Open Access

Heterogeneity of CD34 and CD38 expression in acute B lymphoblastic leukemia cells is reversible and not hierarchically organized

  • Zhiwu Jiang1, 2, 3,
  • Manman Deng4, 5,
  • Xinru Wei1, 2, 3,
  • Wei Ye1, 2, 3,
  • Yiren Xiao1, 2, 3,
  • Simiao Lin1, 2, 3,
  • Suna Wang1, 2, 3,
  • Baiheng Li1, 2, 3,
  • Xin Liu6,
  • Gong Zhang7,
  • Peilong Lai8,
  • Jianyu Weng8,
  • Donghai Wu1, 2,
  • Haijia Chen9,
  • Wei Wei10,
  • Yuguo Ma11,
  • Yangqiu Li12, 13,
  • Pentao Liu14,
  • Xin Du8,
  • Duanqing Pei1, 2,
  • Yao Yao15,
  • Bing Xu4, 5Email author and
  • Peng Li1, 2, 3Email author
Contributed equally
Journal of Hematology & Oncology20169:94

https://doi.org/10.1186/s13045-016-0310-1

Received: 3 August 2016

Accepted: 25 August 2016

Published: 22 September 2016

Abstract

The existence and identification of leukemia-initiating cells in adult acute B lymphoblastic leukemia (B-ALL) remain controversial. We examined whether adult B-ALL is hierarchically organized into phenotypically distinct subpopulations of leukemogenic and non-leukemogenic cells or whether most B-ALL cells retain leukemogenic capacity, irrespective of their immunophenotype profiles. Our results suggest that adult B-ALL follows the stochastic stem cell model and that the expression of CD34 and CD38 in B-ALL is reversibly and not hierarchically organized.

Keywords

B-ALL Leukemia stem cell Heterogeneity Xenografts

Currently, the long-term survival of adult B-ALL patients is less than 50 % [14]. To improve the cure and survival rates of adults, there is an increasing need to understand the biology of B-ALL and to characterize the leukemia-initiating cells (LICs) in B-ALL if they exist [5, 6]. Primary B-ALL cells from 25 adult patients (Additional file 1: Table S1) were intravenously transplanted into groups of adult NSI mice [79] that had undergone preconditioning total body irradiation. Twelve of the 25 samples engrafted successfully (Additional file 2: Table S2). In the 12 cases of successful engraftment, the mice died or developed severe clinical signs suggestive of leukemia and requiring euthanasia (Additional file 3: Table S3). Consistent with primary xenografts, the human B-ALL cells that expressed CD19, CD34, CD38, and CD45 in serial transplanted NSI mice closely recapitulated the immunophenotypes of the original patient (Additional file 4: Figure S1, S2A). The morphology of leukemic cells in the peripheral blood, spleens, and bone marrow (BM) of xenografts resemble the original patient samples (Additional file 5: Figure S2B). The CD34 and CD38 expression profiles of engrafted B-ALL cells from transplanted NSI mice resemble the original patient samples (Additional file 5: Figure S2A and Additional file 6: Figure S3).

CD34 and CD38 molecules had been used as surface markers to distinguish LICs [10, 11]. To identify whether CD34 and CD38 can be used as LICs markers in B-ALL cells, we purified CD34+CD38, CD34+CD38+, and CD34CD38+ fractions from the xenografts of patients #1 and #3. We subsequently performed limited dilution transplantation of these subpopulations in NSI mice. The purities of the subpopulations were 97.3 % ± 0.89 (n = 12, Additional file 7: Figure S4). The xenotransplantation results showed that each fraction of B-ALL cells from xenografts of patients #1 and #3 was capable of engrafting in NSI mice (Additional file 3: Table S3). Each subpopulation from xenografts of patients individually reconstituted B-ALL that contained CD34+CD38, CD34+CD38+, and CD34+CD38 fractions in NSI mice (Fig. 1). Genome-wide expression profile analysis revealed that each population was clustered closely in patients #1 and #3 (Additional file 8: Figure S5). RNA-Seq results were further validated by measuring the messenger RNA (mRNA) levels of oncogenesis-related genes using quantitative RT-PCR (Additional file 9: Figure S6).
Fig. 1

Subpopulations of adult B-ALL cells reconstituted the leukemia in xenografts. Subpopulations of CD34+CD38, CD34+CD38+, and CD34CD38+ from xenografts of patients #1 and #3 were purified and injected into groups of NSI mice. a Representative FACS analysis of gated hCD45+ BM cells from NSI recipients that were transferred with different subpopulations of engrafted B-ALL cells from patient #1. b Representative FACS analysis of gated hCD45+ BM cells from NSI mice that were transferred with CD34+CD38+ and CD34CD38+ fractions of engrafted B-ALL cells from patient #3

