Towards a better characterisation of leukemic cells in chronic lymphocytic leukaemia: Cell-size heterogeneity reflects their activation status and migratory abilities

cance s A icle Towa ds a Be e Cha ac e isa ion o Leukemic Cells in Ch onic Lymphocy ic Leukaemia: Cell-Size He e ogenei y Re lec s Thei Ac i a ion S a us and Mig a o y Abili ies Gayane Manukyan 1,2, Zuzana Mikulko a 1, Pe e Tu csanyi 3, Jakub Sa a a 1,4 , Ma ké a T aje o á 1, Zuzana Kubo a 3, Tomas Papajik 3and E a K iego a 1,*   Ci a ion: Manukyan, G.; Mikulko a, Z.; Tu csanyi, P.; Sa a a, J.; T aje o a, M.; Kubo a, Z.; Papajik, T.; K iego a, E. Towa ds a Be e Cha ac e isa ion o Leukemic Cells in Ch onic Lymphocy ic Leukaemia: Cell-Size He e ogenei y Re lec s Thei Ac i a ion S a us and Mig a o y Abili ies. Cance s 2021,13, 4922. h ps://doi.o g/10.3390/ cance s13194922 Academic Edi o : Ma y F ances McMullin Recei ed: 17 Augus 2021 Accep ed: 27 Sep embe 2021 Published: 30 Sep embe 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Depa men o Immunology, Facul y o Medicine and Den is y, PalackýUni e si y and Uni e si y Hospi al, 77900 Olomouc, Czech Republic; [email p o ec ed] (G.M.); [email p o ec ed] (Z.M.); [email p o ec ed] (J.S.); Ma ke a.T aje [email p o ec ed] (M.T.) 2Labo a o y o Molecula and Cellula Immunology, Ins i u e o Molecula Biology NAS RA, Ye e an 0014, A menia 3Depa men o Hema ology-Oncology, Facul y o Medicine and Den is y, PalackýUni e si y and Uni e si y Hospi al, 77900 Olomouc, Czech Republic; pe e [email p o ec ed] (P.T.); [email p o ec ed] (Z.K.); [email p o ec ed] (T.P.) 4Depa men o Compu e Science, Facul y o Elec ical Enginee ing and Compu e Science, VSB-Technical Uni e si y o Os a a, 70800 Os a a, Czech Republic *Co espondence: [email p o ec ed] Simple Summa y: Ch onic lymphocy ic leukaemia (CLL) is a he e ogeneous ch onic disease cha - ac e ised by he clonal expansion o ma u e CD19 + CD23 + CD5 + B-cells in blood, bone ma ow and lymphoid issue. Despi e he CLL umou cell popula ion showing conside able he e ogenei y in cell size, he unc ional cha ac e is ics o leukemic cells ha di e in size ha e no been explo ed. The esul s o ou s udy demons a e ha di e ences in CLL cell size e lec hei ac i a ion s a e, pola isa ion and mig a o y capaci y, wi h la ge CLL cells being mo e ac i a ed, pola ised and mo ile han he small CLL cells p esen in he CLL cell pool. Ou da a p o ide e idence o he impo - ance o cell-size he e ogenei y wi hin he CLL cell pool and he dynamics o cell-size changes o disease pa hogenesis. Abs ac : Ch onic lymphocy ic leukaemia (CLL) is a gene ically, mo phologically and pheno ypically he e ogeneous ch onic disease wi h clinical a iabili y be ween pa ien s. Whe he he signi ican he e ogenei y o cell size wi hin he CLL popula ion con ibu es o he he e ogeneous ea u es o his disease has no been in es iga ed. The p esen s udy aimed o cha ac e ise he pheno ypic and unc ional p ope ies o wo subpopula ions o ypical CLL cells ha di e in cell size: small (s-CLL) and la ge (l-CLL) CLL cells delinea ed by o wa d sca e cy ome y. The s-CLL cells we e cha ac e ised by he CD5 low CXCR4 hi pheno ype, while he l-CLL cells we e cha ac e ised by he CD5 hi CXCR4 dim pheno ype and indica ed a highe exp ession o CXCR3, CD20, CD38 and HLA-DR. The l-CLL cells displayed highe mig a ion ac i i y owa ds CXCL12, a endency owa ds a highe p oli e a ion a e and an inc eased capaci y o p oduce IgM in he p esence