Identification and characterization of the cellular subclones that contribute to the pathogenesis of mantle cell lymphoma
Authors: Liangsheng Kong
Li Zhang
Ting Ye
Fang Xu
Tiejun Zhou
Jing Guo
Jingyuan Li
Tao Ma
Zhiwei Sun
Xiaoming Li
Li Jing
Hongyun Xing
Junling Tang
Dan Liu
Xiaofeng Zhu
Yan Chen
H Rosie Xing
Shixia Zhou
Min Hu
Jirui Tang
Mei Chen
Jianyu Wang
Hongyan Wu
Yongli Liu
Journal: Genes & Diseases (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: CD19; JeKo-1-spheroid; Mantle cell lymphoma; Pathology; Sub-clone.
Affiliations: Laboratory of Translational Cancer Stem Cell Research, Institute of Life Sciences, Chongqing Medical University, 1 Yixueyuan Rd, Chongqing, 400016, China., Department of Hematology, The Affiliated Hospital of Southwest Medical University, 25 Tai Ping Street, Luzhou, 646000, China., The Affiliated Stomatology Hospital of Southwest Medical University, 2 Jiangyangnan Rd, Luzhou, 646000, China., Department of Pathology, The Affiliated Hospital of Southwest Medical University, 25 Tai Ping Street, Luzhou, 646000, China., Department of Hematology, Mianyang Central Hospital, 12 Changjia Lane, Jingzhong Street, Mianyang, 621000, China., School of Biomedical Engineering, Chongqing Medical University, 1 Yixueyuan Rd, Chongqing, 400016, China.

Abstract:  Mantle cell lymphoma (MCL) is a B-cell malignancy with poor clinical outcome and undefined pathogenesis. Development of clinically relevant cellular models for MCL research is an urgent need. Our preliminary observations lead the development of two novel hypotheses that we tested in this study: 1. multicellular spheroid might be a unique growth mode of early-stage cells in MCL; 2. MCL might be a polyclonal tumor. We made the following original observations that have not been reported: First, we have provided a new experiment method for enriching MCL early-stage cells and characterized the spheroid mode of growth as a unique feature of early-stage MCL cells in cell line as well as in clinical samples. Second, we have established a clinically relevant cellular model of MCL, the JeKo-1-spheroid cell line, that was highly enriched in early-stage sub-clones. JeKo-1-spheroid cells and the spheroid growing cells enriched from MCL patients exhibited comparably enhanced tumorigenic abilities and similar biological features. Third, Immunophenotypic analysis has revealed that MCL may be derived from precursor-B(pre-B), immature-B and mature-B cells, not only the mature-B cells as WHO classified in 2016. Fourth, MCL may be a polyclonal disease composed of CD19-/IgM-, CD19-/IgM+, CD19+/IgM+ three sub-clones, of which the CD19-/IgM+ sub-clone might be the dominant sub-clone with the strongest tumorigenic ability. Fifth, CD19+/IgM- that differentiates MCL and normal B cells may represent a new marker for MCL early detection, minor residual disease monitoring after therapies and prognosis. 

