients (44) supports theclinical relevance of our findings on nuclear size and CIMICS.CIMIC-CS can be considered as an indicator of a recent transfor-mation eventmediatedby theblebbishield emergency programasthis type of chromosomal instability was detected in PBSHMScells but not in RT4P cells, whereas CIMIC-IS could be derivedfrom CIMIC-CS as an effect of prolonged in vitro maintenance(Fig. 4C).

In summary, the blebbishield emergency program drivesCIMICS-type genomic instability, increase in nuclear size, andevasion of phagocytosis by apoptotic cancer stem cells. Theseeffects are made feasible by evasion of p53-mediated genomiccheckpoint to enable galectin-3 and HO-1 expression, which inturn permits evasion of immune checkpoints such as phagocy-tosis of blebbishields, and clonal deletion of blebbishield–immune cell hybrids.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G.G. JineshDevelopment of methodology: G.G. Jinesh

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G.G. Jinesh, A.M. KamatAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G.G. JineshWriting, review, and/or revision of the manuscript: G.G. Jinesh, A.M. KamatAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.G. Jinesh, A.M. KamatStudy supervision: A.M. KamatOther (paid for research reagents in part): G.G. Jinesh

AcknowledgmentsThe authors thank Drs. Lawrence A. Quilliam, Channing Der, and Santosh

Chauhan for plasmids (see Materials and Methods), Elsa Maria Rodarte (TexasA&MUniversity, Houston, TX), andDavid J.McConkey (TheUniversity of TexasMD Anderson Cancer Center, Houston, TX) for various reagents and LNCaPcells, Dr. Robert Langley and Mr. Kenneth Dunner Jr. for help with electronmicroscopy, Drs. David P. Pollock and Sun Siping (MD Anderson CancerCenter, Houston, TX) for quantile-normalizing the microarray data, Ms. Ste-phanieDeming for editorial help inmanuscript preparation, andMs. I.-Ling Leefor technical help.

Grant SupportThis research was supported in part by the NIH through MD Anderson's

Cancer Center Support Grant, CA016672, and used the High ResolutionElectron Microscopy Facility.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 23, 2017; revised June 13, 2017; accepted August 22, 2017;published OnlineFirst August 30, 2017.

References1. Negrini S, Gorgoulis VG,Halazonetis TD.Genomic instability–an evolving

hallmark of cancer. Nat Rev Mol Cell Biol 2010;11:220–8.2. Holland AJ, Cleveland DW. Boveri revisited: chromosomal instability,

aneuploidy and tumorigenesis. Nat Rev Mol Cell Biol 2009;10:478–87.3. Langman RE. Transformation and tumorigenesis. Nature 1980;283:246–8.4. Boveri T. Concerning the origin of malignant tumours by Theodor Boveri.

Translated and annotated by Henry Harris. J Cell Sci 2008;121Suppl 1:1–84.

5. Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D.Cytokinesis failure generating tetraploids promotes tumorigenesis inp53-null cells. Nature 2005;437:1043–7.

6. Delude C. Tumorigenesis: Testing ground for cancer stem cells. Nature2011;480:S43–5.

7. Davidson LA, Callaway ES, Kim E, Weeks BR, Fan YY, Allred CD, et al.Targeted deletion of p53 in Lgr5-expressing intestinal stem cells promotescolon tumorigenesis in a preclinical model of colitis-associated cancer.Cancer Res 2015;75:5392–7.

8. Jinesh GG, Choi W, Shah JB, Lee EK, Willis DL, Kamat AM. Blebbishields,the emergency program for cancer stem cells: sphere formation andtumorigenesis after apoptosis. Cell Death Differ 2013;20:382–95.

9. Jinesh GG, Kamat AM. Endocytosis and serpentine filopodia drive bleb-bishield-mediated resurrection of apoptotic cancer stem cells. Cell DeathDiscov 2016;2:15069.

10. Jinesh GG, Kamat AM. Blebbishield emergency program: an apoptoticroute to cellular transformation. Cell Death Differ 2016;23:757–8.

11. Jinesh GG, Molina JR, Huang L, Laing NM, Mills GB, Bar-Eli M, et al.Mitochondrial oligomers boost glycolysis in cancer stem cells to facilitateblebbishield-mediated transformation after apoptosis. Cell Death Discov2016;2:16003.