o CpG compa ed wi h s-CLL cells. When s imula ed wi h CpG and CXCL12, l-CLL cells we e cha ac e ised by a highe pola isa ion pheno ype and mo ili y han s-CLL cells. Ou s udy e ealed ha he di e ences in CLL cell size e lec ed hei ac i a ion s a us, pola isa ion and mig a o y abili ies. Ou da a p o ide e idence o he impo ance o cell-size he e ogenei y wi hin a CLL pool and he dynamics o cell-size changes o disease pa hogenesis, hus dese ing u he in es iga ion. Keywo ds: ch onic lymphocy ic leukaemia; cell-size he e ogenei y; pool o leukemic cells; pola isa- ion; mig a ion Cance s 2021,13, 4922. h ps://doi.o g/10.3390/cance s13194922 h ps://www.mdpi.com/jou nal/cance s Cance s 2021,13, 4922 2 o 17 1. In oduc ion Ch onic lymphocy ic leukaemia (CLL) is cha ac e ised by he clonal expansion o ma u e CD19 + CD23 + CD5 + B-cells in he blood, bone ma ow and lymphoid issue [ 1 ]. CLL cells a e cha ac e ised by hei pheno ypic, clonal and unc ional he e ogenei y and con ain a pool o es ing and p oli e a ing CLL subpopula ion cells [ 2 – 6 ]. The in e ed exp es- sion o CD5 and CXCR4 has been indica ed as use ul o iden i ying en iched ac ions among ecen ly bo n/di ided and elde ly/quiescen CLL cells [ 2 , 3 , 7 ]. This means ha he ac ion o p oli e a ing, ecen ly bo n/di ided mig a ing CLL cells has been shown o p e e en ially exp ess high le els o CD5, while low le els o CD5 a e associa ed wi h es ing, elde ly/quiescen ci cula ing cells [ 3 , 8 ]. The o e lapping B-cell ecep o (BCR) epe oi es be ween CD5 high and CD5 low cells sugges a dynamic ela ionship be ween hese wo B-CLL cell subpopula ions [ 4 ]. S udies ha e shown ha CXCR4 dim CD5 b igh ‘p o- li e a i e’ cells o e exp ess mo e ‘cell di ision’ genes, while CXCR4 b igh CD5 dim ‘ es ing’ cells exp ess highe le els o ‘an ip oli e a i e’ genes, sugges ing ha he la e subse may ep esen a dis inc sel - enewing ype om which all clonal membe s a e de i ed [3,8]. In addi ion o immunopheno ypic he e ogenei y, CLL cells di e in size and mo phol- ogy. Typical CLL cells a e ma u e small and medium-sized lymphocy es wi h clumped ch oma in su ounded by a hin ing o cy oplasm [ 9 ]. A small numbe o s udies ha ha e analysed he clinical signi icance o a ypical CLL mo phology ha e e ealed he ad e se p ognos ic signi icance o inc eased p olymphocy e numbe s and hei associa ion wi h ma ke s o poo p ognosis, hus p edic ing a sho e p og ession- ee su i al [ 9 , 10 ]. Howe e , no in o ma ion is a ailable on he cha ac e is ics o ypical CLL cells based on hei size. This s udy aimed o analyse he biology o small (s-CLL) and la ge (l-CLL) CLL cells delinea ed by o wa d sca e (FSC) low cy ome y, wi h a p ima y ocus on ac i a ion s a us, immunopheno ype, p oli e a ion, pola isa ion and mig a ion. 2. Ma e ials and Me hods 2.1. Pa ien s and Sampling Pe iphe al blood samples we e collec ed om 23 pa ien s wi h CLL (16 males and se en emales; a median age o 65 yea s). A diagnosis o CLL was es ablished acco d- ing o he In e na ional Wo kshop on Ch onic Lymphocy ic Leukaemia guidelines [ 11 ]. O he en olled pa ien s, eigh we e ea men -naï e, six had been p e- ea ed wi h im- munochemo he apy and nine had been ea ed wi h no el agen s (ib u inib (IBR), idelal- isib). The de ailed clinical cha ac e is ics o he en olled pa ien s a e shown in Table 1. An addi ional eigh CLL pa ien s we e e alua ed be o e he ini ia ion o he IBR ea men and a e 8 weeks o he apy. Table 1. Cha ac e is ics