Chronic lymphocytic leukaemia (CLL) is a genetically, morphologically and phenotypically heterogeneous chronic disease with clinical variability between patients. Whether the significant heterogeneity of cell size within the CLL population contributes to the heterogeneous features of this disease has not been investigated. The present study aimed to characterise the phenotypic and functional properties of two subpopulations of typical CLL cells that differ in cell size: small (s-CLL) and large (l-CLL) CLL cells delineated by forward scatter cytometry. The s-CLL cells were characterised by the CD5lowCXCR4hi phenotype, while the l-CLL cells were characterised by the CD5hiCXCR4dim phenotype and indicated a higher expression of CXCR3, CD20, CD38 and HLA-DR. The l-CLL cells displayed higher migration activity towards CXCL12, a tendency towards a higher proliferation rate and an increased capacity to produce IgM in the presence of CpG compared with s-CLL cells. When stimulated with CpG and CXCL12, l-CLL cells were characterised by a higher polarisation phenotype and motility than s-CLL cells. Our study revealed that the differences in CLL cell size reflected their activation status, polarisation and migratory abilities. Our data provide evidence of the importance of cell-size heterogeneity within a CLL pool and the dynamics of cell-size changes for disease pathogenesis, thus deserving further investigation.

Department of Computer Science Faculty of Electrical Engineering and Computer Science VSB Technical University of Ostrava 70800 Ostrava Czech Republic

Department of Hematology Oncology Faculty of Medicine and Dentistry Palacký University and University Hospital 77900 Olomouc Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacký University and University Hospital 77900 Olomouc Czech Republic

Laboratory of Molecular and Cellular Immunology Institute of Molecular Biology NAS RA Yerevan 0014 Armenia

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Kipps T.J., Stevenson F.K., Wu C.J., Croce C.M., Packham G., Wierda W.G., O’Brien S., Gribben J., Rai K. Chronic lymphocytic leukaemia. Nat. Rev. Dis. Primers. 2017;3:1–22. doi: 10.1038/nrdp.2016.96. PubMed DOI PMC

Calissano C., Damle R.N., Hayes G., Murphy E.J., Hellerstein M.K., Moreno C., Sison C., Kaufman M.S., Kolitz J.E., Allen S.L., et al. In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia. Blood. 2009;114:4832–4842. doi: 10.1182/blood-2009-05-219634. PubMed DOI PMC

Calissano C., Damle R.N., Marsilio S., Yan X.J., Yancopoulos S., Hayes G., Emson C., Murphy E.J., Hellerstein M.K., Sison C., et al. Intraclonal complexity in chronic lymphocytic leukemia: Fractions enriched in recently born/divided and older/quiescent cells. Mol. Med. 2011;17:1374–1382. doi: 10.2119/molmed.2011.00360. PubMed DOI PMC

Bashford-Rogers R.J., Palser A.L., Hodkinson C., Baxter J., Follows G.A., Vassiliou G.S., Kellam P. Dynamic variation of CD5 surface expression levels within individual chronic lymphocytic leukemia clones. Exp. Hematol. 2017;46:31–37. doi: 10.1016/j.exphem.2016.09.010. PubMed DOI PMC

Manukyan G., Papajik T., Mikulkova Z., Urbanova R., Smotkova Kraiczova V., Savara J., Kudelka M., Turcsanyi P., Kriegova E. High CXCR3 on Leukemic Cells Distinguishes IgHVmut from IgHVunmut in Chronic Lymphocytic Leukemia: Evidence from CD5high and CD5low Clones. J. Immunol. Res. 2020;2020:7084268. doi: 10.1155/2020/7084268. PubMed DOI PMC

Mikulkova Z., Manukyan G., Turcsanyi P., Kudelka M., Urbanova R., Savara J., Ochodkova E., Brychtova Y., Molinsky J., Simkovic M., et al. Deciphering the complex circulating immune cell microenvironment in chronic lymphocytic leukaemia using patient similarity networks. Sci. Rep. 2021;11:1–13. PubMed PMC

