Tumor analysis: freeze–thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells

BMC Research Notes, Jun 2018

Objective The use of novel methods to characterize living tumor cells relies on well-conceived biobanks. Herein, we raised the question of whether the composition of fresh and freeze/thawed dissociated tumor samples is comparable in terms of quantitative and qualitative profiling. Results Breast cancer is a heterogeneous disease, encompassing luminal A and B, basal/triple-negative breast cancer (TNBC), and ERBB2-like tumors. We examined living cells dissociated from TNBC and found that a classical freeze/thaw protocol leads to a marked reduction in the number of CD45−CD44LowCD24Low tumor cells. This, in turn, changed the percentage of tumor cells with certain CD44/CD24 expression patterns and changed the percentage of tumor-infiltrating immune cells. These cryopreservation-driven alterations in cellular phenotype make it impossible to compare fresh and frozen samples from the same patient directly. Moreover, the freeze/thaw process changed the transcriptomic signatures of triple-negative cancer stem cells in such a manner that hierarchical clustering no longer ranked them according to expected inter-individual differences. Overall, this study suggests that all analyses of living tumor cells should be conducted only using freshly dissociated tumors if we are to generate a robust scoring system for prognostic/predictive markers.

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Tumor analysis: freeze–thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells

Tumor analysis: freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells tumorcells.This 0 inturn 0 0 UnivRennes , CLCCEugèneMarquis,RueBatailleFlandresDunkerque raisedthequestionofwhetherthecompositionoffreshandfreeze/thaweddissociatedtumorsamplesiscomparable tumor-infiltratingimmunecells.Thesecryopreservation-drivenalterationsincellularphenotypemakeitimpossible transcriptomicsignaturesoftriple-negativecancerstemcellsinsuchamannerthathierarchicalclusteringnolonger - 1,2,3 1,2 1,2* JeanLeveque ,FlorenceGodey andPatrickLegembre intermsofquantitativeandqualitativeprofiling. Introduction concerns related to over-treatment of indolent cancers aggressive and prognoses and clinical outcomes are poor characterization of tumor-infiltrating immune cells and Page2of20 tumor cell heterogeneity is crucial if we are to identify Flowcytometry new prognostic and predictive biomarkers and therapeu- Tumorcells(50,000cells)weresuspendedinPBSsuppletictargets. mented with 2% BSA, 2% FCS, and FcR block (Miltenyi Most,ifnotall,genomicandproteomicstudiesareper- BiotecGmbH)at4 °Cfor20 min.Cellswerethenstained formed using flash-frozen tumor tissues; hopefully, such for 30 min at 4 °C with anti-CD24 PE (clone ML5, BD tissues will yield transcriptomic and/or protein signa- Biosciences), anti-CD44 APC (clone G44-26, BD Bioturesthatcanbeusedtodevelopapersonalizedmedicine sciences), and anti-CD45 PE-Vio770 (clone 5B1, Miltenyi approach. However, the complexity and heterogeneity of Biotec GmbH) antibodies. Isotypic antibodies were used TNBC tumors mean that clinicians have yet to achieve as a control for each fluorochrome (obtained from the this goal. We hypothesized that the use of novel methods same manufacturers). Cells were then washed twice in to exhaustively characterize dissociated and living tumor PBS supplemented with 2% BSA and 2% FCS and resuscells may move us a step closer. For instance, multipa- pended in PBS. To assess cell viability, cells were incurameter flow cytometry (i.e., cyTOF, single cell sequenc- bated with 7-AAD (BD