1. Volume 30, Issue 4, Pages 610-622 (October 2016)
LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia 
Phuong-Hien Nguyen, Oleg Fedorchenko, Natascha Rosen, Maximilian Koch, Romy Barthel, Tomasz Winarski, Alexandra Florin, F. Thomas Wunderlich, Nina Reinart, Michael Hallek 
Cancer Cell 
Volume 30, Issue 4, Pages (October 2016)
DOI: /j.ccell
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2. Cancer Cell 2016 30, 610-622DOI: (10.1016/j.ccell.2016.09.007)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Lyn Deletion Reduces CLL Burden In Vivo
(A) Flow cytometric analysis of CD19+CD5+ CLL cells in the peripheral blood of TCL1tg/wtLynwt/wt and TCL1tg/wtLyn−/− mice over 1 year.
(B) Spleen weights of moribund mice.
(C) Liver weights of moribund mice.
(D) Flow cytometric analysis of CLL cells in spleens of age-matched and moribund mice.
(E) Flow cytometric analysis of CLL cells in bone marrow samples of age-matched and moribund mice.
(F) Representative immunohistochemical stainings of CD45R from sections of spleen, liver, bone marrow, and lymph node from moribund mice. Scale bars represent 50 μm.
See also Figure S1.
Cancer Cell  , DOI: ( /j.ccell )
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4. Figure 2 Lyn Deletion Leads to a Severe Phenotype in TCL1tg Mice
(A) Kaplan-Meier curves representing the overall survival of mice from birth to moribund; p values were calculated by log rank test.
(B) Representative H&E staining of kidney sections from moribund mice. Scale bars represent 50 μm.
(C) Percentage of monocytes per leukocytes in the peripheral blood over 1 year.
(D) Flow cytometric analysis of CD11b+F4/80+ macrophages in the blood, spleen, and bone marrow of moribund mice.
(E) Flow cytometric analysis of CD11b+ cells in blood of WT recipients after transplantation with splenic mononuclear cells from TCL1tg/wtLyn−/− donors. Spleen cells were purified with Ficoll gradient centrifugation, and total harvested cells containing up to 60% CD11b+ cells were injected intraperitoneally into littermate WT recipients. Data are presented as means ± SEM.
(F) Cytokine profile in sera of two TCL1tg/wtLynwt/wt and three TCL1tg/wtLyn−/− mice between 3 and 4 months old. Data represent mean values (pg/mL).
(G) Cytokine profile in sera of two TCL1tg/wtLynwt/wt and three TCL1tg/wtLyn−/− mice between 10 and 12 months old. Data represent mean values (pg/mL).
See also Figure S2.
Cancer Cell  , DOI: ( /j.ccell )
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5. Figure 3 Lyn Deletion in TCL1tg B Cells Does Not Affect CLL Malignancy
(A) Proportion of CLL cells from the B cell compartment in spleens of 3-month-old mice.
(B) Flow cytometric analysis of in vivo bromodeoxyuridine (BrdU) incorporation rate in splenic B cells of mice in all age groups 16 hr after injection of BrdU.
(C) Western blot analysis of purified B cells isolated from 3-month-old mice. Cells were kept untreated or stimulated with 20 μg/mL IgM for 15 min. The TCL1tg/wtLynwt/wt sample is a pool from three mice; the TCL1tg/wtLyn−/− sample is a pool from four mice.
(D) Flow cytometric analysis of CLL cells in the blood of WT recipients after transplantation with splenic cells from 3-month-old TCL1tg/wtLynwt/wt and 3- to 9-month-old TCL1tg/wtLyn−/− mice. Each donor's spleen containing different proportion of CLL cells (0.4%–40%) was purified with Ficoll gradient centrifugation and injected intraperitoneally into one recipient. Time point 0 was assigned when a distinct CLL population could be detected in the peripheral blood of each recipient (2%–4% of peripheral bone marrow cells).
All data are presented as means ± SEM. See also Figure S3.
Cancer Cell  , DOI: ( /j.ccell )
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6. Figure 4
No Significant Difference in the Survival Signaling Pathways of TCL1tg/wtLynwt/wt and TCL1tg/wtLyn−/− CLL Cells
(A) Western blot analysis of purified B cells isolated from 9-month-old mice. Cells were kept untreated or stimulated with 20 μg/mL IgM for the indicated time before protein lysis. Each sample is a pool of B cells from three mice.
(B) Western blot analysis of purified, untreated B cells isolated from individual 9-month-old mice.
See also Figure S4.
Cancer Cell  , DOI: ( /j.ccell )
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7. Figure 5
Lyn Deletion in the Microenvironmental Cells Hindered CLL Progression
(A) Flow cytometric analysis of CLL development in blood of young WT and Lyn−/− recipients between 8 and 12 weeks old after transplantation with CLL cells. Each CLL clone was injected into at least two recipients of each genotype.
(B) Flow cytometric analysis of CLL cells in the blood of old WT and Lyn−/− recipients between 10 and 12 months of age after transplantation with CLL cells. Each CLL clone was injected into two recipients of each genotype.
(C) Kaplan-Meier curves representing the survival of WT and Lyn−/− recipients from the day of CLL transplantation until moribund; p values were determined by log rank test.
(D) Spleen weights of moribund WT and Lyn−/− recipient mice after CLL transplantation.
(E) Flow cytometric analysis of CLL development in the blood of young WT and Btk−/− littermates between 6 and 10 weeks of age after transplantation with CLL cells. Each CLL clone was injected into four recipients of each genotype.
(F) Kaplan-Meier curves representing the survival of WT and Btk−/− recipients from the day of CLL transplantation until moribund; p values were determined by log rank test.
(G) Spleen weights of moribund WT and Btk−/− recipient mice after CLL transplantation.
All data are presented as means ± SEM. See also Figure S5.
Cancer Cell  , DOI: ( /j.ccell )
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8. Figure 6
Lyn deletion in the Immune Cells Partially Contributed to Delayed CLL Progression
(A) Immune phenotype in the peripheral blood of chimeric mice at week 5 after bone marrow reconstitution.
(B) Flow cytometric analysis of CLL engraftment in the blood of WT and Lyn−/− chimeric recipients after transplantation with CLL cells. Littermate WT mice were irradiated and reconstituted with WT or Lyn−/− bone marrow cells for 8 weeks. Each CLL clone was transplanted into at least two littermates of each chimeric group.
(C) Kaplan-Meier curves representing the survival of all chimeric recipients from the day of CLL transplantation until moribund.
(D) Spleen and liver weight of moribund chimeric recipients.
(E) Flow cytometric analysis of viable TCL1tg/wtLynwt/wt and TCL1tg/wtLyn−/− CLL cells cocultured with thioglycollate-elicited Lynwt/wt or Lyn−/− macrophages in vitro.
(F) Flow cytometric analysis of viable primary CLL cells cocultured with murine Lynwt/wt or Lyn−/− bone-marrow-derived macrophages.
(G) Flow cytometric analysis of viable primary CLL cells cocultured with murine Lynwt/wt or Lyn−/− bone-marrow-derived macrophages, with or without 0.4 μm pore inserts.
All data are presented as means ± SEM. See also Figure S6.
Cancer Cell  , DOI: ( /j.ccell )
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