12. Jinesh GG, Laing NM, Kamat AM. Smac mimetic with TNF-alpha targetsPim-1 isoforms and reactive oxygen species production to abrogate trans-formation from blebbishields. Biochem J 2016;473:99–107.

13. Jinesh GG, Taoka R, Zhang Q, Gorantla S, Kamat AM. Novel PKC-zeta top47 phox interaction is necessary for transformation from blebbishields.Sci Rep 2016;6:23965.

14. Jinesh GG, Kamat AM. Blebbishields and mitotic cells exhibit robustmacropinocytosis. Biofactors 2016;43:181–6.

15. Jinesh GG, Mokkapati S, Zhu K, Morales EE. Pim kinase isoforms: devilsdefending cancer cells from therapeutic and immune attacks. Apoptosis2016;21:1203–13.

16. Goodwin Jinesh G, Willis DL, Kamat AM. Bladder cancer stem cells:biological and therapeutic perspectives. Curr Stem Cell Res Ther 2014;9:89–101.

17. Jinesh GG, Kamat AM. RalBP1 and p19-VHL play an oncogenic role, andp30-VHL plays a tumor suppressor role during the blebbishield emergencyprogram. Cell Death Discov 2017;3:17023.

18. Taoka R, Jinesh GG, Xue W, Safe S, Kamat AM. CF3DODA-Meinduces apoptosis, degrades Sp1, and blocks the transformationphase of the blebbishield emergency program. Apoptosis 2017;22:719–29.

19. Arandjelovic S, Ravichandran KS. Phagocytosis of apoptotic cells inhomeostasis. Nat Immunol 2015;16:907–17.

20. Lee EK, Jinesh GG, Laing NM, Choi W, McConkey DJ, Kamat AM. A Smacmimetic augments the response of urothelial cancer cells to gemcitabineand cisplatin. Cancer Biol Ther 2013;14:812–22.

21. Jackman J,O'ConnorP.Methods for synchronizing cells at specific stages ofthe cell cycle. Curr Protoc Cell Biol 2001;Unit-8.3.

22. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI geneexpression and hybridization array data repository. Nucleic Acids Res2002;30:207–10.

23. Fehrmann RS, Karjalainen JM, Krajewska M, Westra HJ, Maloney D,Simeonov A, et al. Gene expression analysis identifies global gene dosagesensitivity in cancer. Nat Genet 2015;47:115–25.

24. Asuri S, Yan J, Paranavitana NC, Quilliam LA. E-cadherin dis-engagementactivates the Rap1 GTPase. J Cell Biochem 2008;105:1027–37.

25. Brtva TR, Drugan JK, Ghosh S, Terrell RS, Campbell-Burk S, Bell RM, et al.Two distinct Raf domains mediate interaction with Ras. J Biol Chem1995;270:9809–12.

26. Fletcher J. Metaphase harvest and cytogenetic analysis of solid tumorcultures. Curr Protoc Hum Genet 2001;Chapter 10:Unit 10.3.






27. Dao VT, Dupuy AG, Gavet O, Caron E, de Gunzburg J. Dynamic changes inRap1 activity are required for cell retraction and spreading during mitosis.J Cell Sci 2009;122:2996–3004.

28. Lakshmikanthan S, Sobczak M, Chun C, Henschel A, Dargatz J, Ramchan-dran R, et al. Rap1 promotes VEGFR2 activation and angiogenesis by amechanism involving integrin alphavbeta(3). Blood 2011;118:2015–26.

29. Cecchinelli B, Lavra L, Rinaldo C, Iacovelli S, Gurtner A, Gasbarri A, et al.Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-inducedapoptosis. Mol Cell Biol 2006;26:4746–57.

30. Grim JE, Knoblaugh SE, Guthrie KA, Hagar A, Swanger J, Hespelt J, et al.Fbw7 and p53 cooperatively suppress advanced and chromosomallyunstable intestinal cancer. Mol Cell Biol 2012;32:2160–7.

31. Ravichandran KS. Find-me and eat-me signals in apoptotic cell clearance:progress and conundrums. J Exp Med 2010;207:1807–17.

32. Zubieta MR, Furman D, Barrio M, Bravo AI, Domenichini E, Mordoh J.Galectin-3 expression correlates with apoptosis of tumor-associated lym-phocytes in human melanoma biopsies. Am J Pathol 2006;168:1666–75.