Herndon T.M., Chen S.S., Saba N.S., Valdez J., Emson C., Gatmaitan M., Tian X., Hughes T.E., Sun C., Arthur D.C., et al. Direct in vivo evidence for increased proliferation of CLL cells in lymph nodes compared to bone marrow and peripheral blood. Leukemia. 2017;31:1340–1347. doi: 10.1038/leu.2017.11. PubMed DOI PMC

Pasikowska M., Walsby E., Apollonio B., Cuthill K., Phillips E., Coulter E., Longhi M.S., Ma Y., Yallop D., Barber L.D. Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration. Blood. 2016;128:563–573. doi: 10.1182/blood-2016-01-683128. PubMed DOI

Oscier D., Else M., Matutes E., Morilla R., Strefford J.C., Catovsky D. The morphology of CLL revisited: The clinical significance of prolymphocytes and correlations with prognostic/molecular markers in the LRF CLL4 trial. Br. J. Haematol. 2016;174:767–775. doi: 10.1111/bjh.14132. PubMed DOI PMC

Melo J.V., Catovsky D., Gregory W.M., Galton D.A. The relationship between chronic lymphocytic leukaemia and prolymphocytic leukaemia. IV. Analysis of survival and prognostic features. Br. J. Haematol. 1987;65:23–29. doi: 10.1111/j.1365-2141.1987.tb06130.x. PubMed DOI

Hallek M., Cheson B.D., Catovsky D., Caligaris-Cappio F., Dighiero G., Döhner H., Hillmen P., Keating M.J., Montserrat E., Rai K.R., et al. International Workshop on Chronic Lymphocytic Leukemia. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: A report from the international workshop on chronic lymphocytic leukemia updating the national cancer institute-working group 1996 guidelines. Blood. 2008;111:5446–5456. PubMed PMC

Petrackova A., Vasinek M., Sedlarikova L., Dyskova T., Schneiderova P., Novosad T., Papajik T., Kriegova E. Standardization of Sequencing Coverage Depth in NGS: Recommendation for Detection of Clonal and Subclonal Mutations in Cancer Diagnostics. Front. Oncol. 2019;9:851. doi: 10.3389/fonc.2019.00851. PubMed DOI PMC

Kruzova L., Schneiderova P., Holzerova M., Vatolikova M., Divoka M., Turcsanyi P., Urbanova R., Kudelka M., Radvansky M., Kriegova E., et al. Complex karyotype as a predictor of high-risk chronic lymphocytic leukemia: A single center experience over 12 years. Leuk. Res. 2019;85:106218. doi: 10.1016/j.leukres.2019.106218. PubMed DOI

Obr A., Procházka V., Jirkuvová A., Urbánková H., Kriegova E., Schneiderová P., Vatolíková M., Papajík T. TP53 Mutation and Complex Karyotype Portends a Dismal Prognosis in Patients with Mantle Cell Lymphoma. Clin. Lymphoma Myeloma. Leuk. 2018;18:762–768. doi: 10.1016/j.clml.2018.07.282. PubMed DOI

Manukyan G., Papajik T., Gajdos P., Mikulkova Z., Urbanova R., Gabcova G., Kudelka M., Turcsányi P., Ryznerova P., Prochazka V., et al. Neutrophils in chronic lymphocytic leukemia are permanently activated and have functional defects. Oncotarget. 2017;8:84889–84901. doi: 10.18632/oncotarget.20031. PubMed DOI PMC

Rossi F.M., Del Principe M.I., Rossi D., Irno Consalvo M., Luciano F., Zucchetto A., Bulian P., Bomben R., Dal Bo M., Fangazio M., et al. Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: Mean fluorescence intensity T/B ratio versus percentage of positive cells. J. Transl. Med. 2010;8:1–11. doi: 10.1186/1479-5876-8-23. PubMed DOI PMC