Biosciences) for 10 min prior to ing)canrevealdetailedsignaturesthatareuniquetocells cytometry analysis. Data were acquired using a Novoinside a tumor (e.g., immune, stromal, and tumor cells) cyte cytometer (ACEA Biosciences) and analyzed using and, by so-doing, identify new markers associated with FlowJoorNovoexpresssoftware. relapse, and/or targets for a new generation of therapeutic drugs. Accordingly, laboratories will come to rely on well-conceived biobanks to develop dissociated cancer tissues. Maintext Methodsandpatients Patients Patients (n=15) were diagnosed and treated at the Centre Eugène Marquis between 2017 and 2018. None showed evidence of relapse at the time of diagnosis and none received chemotherapy, endocrine therapy, or radiation therapy prior to surgery. Treatment decisions and follow-up processes were based solely on international recommendations. Tumorsamples Cryopreservationandstorage The freezing process was carried out using standardized freezing procedures, following the guidelines issued by the “Haute Autorité de Santé”, the government agency regulating the French healthcare system, for human tissue and cell samples biobanking. Freshly dissociated tumor cells were frozen in 1 ml of human serum albumin ® (HSA) Vialebex (LFB BIOMEDICAMENTS, Les Ullis, France) supplemented with 10% DMSO (Sigma Aldrich). 6 Each vial contained 2–5×10 cells (depending on the tumor dissociation yield). Briefly, freshly dissociated cell pellets were resuspended in 500 µl of pre-cooled HSA. Then, 500 µl of pre-cooled HSA containing 20% DMSO solution was added drop by drop to the cell suspension. The suspension was then homogenized and transferred to a cryotube. To ensure a standardized and controlled rate of freezing (−1 °C/min), cryotubes were first placed ® Triple-negativebreasttumorswerecollectedbyapathol- in a CoolCell LX Cell Freezing Container (BioCision) at ogistafterresectionbyasurgeonandimmediatelyplaced −80 °C. After 24 h, cells were transferred to a freezer set in RPMI medium. The dissociation process initiated at −150 °C. All freezers were monitored; no critical temwithin 2 h after surgical resection. Tumors were disso- peraturevariationswererecordedduringstorage. ciated using the tumor dissociation kit (Human) (Milte- For thawing, cells were placed in a water bath at 37 °C nyi Biotec GmbH), which is optimized to deliver a high andthentransferredtoRPMI(40 ml)atRTtoallowcomyield of tumor cells and TILs while preserving important plete thawing. After a second wash in 20 ml of RPMI, the cell surface epitopes. TNBC pieces were weighed and cells were counted using a hemocytometer. Cell viability 3 cut into small pieces (<2 mm ), which were then treated wasassessedusingTrypanBlue. withdissociationkit(Human)inagentleMACSDissociator, according to the manufacturer’s recommendations. Briefly, tumors were mechanically dissociated in the gentleMACSdissociatorfor36 sandthenincubatedat37 °C for 30 min under continuous rotation. Next, a cycle of mechanical–chemical–mechanical dissociation was performed and dissociated cells were resuspended in RPMI. ® Macroscopic pieces were removed using a Corning cell strainer (70 μm). Tumor cells were then washed twice in RPMI(20 ml)andcountedusingahemocytometer. Generationofmammospheres Matched freshly dissociated and thawed tumor cells from the same patient were treated in the same way. 6 Cells (1.5×10 ) were seeded in 2  ml of Mammocult medium (StemCell Technologies) supplemented with heparin (4  µg/ml; Stem Cell Technologies), hydrocortisone (480  ng/ml; Stem Cell Technologies), penicillin (100 units/ml), and streptomycin (100 µg/ml) (Gibco) in LeGalloetal.BMCResNotes (2018)11:401 ultra-low binding 6-well plates (Corning). After 15 days of 120 min after surgical resection. Fresh TNBCs were at 37  °C/5% CO , mammospheres were collected and dissociated using the Miltenyi Tumor dissociation kit 2 passed through a cell strainer (40 µm) to separate sus- (Human); 0.8 million cells were analyzed. In paral6 pended cells from mammospheres. Next, mammos- lel, we cultured 1.5×10 cells in serum-free medium in pheres were dissociated with trypsin/EDTA (0.05% low-attachment plates to isolate spheroids, which are trypsin; Gibco) for 5 min. Dissociated cells were washed enriched in CSCs (see “Methods and patients” section). twice in PBS and stained as described above. RNA The remaining cells were frozen using a standardized extracted from mammospheres using the NucleoSpin procedure following the French “Haute Authorité de RNA XS extraction kit (Macherey–Nagel), according to Santé” guidelines to biobanks and kept for 2 weeks in a themanufacturer’srecommendations. Microarrayanalysis freezer at −150 °C. The cells were then thawed and analyzed immediately by flow cytometry; spheroids were also generated. Total RNA was isolated from fresh and RNA quality was assessed using an RNA6000 nano chip thawed spheroids and the transcriptomic signatures (Agilent). For each condition (fresh or freeze/thawed), compared(seeAdditionalfile 1:FigureS1). 9 ng of RNA was reverse transcribed using the Ovation Cell viability was assessed by 7-AAD staining. The PicoSL WTA System V2 (Nugen, Leek, The Nether- results showed that the freezing/thawing procedure for lands).FragmentedcDNAswerehybridizedtoGeneChip dissociated TNBC cells yielded 25.4% dead cells, with Human Gene 2.0 ST microarrays (Affymetrix), which five dissociated tumor samples showing a drop in absowere scanned by a GeneChip Scanner 3000 7G (Affym- lute numbers of 10% or less, and four tumors showing etrix). Raw data and quality-control metrics were gen- more than 50% dead cells (Fig. 1a) after thawing. The erated using Expression Console software (Affymetrix). reasons for this were unknown since all samples were Probes were mapped using Brainarray V23 CDF files treated in a similar manner. Strikingly, most of the cells (http://brainarray.mbni.med.umich.edu/) and normal- that died following the freezing/thawing process origiized by robust multi-array averaging with R software. nated from the CD45− population (Fig. 1b), which in Statistical analyses were performed using the limma R turnimpactedthepercentageoftumor-infiltratingleukoLow Low and a P value of 0.05 were considered significant. Gene oftumorcellswithaCD45−CD24 CD44 phenotype Ontology terms enrichment analyses were performed fell markedly after thawing (Fig. 1c, d). Two hypotheses using the ToppGene Suite (Chen J, Bardes EE, Aronow mayexplaintheselosses:eitherthesecellsarehighlysenBJ, Jegga AG 2009. ToppGene Suite for gene list enrich- sitive to freezing/thawing, or they are reprogrammed to ment analysis and candidate gene prioritization. Nucleic exhibit a different CD24/CD44 phenotype. The percentAcids Research https://doi.org/10.1093/nar/gkp427). age of dead cells observed in Fig. 1a and the short delay Hierarchical clustering was performed using Morpheus between thawing and immunophenotyping are not comMatrix visualization and analysis software (https://softw patible with the latter hypothesis, strongly suggesting are.broadinstitute.org/morpheus/). Accessionnumbers GEOdatabaseaccessionIDGSE114359. Results Low Low that CD45−CD24 CD44 cells do not tolerate the freeze/thaw process well. Loss of this population resulted High Low mainly in artificial enrichment of the CD24 