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.
The online version of this article (doi:10.1186/s13045-016-0310-1) contains supplementary material, which is available to authorized users.
Abkürzungen
B-ALL
Acute B lymphoblastic leukemia
BM
Bone marrow
LICs
Leukemia-initiating cells
NSI
NOD/SCID/IL2−/−
Currently, the long-term survival of adult B-ALL patients is less than 50 % [1‐4]. 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 [7‐9] 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 CD34−CD38+ 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).
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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 CD34−CD38+ (Additional file 10: Table S4). To investigate whether CD34−CD38+ B-ALL cells after culture were still capable of engrafting in mice, we further purified cultured CD34−CD38+ B-ALL cells from patients #1, #4, and #7 and injected them into groups of NSI mice. After 4 weeks transplantation, cultured CD34−CD38+ B-ALL cells from patient reconstituted B-ALL consisting of CD34+CD38−, CD34+CD38+, and CD34−CD38+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.).
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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.
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).
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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.
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Heterogeneity of CD34 and CD38 expression in acute B lymphoblastic leukemia cells is reversible and not hierarchically organized
verfasst von
Zhiwu Jiang Manman Deng Xinru Wei Wei Ye Yiren Xiao Simiao Lin Suna Wang Baiheng Li Xin Liu Gong Zhang Peilong Lai Jianyu Weng Donghai Wu Haijia Chen Wei Wei Yuguo Ma Yangqiu Li Pentao Liu Xin Du Duanqing Pei Yao Yao Bing Xu Peng Li