Kumar S., Saxena N., Sarkar M., Barai A., Sen S. Combined heterogeneity in cell size and deformability promotes cancer invasiveness. J. Cell Sci. 2021;134:jcs250225. doi: 10.1242/jcs.250225. PubMed DOI

Lüönd F., Tiede S., Christofori G. Breast cancer as an example of tumour heterogeneity and tumour cell plasticity during malignant progression. Br. J. Cancer. 2021;125:164–175. doi: 10.1038/s41416-021-01328-7. PubMed DOI PMC

Dampmann M., Görgens A., Möllmann M., Murke F., Dührsen U., Giebel B., Dürig J. CpG stimulation of chronic lymphocytic leukemia cells induces a polarized cell shape and promotes migration in vitro and in vivo. PLoS ONE. 2020;15:e0228674. doi: 10.1371/journal.pone.0228674. PubMed DOI PMC

Seda V., Vojackova E., Ondrisova L., Kostalova L., Sharma S., Loja T., Mladonicka Pavlasova G., Zicha D., Kudlickova Peskova M., Krivanek J., et al. FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia. Blood. 2021;138:758–772. doi: 10.1182/blood.2020008101. PubMed DOI PMC

Brandes M., Legler D.F., Spoerri B., Schaerli P., Moser B. Activation-dependent modulation of B lymphocyte migration to chemokines. Int. Immunol. 2000;12:1285–1292. doi: 10.1093/intimm/12.9.1285. PubMed DOI

Gary-Gouy H., Harriague J., Bismuth G., Platzer C., Schmitt C., Dalloul A.H. Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production. Blood. 2002;100:4537–4543. doi: 10.1182/blood-2002-05-1525. PubMed DOI

Deaglio S., Vaisitti T., Aydin S., Bergui L., D’Arena G., Bonello L., Omedé P., Scatolini M., Jaksic O., Chiorino G., et al. CD38 and ZAP-70 are functionally linked and mark CLL cells with high migratory potential. Blood. 2007;110:4012–4021. doi: 10.1182/blood-2007-06-094029. PubMed DOI

Stamatopoulos B., Haibe-Kains B., Equeter C., Meuleman N., Sorée A., De Bruyn C., Hanosset D., Bron D., Martiat P., Lagneaux L. Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA. Haematologica. 2009;94:790–799. doi: 10.3324/haematol.2008.002626. PubMed DOI PMC

Calpe E., Purroy N., Carpio C., Abrisqueta P., Carabia J., Palacio C., Castellví J., Crespo M., Bosch F. ZAP-70 promotes the infiltration of malignant B-lymphocytes into the bone marrow by enhancing signaling and migration after CXCR4 stimulation. PLoS ONE. 2013;8:e81221. doi: 10.1371/journal.pone.0081221. PubMed DOI PMC

Laufer J.M., Lyck R., Legler D.F. ZAP70 expression enhances chemokine-driven chronic lymphocytic leukemia cell migration and arrest by valency regulation of integrins. FASEB J. 2018;32:4824–4835. doi: 10.1096/fj.201701452RR. PubMed DOI

Kozlova V., Ledererova A., Ladungova A., Peschelova H., Janovska P., Slusarczyk A., Domagala J., Kopcil P., Vakulova V., Oppelt J., et al. CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells. PLoS ONE. 2020;15:e0229170. doi: 10.1371/journal.pone.0229170. PubMed DOI PMC

Purroy N., Abrisqueta P., Carabia J., Carpio C., Palacio C., Bosch F., Crespo M. Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo. Oncotarget. 2015;6:7632–7643. doi: 10.18632/oncotarget.2939. PubMed DOI PMC

Schleiss C., Ilias W., Tahar O., Güler Y., Miguet L., Mayeur-Rousse C., Mauvieux L., Fornecker L.M., Toussaint E., Herbrecht R., et al. BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo. Sci. Rep. 2019;9:701. doi: 10.1038/s41598-018-36853-8. PubMed DOI PMC