o he CLL pa ien s. Pa ame e s CLL Pa ien s (n= 23) Age in yea s: median (min–max) 65 (51–84) Gende : male/ emale 16/7 Whi e blood cell coun (×109/L): median (min–max) 112.3 (26.7–498.3) Pe cen age o lymphocy es in he pe iphe al blood: median (min–max) 92.8 (75.7–98.8) Time om diagnosis o sampling (in mon hs): median (min–max) 96 (6–200) Bine s age: A/B/C 6/5/12 Bulky lymphadenopa hy ≥5cm: yes/no 5/18 Splenomegaly: yes/no 4/19 IgHV mu a ional s a us: unmu a ed/mu a ed 15/8 Cance s 2021,13, 4922 3 o 17 Table 1. Con . Pa ame e s CLL Pa ien s (n= 23) Gene ic cha ac e is ics: TP53 dis up ion (dele ion 17p and/o TP53 mu a ions): yes/no/n.a. 6/13/4 del(11q22): yes/no/n.a. 7/15/1 del(13q14): yes/no/n.a. 3/18/2 T ea men : yes/no 15/8 P e ious immunochemo he apy: 6 Time om he end o ea men o sampling (in mon hs): median (min–max) 22 (4–68) Cu en ea men wi h iBCR (ib u inib/idelalisib): 9 (8/1) Time om he s a o iBCR ea men o sampling (in mon hs): median (min–max) 3 (1–10) Legend: n.a. = no a ailable; iBCR = B-cell ecep o (BCR) signalling inhibi o s. Nex -gene a ion sequencing was used o he de ec ion o TP53 mu a ions, Sange sequencing o IgHV mu a ional s a us and cy ogene ics and FISH analysis o dele ion 17p and o he abe a ions, as p e iously epo ed [12–14]. 2.2. Cell-Size E alua ion o CLL Cells The CLL cells o he pa ien s we e e alua ed by pe iphe al blood smea s a e s aining using he May–G ünwald–Giemsa p o ocol. The smea s we e e alua ed wi h cellSens En y (Olympus Li e Science) imaging so wa e and he CellaVision DM96 (Sysmex Co - po a ion, Kobe, Japan) au oma ed image analysis sys em a magni ica ions o 600 × and 1000×, espec i ely. 2.3. Su ace Exp ession o Ma ke s on Small and La ge CLL Cells The CLL cells om all he blood samples we e s ained wi h op imal concen a ions o an ibody combina ions and di ec ed agains he ollowing su ace an igens: CD183(CXCR3)- FITC, CD20-PE, CD5-Pe CP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all p ocu ed om BioLegend), as p e iously e- po ed [5,15]. Iso ype-ma ched an ibodies (BioLegend) we e used as nega i e con ols. The de e mina ion o s-CLL and l-CLL cells was conduc ed using FSC da a and a back- ga ing s a egy. The analysis was pe o med using a BD FACSCan o II (Bec on Dickinson) ins umen , and da a acquisi ion was pe o med using BD FACSDi a so wa e ( .8.0.2; Bec on Dickinson). Flow cy ome y da a we e analysed using FlowJo .X0.7 so wa e (T ee S a , Inc., San Ca los, CA, USA). In all he expe imen s, a minimum o 10,000 e en s was coun ed. The esul s we e exp essed as a pe cen age and mean luo escence in ensi y (MFI) . 2.4. In acellula ZAP-70 S aining Whole blood cells we e labelled wi h CD3-FITC, CD5-Pe CP-Cy5.5 and CD19-APC- Cy7 an ibodies. The ea e , hey we e ea ed wi h ixing and pe meabilising eagen s (BioLegend) acco ding o he manu ac u e ’s ins uc ions, and hey we e hen s ained wi h he PE-conjuga ed ZAP-70 p o ein (clone 1E7.2, BioLegend). The quan i y o ZAP- 70-posi i e CLL cells was es ima ed using iso ype con ol and esidual ZAP-70-posi i e T-cells (T me hod). Th ough he T me hod, ZAP-70 was posi ioned close o he le edge o he T-cell clus e in a ZAP-70/CD3 plo , which was applied o calcula e he pe cen age o CLL-posi i e cells [16]. 