Retracted: No

PubPeer comments:
 The first two plots and figure 2A appear to be the same. *** ![file] *** Full figure ![file] , 
 Figure 6C appears to have at least one repeated image. Perhaps the authors or peer reviewers could check for more. Apparently peer review at this journal is done by the affiliated institution: u201cPeer review under responsibility of Chongqing Medical University.u201d *** ![file] , 
 https: retractionwatch.com 2016 02 08 2014-ori-finding-results-in-retraction-of-cancer-paper-with-manipulated-images , 
 Images in Figure 3C and 6A appear to be reused. *** ![file] , 
 https: retractionwatch.com 2013 11 27 cancer-researchers-retract-two-papers-in-the-jbc https: retractionwatch.com 2016 02 08 2014-ori-finding-results-in-retraction-of-cancer-paper-with-manipulated-images https: retractionwatch.com 2014 12 14 ori-sanctions-former-university-chicago-ucsf-scientists-faking-findings , 
 #1 First of all, the comment that the inquiry regarding Figure 2A was raised in July 2020 caught our attention. Since this is the first time we aware of this matter by the email alert from pubpeer, we looked through the mailbox and found an email sent by pubpeer on July 4, 2020 in the Junk Mail Box. We feel very sorry that we have missed an opportunity to respond when the concern was raised in July 2020. For the two indicated sub-Figures in the Figure 2A, which are the graphic exhibition of the Isotype IgG-ECD and Isotype IgG-PE, the control IgG we used in the FCS analysis. The corresponding antibodies were CD34-ECD with CD10-PE and CD22-PE, respectively. In fact, we knew these two control sub-Figures are identical. However, we failed to indicate it clearly in the figure legend thus led to readeru2019s misunderstanding and caused the inquiry. Please let us to explain: (1) this FCS analysis was repeated 3 times which yielded similar observations. We chose a representative set and made into Figure 2A for the manuscript. (2) The antibodies used in this experiment were from the same manufacturer, so the isotypes are the same, except that the vertical coordinates are CD34-ECD, and the detection markers from left to right are CD10-PE, CD22-PE in the abscissa, respectively. Since the isotope for CD10-PE and CD22-PE is the same (PE), the control isotope graph for CD10-PE and CD22-PE is the same. However, we did not make proper identification and explanation in the Figure legend or the in the article, which led to readers misunderstanding. We will contact the Journal and make a revision to the Figure 2A (see below) and the figure legend. We found the original lab records in the lab note book and present the raw data for your review (see the inserted photocopy of the lab record). This set of experiment was performed on April 11, 2016. Since the lab notes were written in Chinese, we made a line-by-line translation of the lab note for your comprehension and review. Translated version: Line1: Experimental Date: April 11, 2016 Line2: Experiment: The JeKo-1cells were analyzed for CD10, CD19, CD34. And Cryopreserved cells. Line3: JeKo-1 and 293 T cells were sub-cultured and cryopreserved. Line4: Observe the cell growth status. Line5: Staffs: Tang Junling and Xu Lihong. Line6: Location: 406 Cell Culture Room, Flow Cytometry Laboratory, Department of Biological Sciences. Line7: 1u3001Jeko-1 cells were sub-cultured into 2 T25 and T75 culture flask at a ratio of 1:20, Line8: and set 4 tubes of cells as follows: 3: blank, 4: ISO, 5:CD10-PE, CD19-FITC Line9: and CD34-ECD. 2u3001293T and SKOV3 cells as negative control group, numbered 1:293T ISO, 2 Line10: 293T CD19, 6: SKOV3 ISO, 7: SKOV3 CD19. 3u3001Frozen the 2 tubes of 293 T and SKOV3 cells, respectively. ![file] Revised Figureuff1a ![file] , 