Longo P.G., Laurenti L., Gobessi S., Petlickovski A., Pelosi M., Chiusolo P., Sica S., Leone G., Efremov D.G. The Akt signaling pathway determines the different proliferative capacity of chronic lymphocytic leukemia B-cells from patients with progressive and stable disease. Leukemia. 2007;21:110–120. doi: 10.1038/sj.leu.2404417. PubMed DOI

Lúcio P., Parreira A., van den Beemd M.W., van Lochem E.G., van Wering E.R., Baars E., Porwit-MacDonald A., Bjorklund E., Gaipa G., Biondi A., et al. Flow cytometric analysis of normal B cell differentiation: A frame of reference for the detection of minimal residual disease in precursor-B-ALL. Leukemia. 1999;13:419–427. doi: 10.1038/sj.leu.2401279. PubMed DOI

Burger J.A., Gribben J.G. The microenvironment in chronic lymphocytic leukemia (CLL) and other B cell malignancies: Insight into disease biology and new targeted therapies . Semin. Cancer Biol. 2014;24:71–81. doi: 10.1016/j.semcancer.2013.08.011. PubMed DOI

Donahue A.C., Fruman D.A. Proliferation and survival of activated B cells requires sustained antigen receptor engagement and phosphoinositide 3-kinase activation. J. Immunol. 2003;170:5851–5860. doi: 10.4049/jimmunol.170.12.5851. PubMed DOI

Waters L.R., Ahsan F.M., Wolf D.M., Shirihai O., Teitell M.A. Initial B Cell Activation Induces Metabolic Reprogramming and Mitochondrial Remodeling. iScience. 2018;5:99–109. doi: 10.1016/j.isci.2018.07.005. PubMed DOI PMC

Vanhee S., Åkerstrand H., Kristiansen T.A., Datta S., Montano G., Vergani S. Lin28b controls a neonatal to adult switch in B cell positive selection. Sci. Immunol. 2019;4:eaax4453. doi: 10.1126/sciimmunol.aax4453. PubMed DOI

Hamilton E., Pearce L., Morgan L., Robinson S., Ware V., Brennan P., Thomas N.S., Yallop D., Devereux S., Fegan C., et al. Mimicking the tumour microenvironment: Three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells. Br. J. Haematol. 2012;158:589–599. doi: 10.1111/j.1365-2141.2012.09191.x. PubMed DOI

Solman I.G., Blum L.K., Burger J.A., Kipps T.J., Dean J.P., James D.F., Mongan A. Impact of long-term ibrutinib treatment on circulating immune cells in previously untreated chronic lymphocytic leukemia. Leuk. Res. 2021;102:106520. doi: 10.1016/j.leukres.2021.106520. PubMed DOI

Cheng S., Ma J., Guo A., Lu P., Leonard J.P., Coleman M., Liu M., Buggy J.J., Furman R.R., Wang Y.L. BTK inhibition targets in vivo CLL proliferation through its effects on B-cell receptor signaling activity. Leukemia. 2014;28:649–657. doi: 10.1038/leu.2013.358. PubMed DOI

Herman S.E., Mustafa R.Z., Gyamfi J.A., Pittaluga S., Chang S., Chang B., Farooqui M., Wiestner A. Ibrutinib inhibits BCR and NF-κB signaling and reduces tumor proliferation in tissue-resident cells of patients with CLL. Blood. 2014;123:3286–3295. doi: 10.1182/blood-2014-02-548610. PubMed DOI PMC

Chen S.S., Chang B.Y., Chang S., Tong T., Ham S., Sherry B., Burger J.A., Rai K.R., Chiorazzi N. BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia. Leukemia. 2016;30:833–843. doi: 10.1038/leu.2015.316. PubMed DOI PMC

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