2.5. Cell-Cul u e Expe imen s Pe iphe al blood mononuclea cells we e isola ed using densi y-g adien cen i uga- ion. CLL cells we e hen u he sepa a ed by luo escence-ac i a ed cell so ing (FACS) using a FACSA ia Fusion cell so e (BD Biosciences), which is used o cul u ing, apop osis, Cance s 2021,13, 4922 4 o 17 mig a ion, pola isa ion and p oli e a ion s udies. CLL cell so ing esul ed in pu i y > 95% o CD19 + CD5 + cells, as de e mined by low cy ome y. So ed and pai ed s-CLL cells ( he smalles 15% o he CLL cells in he CLL pool) and l-CLL cells ( he la ges 15% o he CLL cells in he CLL pool) we e cul u ed in he absence o p esence o CpG (3 µ g/mL) and CD40L (400 ng/mL) in a comple e RPMI-1640 medium, which was supplemen ed wi h 10% hea -inac i a ed oe al bo ine se um, 2mM o L-glu amine, 100U/mL o penicillin and 100µg/mL o s ep omycin o 24 and 48 h. 2.6. Mig a ion Analysis T ansmig a ion o he CLL cells was assessed using polyca bona e T answell inse s wi h a 5 µ m po e size (Co ning Cos a ). B ie ly, he s-CLL and l-CLL cells so ed a 1×106/mL each we e placed in he uppe chambe in he RPMI-1640 medium con aining 1% bo ine se um albumin (BSA). Inse s we e placed in o he lowe chambe con aining RPMI-1640 wi h 1% BSA in he p esence o absence o 200 ng/mL o CXCL12 (BioLegend). A e 3 h a 37 ◦ C in 5% CO 2 , he cells mig a ed in o he lowe chambe and we e coun ed using a BD FACSCan o II ins umen . The mig a ion a e was calcula ed as he a io o CXCL12- ea ed o CXCL12-un ea ed cells ha ansmig a ed h ough he inse . 2.7. P oli e a ion Assay The p oli e a ion a e was measu ed using a colo ime ic cell cy o oxici y assay (Cell Cy o oxici y Assay Ki ; Abcam); 0.5 × 10 5 so ed s-CLL and l-CLL cells we e sepa a ely cul u ed wi h CpG and CD40L in duplica es in a 96-well pla e. The a io o OD 570 o OD 605 was used o de e mine cell iabili y. 2.8. Assessmen o Apop osis The apop o ic a e o he cul u ed s-CLL and l-CLL cells was assessed by analysing he binding o annexin V-FITC and he inco po a ion o p opidium iodide (PI). Annexin V/PI double-nega i e cells we e conside ed iable cells, and annexin V+/PI-cells we e classi ied as apop o ic cells. 2.9. Fluo escen Mic oscopy A 1:1 mix u e o so ed s-CLL and l-CLL cells in a liquid suspension was luo escen ly s ained o mic oscopy (simila o he p o ocol o immuno luo escence in he case o sus- pension cells p o ided by The moFishe ). The cells we e ixed wi h 4% pa a o maldehyde o 10 min and pe meabilised by 0.5% T i on X-100 in phospha e-bu e ed saline (PBS) o 4 min . Unspeci ic binding was blocked wi h 1% BSA in PBS o 30 min. Then, he cells we e incuba ed o 90 min wi h p ima y an ibodies, i.e., mouse an i-human CD5 (1:300, clone CD5/54/F6, Abcam) and abbi an i-human CXCR3 (1:300, polyclonal, Sigma-Ald ich). This was ollowed by incuba ion wi h seconda y an ibodies, i.e., goa p e-abso bed an i- mouse IgG (H&L, 1:800, Cy5, polyclonal, Abcam) and goa an i- abbi IgG (H&L, 1:800, Alexa Fluo 488, polyclonal, Abcam) o 60 min. The CLL cells we e washed wi h PBS and cen i uged ollowing he comple ion o all o he abo e s eps. Finally, he cell pelle was moun ed using Fluo omoun -G moun ing media wi h 4 0 ,6-diamidino-2-phenylindole (DAPI; In i ogen) o label he nuclea DNA. Images we e aken a a inal magni ica ion o 600 × using Gen5 so wa e ( .3.08.01; BioTec Ins umen s, Winooski, VT, USA) and a BioTec Cy a ion 5MV (BioTec Ins umen s, Winooski, VT, USA) cell-imaging mul i-mode eade . 