CD44 Low High (Fig.  1d). Because the CD24 CD44 lineage was enriched after the freeze/thaw cycle, and this cell subset Raw and normalized data have been deposited to the cell population and, albeit to a lesser extent, the We raised the question of whether the composition of seems to correspond to stem/progenitor cancer cells [5, fresh and freeze/thawed samples from the same patients 6], we next wondered whether the transcriptomic sigwassufficientlysimilarintermsofquantitativeandquali- nature of these enriched CSCs varied after the freezing/ tative profiling and asked whether both could be used to thawing process. To compare the transcriptome of CSCs investigate TILs (CD45-positive cells) and the pheno- derivedfromfreshandfrozensamples,weusedstringent neg Low High type (CD45 CD24 CD44 ) of CSCs. To address cell culture procedures to culture spheroid cells. Comthis, we used multiparameter flow cytometry to com- pared with the bulk of dissociated tumor cells, spheroids Low High pare the phenotypes of 15 freshly dissociated TNBCs were enriched for CD24 CD44 cells (Fig. 2a). Next, and their matched cryopreserved counterparts. Briefly, we examined freshly dissociated TNBC cells from three to minimize mortality due to sample processing time, different TNBC patients, along with the cells from the the pathologist collected samples in RPMI medium and same patients frozen immediately after dissociation and the dissociation process was started within a maximum cryopreserved for 15 days before thawing. Both sets of a ) % ( LeGalloetal.BMCResNotes (2018)11:401 CD24LowCD44Low; P<0.0001**** o s i iso Fig.1 Flowcytometryanalysisoffreshlydissociatedandfrozentumorsamples.aLeftpanelsviabilityoffresh(upper)orfreeze/thawed(lower) tumorcellswasassessedusingthenon-permeantfluorescentDNAmarker7-ADD.Thegateshowsthepercentageoflivingcells.Rightpanels (n=15).DensityplotshowingthepercentageoflivingtumorcellsexpressingCD24andCD44beforeandaftercryopreservation CD45−tumorcellsshowingCD24andCD44beforeandafterthefreeze/thawprocess.PvalueswerecalculatedusingapairedStudent’sttest cells were cultured for 15 days in serum-free medium in role in organization of cell junctions and are lost during High Low low-adherence plates to select CSCs (CD44 CD24 ) freeze/thaw of CSCs, we suggest that cryopreservation and the transcriptomic signatures were compared either spares breast cancer stem cells showing the most (Fig. 2b). Unexpectedly, unsupervised hierarchical clus- dedifferentiated mesenchymal profiles or favors EMT tering of these tumor cells ranked tumors according to reprogramming(Fig. 2candAdditionalfile 2:Table S1). whether they had been subjected to freeze/thaw treatment and not according to their inter-individual dif- Discussion ferences; this shows that freeze/thawing the cells had Although one study did examine the impact of cryoa deleterious effect in their transcriptomic signatures. preservation on gametes and embryos, this study appears Gene Ontology terms enrichment analysis revealed that to be the first to examine phenotypic changes caused by the freeze/thaw process drove deregulation of 274 genes freeze/thawing living cells isolated from cancer patients (-fold change>2, P<0.05); most of these were associated [7]. to epithelial to mesenchymal transition (EMT), tissues Although molecular oncology has opened new avemorphogenesis and cell junction and adhesion (Fig. 2c). nues to classifying human cancers from a molecular Of these, 224 genes (82%) in freeze/thaw spheroids were standpoint, a number of issues associated with hetsignificantly down-regulated when compared with those erogeneous genomic platforms limit their