Next, we investigated whether expanded B-ALL cells in vitro still maintain original expression profiles of CD34 and CD38 and the LIC capacity. B-ALL cells from 11 of the 12 patient samples that successfully engrafted in NSI mice attached to OP9 cells and proliferated vigorously for at least 2 months (Additional file 10: Table S4). We then monitored the expression profiles of CD34 and CD38 in B-ALL cells in differential time. To our surprise, CD34+CD38 and CD34+CD38+ subpopulations from patient #1 disappeared gradually in culture (Fig. 2a). Six weeks after co-culture with OP9 cells, all remaining leukemic cells were CD34CD38+ (Additional file 10: Table S4). To investigate whether CD34CD38+ B-ALL cells after culture were still capable of engrafting in mice, we further purified cultured CD34CD38+ B-ALL cells from patients #1, #4, and #7 and injected them into groups of NSI mice. After 4 weeks transplantation, cultured CD34CD38+ B-ALL cells from patient reconstituted B-ALL consisting of CD34+CD38, CD34+CD38+, and CD34CD38+subpopulations in mice (Fig. 2b and Additional file 11: Table S5). Whole exome-sequencing analysis [12] showed that B-ALL cells from co-culture and B-ALL cells from xenografts shared similar SNP profiles (Additional file 12: Figure S7). This result indicates B-ALL cells maintain stable genetic characteristics irrespective of phenotypes. Our results also showed that individual B-ALL cells successfully engrafted in 4 of the 70 hosts and repopulated original surface profiles (Additional file 13: Figure S8 and Additional file 14: Table S6, detailed  methodological information was included in Additional file 17: supplementary methods.).
Fig. 2

Cultured leukemic cells maintain the stem cell capacity. a Representative FACS analysis of CD34 and CD38 expression profiles in primary B-ALL cells from patient #1 in OP9 co-culture at indicated time points. b B-ALL cells from xenografts of patients #1, #4, and #7 were co-cultured with OP9 stromal cells. After 6 weeks, cultured B-ALL cells were subjected to FACS analysis. Then CD34CD38+ populations were enriched from cultured B-ALL cells and were subsequently injected into groups of NSI mice for serial transplantations. Eight weeks after transplantation, BM cells from xenografts were subjected for FACS analysis. Representative FACS analysis of gated CD45+ cells from xenografts or co-cultures

In conclusion, our results demonstrate that leukemic blasts, irrespective of CD34 and CD38 expression, are able to engraft immunodeficient mice and reconstitute the original leukemia. Furthermore, we provide evidence that the heterogeneity of CD34 and CD38 expression in B-ALL obtained from patients reverses in different microenvironments. This phenotypic plasticity contrasts the cancer stem cell model, which largely attributes heterogeneity to irreversible epigenetic changes.

Abbreviations

B-ALL: 

Acute B lymphoblastic leukemia

BM: 

Bone marrow

LICs: 

Leukemia-initiating cells

NSI: 

NOD/SCID/IL2−/−

Declarations

Acknowledgements

Not applicable.

Funding

This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No.: XDA01020310), the National Natural Science Foundation of China (Grant No.: 81272329, 81522002, and 81327801), the Natural Science Fund for Distinguished Young Scholars of Guangdong Province (Grant No.: 2014A030306028), the Guangdong Provincial Outstanding Young Scholars Award (Grant No.: 2014TQ01R068), the Guangdong Provincial Basic Research Program (Grant No.: 2015B020227003), the Guangdong Provincial Research and Commercialization Program (Grant No.: 2014B090901044), the Guangdong Province and Chinese Academy of Sciences Joint Program for Research and Commercialization Program (Grant No.: 2013B091000010), the Guangzhou Basic Research Program (Grant No.: 201510010186), the MOST funding of the State Key Laboratory of Respiratory Disease, and the National Basic Research Program of China (973 Program) (2011CB504004 and 2010CB945500), the Major Scientific and Technological Project of Guangdong Province (2014B020225005).

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and additional files.