33. Dalton P, Christian HC, Redman CW, Sargent IL, Boyd CA. Membranetrafficking of CD98 and its ligand galectin 3 in BeWo cells–implication forplacental cell fusion. FEBS J 2007;274:2715–27.

34. Berfield AK, Andress DL, Abrass CK. IGFBP-5(201-218) stimulatesCdc42GAP aggregation and filopodia formationin migrating mesangialcells. Kidney Int 2000;57:1991–2003.

35. Dellinger MT, Brekken RA. Phosphorylation of Akt and ERK1/2 is requiredfor VEGF-A/VEGFR2-induced proliferation and migration of lymphaticendothelium. PLoS One 2011;6:e28947.

36. Kwong J, Kulbe H,WongD, Chakravarty P, Balkwill F. An antagonist of thechemokine receptor CXCR4 induces mitotic catastrophe in ovarian cancercells. Mol Cancer Ther 2009;8:1893–905.

37. Martins R, Maier J, Gorki AD, Huber KV, Sharif O, Starkl P, et al.Heme drives hemolysis-induced susceptibility to infection viadisruption of phagocyte functions. Nat Immunol 2016;17:1361–72.

38. Sioh-Yang T, Wolfgang W. Phagocyte mayHEME caused by severe hemo-lysis. Nat Immunol 2016;17:1335–7.

39. Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al.Selective depletion of Foxp3þ regulatory T cells induces a scurfy-likedisease. J Exp Med 2007;204:57–63.

40. Nie Y, Waite J, Brewer F, Sunshine MJ, Littman DR, Zou YR. The role ofCXCR4 in maintaining peripheral B cell compartments and humoralimmunity. J Exp Med 2004;200:1145–56.

41. Fermino ML, Dias FC, Lopes CD, Souza MA, Cruz AK, Liu FT, et al.Galectin-3 negatively regulates the frequency and function of CD4(þ) CD25(þ) Foxp3(þ) regulatory T cells and influences thecourse of Leishmania major infection. Eur J Immunol 2013;43:1806–17.

42. KovtunovychG, EckhausMA,GhoshMC,Ollivierre-WilsonH, Rouault TA.Dysfunction of the heme recycling system in heme oxygenase 1-deficientmice: effects on macrophage viability and tissue iron distribution. Blood2010;116:6054–62.

43. AlexandrovLB,Nik-Zainal S,WedgeDC,Aparicio SA, Behjati S, BiankinAV,et al. Signatures of mutational processes in human cancer. Nature2013;500:415–21.

44. van Velthoven R, Petein M, Oosterlinck WJ, Zandona C, Zlotta A, Van derMeijden AP, et al. Image cytometry determination of ploidy level, prolif-erative activity, and nuclear size in a series of 314 transitional bladder cellcarcinomas. Hum Pathol 1995;26:3–11.

45. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasisreveals an essential role for RhoC. Nature 2000;406:532–5.

46. Kruczynski A, Pauwels O, Wright M, Delsol G, Kiss R. Spontaneousevolution of nuclear size and DNA content of non-small-cell-lung cancersgrafted onto nude mice. Cytometry 1992;13:586–94.

47. Beheshti B, Park PC, Sweet JM, Trachtenberg J, Jewett MA, Squire JA.Evidence of chromosomal instability in prostate cancer determined byspectral karyotyping (SKY) and interphase fish analysis. Neoplasia2001;3:62–9.

48. Pedersen-Bjergaard J, Ersboll J, Hansen VL, Sorensen BL, Christoffersen K,Hou-Jensen K, et al. Carcinoma of the urinary bladder after treatment withcyclophosphamide for non-Hodgkin's lymphoma.NEngl JMed1988;318:1028–32.

49. Khan MA, Travis LB, Lynch CF, Soini Y, Hruszkewycz AM, Delgado RM,et al. p53 mutations in cyclophosphamide-associated bladder cancer.Cancer Epidemiol Biomarkers Prev 1998;7:397–403.

50. Millard TH, Martin P. Dynamic analysis of filopodial interactions duringthe zippering phase of Drosophila dorsal closure. Development 2008;135:621–6.


Jinesh and Kamat




2017;77:6144-6156. Published OnlineFirst August 30, 2017.Cancer Res Goodwin G. Jinesh and Ashish M. Kamat Instability and Phagocytosis Checkpoints in Cancer Stem CellsThe Blebbishield Emergency Program Overrides Chromosomal

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