ability to in fresh spheroids( Table 1). Because these genes play a identifying signatures capable of predicting biological LeGalloetal.BMCResNotes (2018)11:401 Dissociatedtumorcells Spheroidcells 0.2 1 0.3 a 4 2 D C Fig.2 Transcriptomicsignaturesoffreshandfreeze/thawedCSCs.aFlowcytometryanalysisoffreshlydissociatedtumorcellsandspheroids derivedfromthetumorafter15daysofcultureinlowserum/lowadherenceconditions.Thedotplotisrepresentativeofdataobtainedfromall 15tumors.bHeatmapshowingRNAexpressionbyspheroidsderivedfromfreshorfrozentumorcellsfromthreedifferentpatients.Hierarchical clusteringwasperformedonRobustMulti-ArrayAverage(rma)-normalizedexpressiondatausingtheOneminusPearson’scorrelationmethod. cOverall,274genesshoweddifferentialexpressioninspheroidsisolatedfromfreshandfrozentumors.NetworkrepresentationofGOterms termswerespatiallyclusteredandcolorsrepresentcorrectedp-values behavior and/or identifying new molecular targets for should be conducted using fresh tumors only; in paralmore effective and less toxic therapeutic interventions lel, tumor biobanks should develop validate methods of [8]. We identified a novel issue using data meta-analysis freezing living cells isolated from resected tumors that based on profiling of fresh and frozen tumor samples; preserves tumor heterogeneity. In this way, we may be our findings suggest that it will be difficult to identify able to generate robust scoring systems for prognosrobust signatures transposable to patients if freeze/ tic, predictive, and therapeutic markers. Such a system thawed dissociated cancer tissues are used. is an unmet clinical need with respect to patients with We strongly discourage the use of frozen dissoci- TNBC. ated tumor sample for accurate cell population characterization, as thawing creates a disequilibrium in the Limitations proportion of immune and tumor cell subpopulations. Although, the freeze–thaw shock seems mainly to In addition, because the transcriptomic signatures of affect immune cells, a more detailed analysis would be mammospheres derived from frozen/thawed samples required to further investigate whether some minor are not representative of those of initial CSCs, this immune subsets could be affected by this stress and study rules out their use for identification of new prog- thereby, could also alter the conclusions drawn from nostic and/or therapeutic targets. We strongly suggest analyses performed using freeze/thaw living cells. that future studies involving dissociated tumor cells .386 /x14obCAD iiifrrtcee404onnnZgp ,llllrcaaSeRuonANm -/x3411obCD .433− .483− ,llllrcaaSeRuonANm -/x24611obCD .443− .424− ilfryaee1Bbmmm iiirrtttcaeeonnngp iiifrrtcee605onnnZgp ,llllrcaaSeRuonANm -/x26811obCD ilkSe1PRCA ta ta ta ta ta iillffsyyaa1ubQmm irc5187RoANM ,seee49uondgp .424− .453− frrttcaoohgw illrttvaeeeePdd itsuunbA iir-tceooonnnndgp 461RAN lfrrtssycaaaeen iiilllf-kyae62ongAGm iillfrrrtycaaSee22uom irte5onp r-tssaSee62Puuoobm iiifrrtcee564onnnZgp iiirrttsscaaaeeVonndU iillr-ttsccLeeeoohhn .058 iillfrrrtycaaSee31uom .483− .058 illfrrtssycaaaeee2nn il-tssyycLaooohhhdpp iifrre2uoonbNm iilrrtttskycaaaEuoonn .058 iiiifrtttcaaE4oonn iiirte1onnndgpb ir-c17RoANM .254− .240− .130− 2PRRD ir-txFe3oonpb iitca2onnng irttssaaee6Poohhnpp .483− .058 .030 .288− .250− .140− 62PPPR ir-aeRondpANmm iitca2onnng ilrrttsyae2uuuongb iil-caSeuoohnnddmm iilfrrttsycaaPooonbAD iiltcaeeeuuonnndg frtxccaaee2ohng ifre1 irrsycaaae13ohnndbC illr-tssycaauonngm :ll-tccaaePbAGGUND ,l----ttycaaee13bN ree2bmm irttsyae1onhnpb frsaee2 iiiirttsaee15Pohndbp rrsaaTee42nnbpmm iiiffrrttkcae4onngp iillfrrrtycaaSee12uom .483− .058 .030 .288− .260− .140− .404− .276− .211− .180− iiillfrr-tsycaaaSnnpm