2.10. Quan i ica ion o IFN-γand IgM in Cell-Cul u e Supe na an s by Enzyme-Linked Immunoso ben Assay The in e e on gamma (IFN- γ ) le els we e measu ed in he supe na an s o cul u ed s-CLL and l-CLL cells using he MAX ™ Deluxe Se Human IFN- γ enzyme-linked im- munoso ben assay (ELISA; BioLegend). The o al IgM le els we e de e mined using a human IgM ELISA ki (Mab ech AB, Sweden). Cance s 2021,13, 4922 5 o 17 2.11. Time-Lapse Video Mic oscopy The s-CLL and l-CLL cells we e sepa a ely ea ed wi h (i) CpG (3 µ g/mL) and cul u ed in a clima e-con olled chambe (37 ◦ C and 5% CO 2 ) o 24 h o (ii) CXCL12 ( 200 ng/mL ) and placed on a hea ed (37 ◦ C and 5% CO 2 ) mic oscope s age. Cell pola - isa ion and mo ili y we e moni o ed by he ime-lapse ideo mic oscopy o cells o e 60 min and eco ded using Gen5 so wa e and he BioTec Cy a ion 5MV eade a a i- nal magni ica ion o 200 × . The ime-dependen changes in cell shape and mo ili y we e isually e alua ed. 2.12. S a is ical Analysis The di e ence be ween he g oups was analysed using he Mann–Whi ney es . A Wilcoxon signed- ank es was used o es ima e he e ec o induce s wi hin hein es iga ed g oups, and co ela ions we e assessed using Spea man’s co ela ion analysis. The esul s a e exp essed as mean ± s anda d e o o he mean. All s a is ical analyses and he unsupe ised p incipal componen analysis (PCA) we e pe o med using R so wa e (www. -p ojec .o g, e sion 4.1.1, accessed on 8 Augus 2021). 3. Resul s 3.1. The Exp ession o Su ace and In acellula Ma ke s The size o ypical CLL cells anged om 6.0–9.5 µ m; hei mo phology and size dis ibu ion wi hin he CLL cell pool a e shown in Figu e 1. Cance s 2021, 13, x FOR PEER REVIEW 5 o 16 2.11. Time-Lapse Video Mic oscopy The s-CLL and l-CLL cells we e sepa a ely ea ed wi h (i) CpG (3 µg/mL) and cul- u ed in a clima e-con olled chambe (37 °C and 5% CO2) o 24 h o (ii) CXCL12 (200 ng/mL) and placed on a hea ed (37 °C and 5% CO2) mic oscope s age. Cell pola isa ion and mo ili y we e moni o ed by he ime-lapse ideo mic oscopy o cells o e 60 min and eco ded using Gen5 so wa e and he BioTec Cy a ion 5MV eade a a inal magni ica- ion o 200×. The ime-dependen changes in cell shape and mo ili y we e isually e alu- a ed. 2.12. S a is ical Analysis The di e ence be ween he g oups was analysed using he Mann–Whi ney es . A Wilcoxon signed- ank es was used o es ima e he e ec o induce s wi hin he in es i- ga ed g oups, and co ela ions we e assessed using Spea man’s co ela ion analysis. The esul s a e exp essed as mean ± s anda d e o o he mean. All s a is ical analyses and he unsupe ised p incipal componen analysis (PCA) we e pe o med using R so wa e (www. -p ojec .o g, e sion 4.1.1 accessed on 08/08/2021). 3. Resul s 3.1. The Exp ession o Su ace and In acellula Ma ke s The size o ypical CLL cells anged om 6.0–9.5 µm; hei mo phology and size dis- ibu ion wi hin he CLL cell pool a e shown in Figu e 1. Figu e 1. The mo phological cha ac e is ics o CLL cells. (A) Pe iphe al blood smea s om pa- ien s wi h CLL (magni ica ion 600×). (B) The s-CLL and l-CLL cells om a pool o ypical CLL cells wi h scan cy oplasm, small ound nuclei wi h condensed ch oma in and inconspicuous nu- cleoli (magni ica ion 1000×). Du ing smea p epa a ion, CLL cells a e easily dis up ed, c ea ing smudge cells (P1 and P3). (C) The dis ibu ion o CLL cell sizes wi hin a CLL cell pool based on he o wa d-sca e (FSC) da a using a back-ga ing s a egy. (D) The size dis ibu ion o CLL cells in a pa icula pa ien . Rep esen a i e examples a e shown o h ee CLL pa ien s (P1–P3). In his s udy, wo CLL cell subpopula ions wi h di e en cell sizes we e selec ed based on he back-ga ing s a egy (Figu e 2). Each