 #4 3 pairs of sub figures were alleged of u201creuse of figuresu201d: (1) Figure 3C, upper panel, lane 3 (CD79a, IgM- group) vs Figure 6A, lower panel, lane 1 (CCND1, CD19- IgM- group); (2) Figure 3C, upper panel, lane 6 (CD79a, IgM+ group) vs Figure 6B, lower panel, lane 3 (CCND1, CD19+ IgM+ group); (3) Figure 6C(ii), upper panel, lane 4 (IgM) vs lane 5 (Pax5). We examined the alleged pairs closely by enlarging the high-resolution images in the published pdf of this manuscript. We agree with the assessment of this reader. We contacted the first author of this manuscript who conducted most part of the study, Dr, Junling TANG to seek her response. Dr. Tang continued this line of research when she became an independent researcher and has obtained experimental proof of the polyclonal origin of Jeko-1-spheroid at the single cell level. It took her several days to examine the original records (lab book notes and the electronic data). She provided the original lab records and original images (dated back in 2017) related to the alleged sub-figure panels. She also reflected on what had caused such mistake: (1) at the time of writing up the manuscript and revision of the manuscript, she was not familiar with Photoshop. As a result, she asked the favor of another graduate student in the lab to make all the figures for the publication; (2) this manuscript had undergone a lengthy initial submission and tried different journals. Since the number of figures allowed by each journal differs, the figures had been modified and re-organized accordingly; 3) this manuscript had undergone extensive revision both experimental wise and the write up. The mistakes had most likely occurred during this process and might be caused by miscommunication between Dr. Tang and the student who helped her with making the figures. Based on the original lab record, Dr. Tang first identified which sub-figures contain the re-used image: (1)t Figure 3C is correct. The images of Figure 6A, lower panel, lane 1 (CCND1, CD19- IgM- group), and Figure 6B, lower panel, lane 3 (CCND1, CD19+ IgM+ group) were incorrect. While Figure 3(C) is the H E staining and IHC of JeKo-1-spheroid or JeKo-1- parental-derived tumour xenografts, Figure 6 (A-B) are the H E staining and IHC of tumors derived from three sub-clones isolated from JeKo-1-spheroid cell line or patient sample. (2)tFigure 6C(ii) lane 5 (Pax5) is correct and lane 4 (IgM) is incorrect. We found the original lab records in the lab note book that summarized the xenotransplantation assays in vivo of the JeKo-1-spheroid and JeKo-1-parental cells (see the inserted photocopy of the lab record). Since the lab notes were written in Chinese, we made a line-by-line translation of the lab note for your comprehension and review. Translated version: Line1: Experimental Date: September 13, 2016 Line2: Experiment Item: Termination the xenotransplantation assays from spheres cells and JeKo-1-parental cells on August 17, 2016. Line3: Staffs: Tang Junling, Sun Zhiwei,Zhou Shixia,Zhang Li Line4: Labs: 406 Cell Culture Room, IVC Animal Laboratory Center Line5: Experimental content: Today, we found that some mice had reduced exercise and Poor eatinguff0c two mice had significantly Line6: decreased weight, so the experiment was terminated. The mice were killed by neck breaking and gross anatomy was performed. 11 tissues and organs were collected Line7: and fixed with 4% paraformaldehyde and sent to the Department of Pathology, Affiliated Hospital of Southwest Medical University for microscopic examination. Line8: To determine the incidence of bleeding, infection, and Xenograft tumor growth. The summaries are as followsuff1a Line15: The expression of CD79a, CCND1 and IgM was observed: spheres cells uff1eJeKo-1-parental cells, but the results were not quantifiable. ![file] We also found the original lab records in the lab note book that summarized the xenotransplantation assays using the subclones sorted from JeKo-1-spheroid and patient sample (pt4). Since the lab notes were written in Chinese, we made a line-by-line translation of the lab note for your comprehension and review. Highly purified CD19- IgM-, CD19- IgM+, and CD19+ IgM+ cells from JeKo-1-spheroid (n =3, 19 days) and pt4 (n=4, 9 days) were injected intraperitoneally at two doses 102 and 104, respectively. In order to confirm that the three sub-populations from JeKo-1-spheroid and pt4 cells whether have different tumor biology, we conducted xenotransplantation assays. We further confirm every slide so as to confirm that while the monoclones retained CCND1-specific high expression, the negative expression of CD20, CD79a, PAX 5, Ki-67 and IgM. Translated version: Line1: Experimental Date: January 23, 2017 Line2: Experiment Item: Termination the xenotransplantation assays in vivo on the sorting sub-population from spheres cells on January 4, 2017. Line3: Staffsuff1aTang Junling, Zhang Li, Sun Zhiwei, Xu Lihong, Kong