Authors’ contributions

ZJ and YY contributed to the conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing. MD contributed to the provision of study material or patients, collection and/or assembly of data. XW, WY, and YX contributed to the collection and/or assembly of data, and data analysis and interpretation. SL provided administrative support and is responsible for the collection and/or assembly of data. SW and BL provided administrative support. XL, DW, and DP contributed to the conception and design and provided financial support. GZ contributed to the data analysis and interpretation. PLai, JW, HC, WW, and YM are responsible for the provision of study material or patients. YL and PLiu contributed to the conception and design. XD contributed to the conception and design and provision of study material or patients. BX contributed to the conception and design, provision of study material or patients, and final approval of manuscript and provided financial support. PLi contributed to the conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript and provided financial support. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Consent to publish has been obtained from the participants.

Ethics approval and consent to participate

All experimental protocols were performed in accordance with instruction guidelines from the China Council on Animal Care and approved by the guidelines of the Ethics Committee of Animal Experiments at Guangzhou Institutes of Biomedicine and Health (GIBH). Samples were obtained with informed consent for research purposes, and the procedures were approved by the Research Ethics Board of GIBH. Consent to publish has been obtained from the participant to report individual patient data.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences
(2)
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences
(3)
Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences
(4)
Department of Hematology, The First Affiliated Hospital of Xiamen University
(5)
Department of Hematology, Nanfang Hospital, Southern Medical University
(6)
Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences
(7)
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University
(8)
Department of Hematology, Guangdong Provincial People’s Hospital
(9)
Guangzhou SALIAI Stem Cell Science and Technology Co. Ltd
(10)
Guangdong Cord Blood Bank
(11)
Yikang Tailai Technology Co. Ltd
(12)
Department of Hematology, Medical College, Jinan University
(13)
Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University
(14)
Wellcome Trust Sanger Institute
(15)
Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

References

  1. Liew E, Atenafu EG, Schimmer AD, Yee KW, Schuh AC, Minden MD, Gupta V, Brandwein JM. Outcomes of adult patients with relapsed acute lymphoblastic leukemia following frontline treatment with a pediatric regimen. Leuk Res. 2012;36(12):1517–20.View ArticlePubMedGoogle Scholar
  2. Larson S, Stock W. Progress in the treatment of adults with acute lymphoblastic leukemia. Curr Opin Hematol. 2008;15(4):400–7.View ArticlePubMedGoogle Scholar
  3. Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med. 2006;354(2):166–78.View ArticlePubMedGoogle Scholar
  4. Nagafuji K, Miyamoto T, Eto T, Kamimura T, Taniguchi S, Okamura T, Ohtsuka E, Yoshida T, Higuchi M, Yoshimoto G, et al. Monitoring of minimal residual disease (MRD) is useful to predict prognosis of adult patients with Ph-negative ALL: results of a prospective study (ALL MRD2002 Study). J Hematol Oncol. 2013;6:14.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Kong Y, Yoshida S, Saito Y, Nagatoshi Y, Fukata M, Saito N, Yang S, Iwamoto C, Okamura J, Liu K. CD34+ CD38+ CD19+ as well as CD34+ CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL. Leukemia. 2008;22(6):1207–13.Google Scholar
  6. le Viseur C, Hotfilder M, Bomken S, Wilson K, Röttgers S, Schrauder A, Rosemann A, Irving J, Stam RW, Shultz LD. In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties. Cancer Cell. 2008;14(1):47–58.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Ye W, Jiang Z, Li GX, Xiao Y, Lin S, Lai Y, Wang S, Li B, Jia B, Li Y, et al. Quantitative evaluation of the immunodeficiency of a mouse strain by tumor engraftments. J Hematol Oncol. 2015;8:59.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Deng M, Jiang Z, Li Y, Zhou Y, Li J, Wang X, Yao Y, Wang W, Li P, Xu B. Effective elimination of adult B-lineage acute lymphoblastic leukemia by disulfiram/copper complex in vitro and in vivo in patient-derived xenograft models. Oncotarget. 2016. Online ISSN: 1949-2553.Google Scholar
  9. Xiao Y, Jiang Z, Li Y, Ye W, Jia B, Zhang M, Xu Y, Wu D, Lai L, Chen Y, et al. ANGPTL7 regulates the expansion and repopulation of human hematopoietic stem and progenitor cells. Haematologica. 2015;100(5):585–94.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Wang L, Gao L, Xu S, Gong S, Chen L, Lu S, Chen J, Qiu H, Xu X, Ni X, et al. FISH + CD34 + CD38- cells detected in newly diagnosed acute myeloid leukemia patients can predict the clinical outcome. J Hematol Oncol. 2013;6(1):85.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367(6464):645–48.Google Scholar
  12. Woo JS, Alberti MO, Tirado CA. Childhood B-acute lymphoblastic leukemia: a genetic update. Exp Hematol Oncol. 2014;3:16.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s). 2016

Advertisement