s-CLL and l-CLL cell popula ion so ed ep esen ed ~15% o he CLL cell pool. Figu e 1. The mo phological cha ac e is ics o CLL cells. ( A ) Pe iphe al blood smea s om pa ien s wi h CLL (magni ica ion 600 × ). ( B ) The s-CLL and l-CLL cells om a pool o ypical CLL cells wi h scan cy oplasm, small ound nuclei wi h condensed ch oma in and inconspicuous nucleoli (magni ica ion 1000 × ). Du ing smea p epa a ion, CLL cells a e easily dis up ed, c ea ing smudge cells (P1 and P3). ( C ) The dis ibu ion o CLL cell sizes wi hin a CLL cell pool based on he o wa d- sca e (FSC) da a using a back-ga ing s a egy. ( D ) The size dis ibu ion o CLL cells in a pa icula pa ien . Rep esen a i e examples a e shown o h ee CLL pa ien s (P1–P3). Cance s 2021,13, 4922 6 o 17 In his s udy, wo CLL cell subpopula ions wi h di e en cell sizes we e selec ed based on he back-ga ing s a egy (Figu e 2). Each s-CLL and l-CLL cell popula ion so ed ep esen ed ~15% o he CLL cell pool. Cance s 2021, 13, x FOR PEER REVIEW 6 o 16 Figu e 2. The cy ome ic analysis o he iden i ica ion o CLL subpopula ions wi h small and la ge cell sizes. (A) The back-ga ing s a egy o de ining s-CLL and l-CLL cells (each cons i u ed ~15% o he CLL cell pool). (B) Do plo s showing he posi ioning o CD5high and CD5low cells (de ined by CD5/CXCR4 ma ke s) on he SSC/FSC plo . The immunopheno yping o bo h cell popula ions e ealed ha s-CLL cells exhibi ed low le els o CD5 exp ession (p < 0.001) and high le els o CXCR4 exp ession (p < 0.05) compa ed wi h l-CLL cells. Addi ionally, he pe cen age o CXCR3+, CD20+ and HLA-DR+ cells was ound o be lowe in he g oup o s-CLL cells han in he l-CLL g oup (Figu e 3A). The pe cen age o CD38+ and CD49d+ cells did no di e be ween he s udied g oups. The in acellula labelling o he cells e ealed a lowe exp ession o ZAP-70 in he s-CLL cells han in he l-CLL cells (p < 0.01; Figu e 3B). Figu e 2. The cy ome ic analysis o he iden i ica ion o CLL subpopula ions wi h small and la ge cell sizes. ( A ) The back-ga ing s a egy o de ining s-CLL and l-CLL cells (each cons i u ed ~15% o he CLL cell pool). ( B ) Do plo s showing he posi ioning o CD5high and CD5low cells (de ined by CD5/CXCR4 ma ke s) on he SSC/FSC plo . The immunopheno yping o bo h cell popula ions e ealed ha s-CLL cells exhibi ed low le els o CD5 exp ession (p< 0.001) and high le els o CXCR4 exp ession (p< 0.05) compa ed wi h l-CLL cells. Addi ionally, he pe cen age o CXCR3 + , CD20 + and HLA-DR + cells was ound o be lowe in he g oup o s-CLL cells han in he l-CLL g oup (Figu e 3A). The pe cen age o CD38 + and CD49d + cells did no di e be ween he s udied g oups. The in acellula labelling o he cells e ealed a lowe exp ession o ZAP-70 in he s-CLL cells han in he l-CLL cells (p< 0.01; Figu e 3B). Co ela ion analyses e ealed a signi ican posi i e associa ion o CD5 high and CD5 low cells wi h he pe cen ages o CXCR3-posi i e cells, as indica ed in ou p e ious s udy [ 5 ]; his was also isualised by immuno luo escen s aining (Figu e 3C). The pe cen age o CLL cells in he pa ien s’ blood nega i ely co ela ed wi h he pe cen age o s-CLL and l-CLL cells ha es ed posi i e o HLA-DR ( = − 0.59, p< 0.01 and = − 0.48, p< 0.05, espec i ely) and CD20 MFI ( = − 0.55, p< 0.01 and = − 0.56, p< 0.01 espec i ely). In he s-CLL cells, CD5 MFI posi i ely co ela ed wi h CXCR3 pe cen age ( = 0.51, p< 0.05); in he l-CLL cells, CD5 MFI posi i ely co ela ed wi h bo h CXCR3 pe cen age ( = 0.51, p< 