rtscaee1ud ilfrrrttssaaaeeennm irrrttFeeeeoonnpCm lr-yyeeeohhddddA ilrrrtssaee1oonp ilfsyaaee81ngm ill-txyyceeuoondD ree1bAmm iiirrtttcaee1onnngp lrrrttsycaaeeeuuogd iifr-ce2PRBnnZgAN its2uunbM iittycae1onnngp ,llllrcaaSeRuonANm ,llllrcaaSeRuonANm Ilrr-tsyaaeeonpm ,l3RRRooohgANNm iifrrttsccaoonp /x79obCAAH /x35BobCD .538 .636 ilfrttccaae1uoonn itscae2onA .203− .102− .274− .233− .122− rr-ttyycEeeohmm iiiilrttsaeeuonngp looohgm ii---ssSe1oonhhngpp rrttcaeee3ohpp L010288069CO irrtzccaaeehndU .493− .493− -saePonnGDm .288− .234− .182− ilfryaeeRbCANmmm ,rSee21BRRbAAmm ilfrrrttaeePuooong ilfycaeeoonngm iitsskyce1on ifr--t/sskcaee2uon .449 .501 .453− .473− .424− fr--tsc6Puooohhp .058 .050 .424− .264− .294− .113− lfrrttycaa1uooodm .445 .521 .582 i-sxyaee53ondg (())saee3ngAND+ .404− .058 .030 .227− .218− .194− .473− .058 .020 .289− .228− .105− BZXD illrtxycaeooppm iiitcaa2oonnnngdm ,ilfsyaaTePAAAAm tceoonnpm iijfrttcauooonnnm frtcao ,ifrsaaeennhddA .058 .020 .380− .299− .185− FAND .370− .126− ir-tsaee18Kuondp rrsaaTeennbmm irte238onp ree1bmm ee10ng .428 .651 .493− .058 .030 .297− .370− .126− 7PMM .400 .660 .663 .295− .380− .126− .424− .493− iillfrrrtycaaSee26uom .483− .058 .030 .209− .371− .176− .337 .552 .235− .364− .197− .684 .785 .414− .443− .493− .148− .350 iIr-tsscaae1BoodpA iilkaE1npp iillffsyyaa1Bubmm iillfrrrtycaaSee44uom .414− .058 .040 .226− .388− .169− .371− .474− .261− .389 .400 .256− .435− .281− irrssxLaaTee4nnbmm ilfryaee1bmmm rrtcFee11op .285− .519− .265− L015376425CO ilfrycaeebmmm irrttsceeeeondp irte8onpA ta ta .442− .404− .463− .058 .000 .537− .681− .236− .391− .697− .267− .276− .752− .268− .443− .058 .010 .353− .882− .350− _T134B _T133B _T133B LEESBNM IEETZRND ESCD B taehdw taehdw taehdw .690 .495 .229− .507− .215− PRBAG ta8526_ .331− .918− .392− 424PCD .443− .058 .010 .-1310 .304− .364− .453− .058 .020 .-2913 .352− .336− .-1624 .218− .424− FSPCD .010 .-7626 .339− .497− Additional files andspheroidprotocols. thefreeze/thawprocessinTNBCs. MLG,TMRandAPconductedtheexperimentsandanalyzeddata.VL,PT,JL PLwrotethepaper.PLdesignedtheexperimentsandsupervisedtheproject. Authordetails FlandresDunkerque,35042Rennes,France.3CHUPontchaillou,Rennes, Acknowledgements WearegratefultotheCRBRennesforitstechnicalassistance. Competinginterests Availabilityofdataandmaterials ThestudywasconductedwithhumansamplesandclinicaldataofRennes NFS96900forreceiptpreparationpreservationandprovisionofbiological Consentforpublication Ethicsapprovalandconsenttoparticipate ThebreastCancerCollectionwasdeclaredandapprovedbytheFrench Funding ThisworkwassupportedbyINCaPLBIO,LigueContreleCancerandFoundaSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpub1. CroninKA,LakeAJ,ScottS,ShermanRL,NooneAM,HowladerN,Henley 2. NegoitaS,FeuerEJ,MariottoA,CroninKA,PetkovVI,HusseySK,BenardV, 3. AliHR,ChlonL,PharoahPD,MarkowetzF,CaldasC.Patternsofimmune 4. OskarssonT,BatlleE,MassagueJ.Metastaticstemcells:sources,niches, 5. Al-HajjM,WichaMS,Benito-HernandezA,MorrisonSJ,ClarkeMF.ProSciUSA.2003;100:3983–8. 6. GinestierC,HurMH,Charafe-JauffretE,MonvilleF,DutcherJ,BrownM, JacquemierJ,ViensP,KleerCG,LiuS,etal.ALDH1isamarkerofnormal 7. GardnerDK,SheehanCB,RienziL,Katz-JaffeM,LarmanMG.Analysisof 8. BianchiF,NicassioF,DiFiorePP.Unbiasedvs.biasedapproachestothe ogy.2007;67:64–72.


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Matthieu Le Gallo, Thibault de la Motte Rouge, Amanda Poissonnier, Vincent Lavoué, Patrick Tas, Jean Leveque, Florence Godey, Patrick Legembre. Tumor analysis: freeze–thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells, BMC Research Notes, 2018, 401, DOI: 10.1186/s13104-018-3504-5