Liangsheng Line4: Labs: IVC Animal Laboratory Center, 310 Lab Line5: Experimental content: Today, we found that many mice had reduced exercise and four mice had significantly decreased weight, so the experiment was terminated Line6: and 11 tissues and organs were collected and fixed with 4% paraformaldehyde and sent to the Department of Pathology, Affiliated Hospital of Southwest Medical University for microscopic examination. Line7:The summaries are as followsuff1a ![file] ![file] Translated version: Line1: Experimental Date: March 15, 2017 Line2: Experiment Item: Sorting the clinical sample from pt4 and performed xenotransplantation assays in vivo. Line3: Staffs: Tang Junling, Zhang Li, Sun Zhiwei, Xu Lihong Line4: Labs: 406 Cell Culture Room, Flow Cytometry Laboratory, IVC Animal Laboratory Center Line5: Experimental content: 1, 5 ml of peripheral blood was collected from a 69-year-old male newly diagnosed patient with MCL-IVB group Line6: in the Department of Hematology, Affiliated Hospital of Southwest Medical University, and blood routine test: WBC: 82.78u00d7109 L,LYM:40.15u00d7109 L , Juvenile cell rate :33.6% Line7:RBC:1.83u00d71012 L, Hb:67g l, PLT:39u00d7109 L, PBMC were extracted, Line8: and the target cells were sorted, and the remained cells were frozen in 8 tubes. The experimental grouping and observation table are as follows :(4 mice *6 cages) Line9-20: ![file] To summarize our analysis and findings: (1)tThe two identical images in Figure 2A represent the true experiment findings. However, adding a line of explanation in the Figure Legend will avoid further misunderstanding to the reader. We will seek the advice of the editorial of Genes and Diseases regarding the amendment of the Figure Legend. (2)tFigure 3C is correct and no corrections are required. (3)tThe images of (i) Figure 6A, lower panel, lane 1 (CCND1, CD19- IgM- group), (ii) Figure 6B, lower panel, lane 3 (CCND1, CD19+ IgM+ group), (iii) Figure 6C(ii), lane 4 (IgM) were incorrect and need to be corrected. We made the revised Figure 6 accordingly (see below). When request for making the corrections, we will provide the Journal with the original data of all images taken to show that the experiments had been carefully done. The revised figure: ![file] We sincerely apologize for our careless that resulted in the unintentional errors present in the published paper. We thank you for the alert you sent us which gave us an opportunity tocorrect the mistakes. We will contact the Journal to make the correction as soon as possible. Finally, we are very honored that the research results of the first stage of this research can be recognized by Genes Diseases. After the publication of this article, Dr. Tang continued this line of research in more depth and has obtained experimental evidence by single-cell RNA-dequencing to substantiate our major conclusion that Mantel Lymphoma is plolyclonal origin, a discovery that will have significant clinical implications. Dr. Tang has provided the summary of her unpublished data for your review: ![file] , 
 #2 Thanks for your comments. The reply to question 2 comes with question 4 , 
 Corrigendum published January 1, 2022 After publication of the article, the authors (sic) recently noticed unintentional errors in Figure 6. Three pairs of sub figures contain misplaced and identical images due to unintentional errors during the preparation of the relevant Figures for publication: (1) Figure 3C, upper panel, lane 3 (CD79a, IgM-group) vs. Figure 6A, lower panel, lane 1 (CCND1, CD19- IgM-group); (2) Figure 3C, upper panel, lane 6 (CD79a, IgM + group) vs. Figure 6B, lower panel, lane 3 (CCND1, CD19+ IgM + group); (3) Figure 6C (ii), upper panel, lane 4 (IgM) vs. lane 5 (Pax5). Nevertheless, the unintentional errors do not change the conclusion or discussion of the article. The authors apologize for any inconvenience caused by these unintentional errors. In addition, there is a set of identical images present in Figure 2A. Since the isotope for CD10-PE and CD22-PE is the same (PE), the control isotope graph for CD10-PE and CD22-PE is the same. Nevertheless, the presentation of the data as it appeared in the original publication may lead to misinterpretation of the results, thus we deleted one image. The corrected Figure 2A and Figure 6 are provided below: ![file] ![file] ,