0.05) and ZAP-70 MFI ( = 0.46, p< 0.05). Cance s 2021,13, 4922 7 o 17 Cance s 2021, 13, x FOR PEER REVIEW 7 o 16 Figu e 3. The immunopheno ype o s-CLL and l-CLL cells om all he blood samples o he CLL pa ien s. (A) The su ace exp ession le els o CD5 and CXCR4, and he pe cen age o posi i e cells o CXCR3, CD20, CD38 and HLA-DR. (B) The pe cen age o ZAP-70-posi i e cells, and ep e- sen a i e do plo s o he in acellula exp ession le els o ZAP-70 in s-CLL and l-CLL cells. (C) The double-immuno luo escence s aining o s-CLL and l-CLL cells o CXCR3 and CD5; DAPI was used o nuclea s aining. Co ela ion analyses e ealed a signi ican posi i e associa ion o CD5high and CD5low cells wi h he pe cen ages o CXCR3-posi i e cells, as indica ed in ou p e ious s udy [5]; his was also isualised by immuno luo escen s aining (Figu e 3C). The pe cen age o CLL cells in he pa ien s’ blood nega i ely co ela ed wi h he pe cen age o s-CLL and l- CLL cells ha es ed posi i e o HLA-DR ( = −0.59, p < 0.01 and = −0.48, p < 0.05, espec- i ely) and CD20 MFI ( = −0.55, p < 0.01 and = −0.56, p < 0.01 espec i ely). In he s-CLL cells, CD5 MFI posi i ely co ela ed wi h CXCR3 pe cen age ( = 0.51, p < 0.05); in he l- CLL cells, CD5 MFI posi i ely co ela ed wi h bo h CXCR3 pe cen age ( = 0.51, p < 0.05) and ZAP-70 MFI ( = 0.46, p < 0.05). To de e mine i he s-CLL and l-CLL subpopula ions we e dis inc wi hin he exp es- sion p o iles, PCA was applied. The analysis esul s e ealed he dense clus e ing o he wo subpopula ions wi h ew dispe sed cases; 66.7% o he cases we e co ec ly classi ied in he s-CLL and l-CLL subse s. O he s udied ma ke s, he CXCR3, CXCR4, CD20 and CD5 ma ke s had he mos consis en abili y o co ec ly classi y s-CLL and l-CLL cells (Figu e 4). Figu e 3. The immunopheno ype o s-CLL and l-CLL cells om all he blood samples o he CLL pa ien s. ( A ) The su ace exp ession le els o CD5 and CXCR4, and he pe cen age o posi i e cells o CXCR3, CD20, CD38 and HLA-DR. ( B ) The pe cen age o ZAP-70-posi i e cells, and ep esen a i e do plo s o he in acellula exp ession le els o ZAP-70 in s-CLL and l-CLL cells. ( C ) The double-immuno luo escence s aining o s-CLL and l-CLL cells o CXCR3 and CD5; DAPI was used o nuclea s aining. To de e mine i he s-CLL and l-CLL subpopula ions we e dis inc wi hin he exp es- sion p o iles, PCA was applied. The analysis esul s e ealed he dense clus e ing o he wo subpopula ions wi h ew dispe sed cases; 66.7% o he cases we e co ec ly classi ied in he s-CLL and l-CLL subse s. O he s udied ma ke s, he CXCR3, CXCR4, CD20 and CD5 ma ke s had he mos consis en abili y o co ec ly classi y s-CLL and l-CLL cells (Figu e 4). Cance s 2021,13, 4922 8 o 17 Cance s 2021, 13, x FOR PEER REVIEW 8 o 16 Figu e 4. The PCA analysis o su ace ma ke exp ession in s-CLL and l-CLL cells. Each do ep e- sen s a sco e o he s-CLL (blue) and l-CLL (pink) cell popula ions de i ed om he s udied CLL pa ien s (n = 23). The model loadings a e ep esen ed by ec o s and indica e how each su ace ma ke con ibu es o he cell a iabili y in a speci ic di ec ion. The pe cen age gi en is he ex- plained a iance pe p incipal componen o a pa ien . 3.2. The E ec s o CpG and CD40L on he Pheno ype o Small and La ge Ch onic Lymphocy ic Leukaemia Cells The sensi i i y o CLL cells o s imula ion may e lec dis inc i e pheno ypic and unc ional cell subse s. To illus a e he pheno yping ea u es o cul u ed s-CLL and l-CLL cells, hey we e le un ea ed o s imula ed wi h CpG and CD40L. The cul u ed s-CLL and l-CLL cells e ained hei exp ession pa e ns, which we e obse ed a he basal le el and esponded o he induce s in a simila manne (Figu e 5). Figu e 4. The PCA analysis o su ace ma ke exp ession in s-CLL and l-CLL cells. Each do ep esen s a sco e o he s-CLL (blue) and l-CLL (pink) cell popula ions de i ed om he s udied CLL pa ien s (n= 23). The model loadings a e ep esen ed by ec o s and indica e how each su ace ma ke con ibu es o he cell a iabili y in a speci ic di ec ion. The pe cen age gi en is he explained a iance pe p incipal componen o a pa ien . 3.2. The E ec s o CpG and CD40L on he Pheno ype o Small and La ge Ch onic Lymphocy ic Leukaemia Cells The sensi i i y o CLL cells o s imula ion may e lec dis inc i e pheno ypic and unc ional cell subse s. To illus a e he pheno yping ea u es o cul u ed s-CLL and l-CLL cells, hey we e le un ea ed o s imula ed wi h CpG and CD40L. The cul u ed s-CLL and l-CLL cells e ained hei exp ession pa e ns, which we e obse ed a he basal le el and esponded o he induce s in a simila manne (Figu e 5). Cance s 2021,13, 4922 9 o 17 Cance s 2021, 13, x FOR PEER REVIEW 9 o 16 Figu e 5. The immunopheno ypes o so ed s-CLL s. l-CLL cells cul u ed o 24 and 48 h wi h CpG and CD40L; CXCR4 MFI and he pe cen age o CXCR3, CD20 and CD38 on cul u ed CLL cells (n = 11). * p < 0.05, ** p < 0.01, *** p < 0.001. In pa icula , an inc eased pe cen age o posi i e l-CLL cells o CXCR3 and CD38 was obse ed o all s imula ed and uns imula ed samples compa ed wi h s-CLL cells. An inc eased pe cen age o CD20 in l-CLL cells was obse ed only when he cells we e le un ea ed o when hey we e ea ed wi h CD40L a bo h 24 and 48 h o cul i a ion. The di e ences in CXCR4 exp ession be ween s-CLL and l-CLL cells we e less signi ican . In con as o o he ma ke s, l-CLL cells, which we e le un ea ed o 24 h, exhibi ed a de- c eased exp ession o CXCR4 (p < 0.05) compa ed wi h s-CLL cells. 3.3. The Mig a ion Ra e o Small and La ge Ch onic Lymphocy ic Leukaemia Cells Nex , we es ima ed he mig a ion a e o he so ed s-CLL and l-CLL cells owa ds CXCL12. We obse ed ha l-CLL cells mig a ed as e han s-CLL cells in his con ex (Figu e 6A). Figu e 5. The immunopheno ypes o so ed s-CLL s. l-CLL cells cul u ed o 24 and 48 h wi h CpG and CD40L; CXCR4 MFI and he pe cen age o CXCR3, CD20 and CD38 on cul u ed CLL cells (n= 11). * p< 0.05, ** p< 0.01, *** p< 0.001. In pa icula , an inc eased pe cen age o posi i e l-CLL cells o CXCR3 and CD38 was obse ed o all s imula ed and uns imula ed samples compa ed wi h s-CLL cells. An inc eased pe cen age o CD20 in l-CLL cells was obse ed only when he cells we e le un ea ed o when hey we e ea ed wi h CD40L a bo h 24 and 48 h o cul i a ion. The di e ences in CXCR4 exp ession be ween s-CLL and l-CLL cells we e less signi ican . In con as o o he ma ke s, l-CLL cells, which we e le un ea ed o 24 h, exhibi ed a dec eased exp ession o CXCR4 (p< 0.05) compa ed wi h s-CLL cells. 3.3. The Mig a ion Ra e o Small and La ge Ch onic Lymphocy ic Leukaemia Cells Nex , we es ima ed he mig a ion a e o he so ed s-CLL and l-CLL cells owa ds CXCL12. We obse ed ha l-CLL cells mig a ed as e han s-CLL cells in his con ex (Figu e 6A). Cance s 2021,13, 4922 16 o 17 13. K uzo a, L.; Schneide o a, P.; Holze o a, M.; Va oliko a, M.; Di oka, M.; Tu csanyi, P.; U bano a, R.; Kudelka, M.; Rad ansky, M.; K iego a, E.; e al. 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