Cancer Cell, Volume 25 Supplemental Information Angiocrine Factors Deployed by Tumor Vascular Niche Induce B Cell Lymphoma Invasiveness and Chemoresistance Zhongwei Cao, Bi-Sen Ding, Peipei Guo, Sharrell B. Lee, Jason M. Butler, Stephanie C. Casey, Michael Simons, Wayne Tam, Dean W. Felsher, Koji Shido, Arash Rafii, Joseph M. Scandura, and Shahin Rafii
Supplemental Data: 1
Figure S1 (related to Figure 1) Aggressive lymphoma initiating cell (LIC) attributes of Eµ-Myc lymphoma cells (LCs) co-cultured with endothelial cells (LC EC ) and maintained in serum supplementation (LC Serum ) (A) Representative images of time course of Myc + LCs co-cultured with monolayers of E4ORF1 transduced human umbilical cord endothelial cells (VeraVec endothelial cells)-referred here to as ECs- or cultured alone in serum-free condition. Scale bar= 200 µm. (B) Flow cytometry analysis of B cell LC marker B220 and CD19 expression in mouse Myc + LCs maintained in serum-containing condition (left panel) and EC co-culture (right panel). (C) 5x10 5 isolated Myc + LCs were subcutaneously transplanted into NSG mice alone (LC alone) or coinjected with 1x10 6 ECs (LC + EC). LCs positioned adjacent to GFP + ECs manifest increased mitosis, as evidenced by Ki67 staining (right panel). Scale bar= 50 µm. (D) Ten days after culture, retrieved LC EC and LC Serum were subcutaneously injected to wild-type C57/B6 mice (2x10 6 cells per mouse). Tumor volume was measured; n= 4. (E) 2x10 6 Myc + LCs were injected into wild type C57/B6 mice. At day 8 and 16 after injection, the lymphoma mass were stained for VE-cadherin. Note that VE-cadherin + host ECs were seen to invade the lymphoma mass on day 16 but not earlier (day 8). Scale bar= 50 µm. (F, G) Markers associated with aggressive attributes were determined in LCs after EC co-culture in serumfree or serum-supplemented condition by quantitative PCR (qpcr) (F) and flow cytometry (G), including CD44, IGF1R, and CSF1R. Expression of CD44 and IGF1R on LCs in serum containing medium without EC co-culture is shown in Figure 1J. (H) Serial colony formation of LCs in methylcellulose was assessed by randomly selecting and serially passing LC EC and LC Serum. Quantification is shown in Figure 1L. Scale bar= 500 µm. (I) Representative images of LC EC and LC Serum in response to treatment with 0.1 µg/ml doxorubicin. Quantification of growth inhibition by doxorubicin is shown in Figure 1M. Scale bar= 500 µm. (J) Flow cytometry analysis of CD44 and IGF1R expression mouse IGF1R + CD44 + CSF1R + LCs during limiting dilution transplantation. Table S1 (related to Figure 1, Provided as an Excel file) Transcriptional comparison between lymphoma cells (LCs) co-cultured with endothelial cells (ECs) and maintained in serum containing medium. 2
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Figure S2 (related to Figure 2) Co-culture with ECs activates the Notch2-Hey1 pathway in indolent LCs, fostering emergence of aggressive LCs with LIC-like attributes (A, B) Serial colony forming capacity of IGF1R + CD44 + CSF1R + subset in LC EC and LC Serum was compared (A). Representative image of formed colonies in methylcellulose after serial passage 3 (P3) is shown in (B); Scale bar= 500 µm. (C) Notch effector Hey1 expression in IGF1R + CD44 + CSF1R + LC clones was compared with CD44 - IGF1R - CSF1R - LC subset in LC Serum by qpcr; n= 4. (D-F) Individual shrna clones were employed to knockdown mouse Hey1 (D), Notch1 (E), Notch2 (F). Protein level of targeted molecules was tested by immunoblot and quantified by densitometry; n= 3. (G-H) After Hey1 (G) and Notch2 (H) knockdown, LC expansion after EC co-culture was quantified; n= 3. (I-J) Effect of shrna against Notch1 (I) and Notch2 (J) on Notch signaling in LCs was compared by chromatin immunoprecipitation (ChIP) analysis of Notch downstream mediator RBPJ. (K-L) Notch2 (K) and Hey1 (L) were silenced in mouse LCs with three independent shrna constructs and intrasplenically injected into mice without EC co-culture. Hepatic lymphoma load was evaluated by H&E staining. 1x10 5 LCs were injected via spleen. Scale bar= 50 µm. (M) Expression of Jag1 in Myc + LCs express negligible level of Jag1, compared with that of ECs; n=3. (N) Approach to investigate the contribution of Notch-Hey1 in maintaining the colony forming capacity in aggressive CD44 + IGF1R + LCs. To block Notch-Hey1 pathway, CD44 + IGF1R + aggressive LCs were incubated with compound E or transduced with shhey1 after co-culture with ECs. (O) Colony formation capacity of CD44 + IGF1R + LCs with or without inhibition of Notch-Hey1 pathway was quantified; n= 3. 4
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Figure S3 (related to Figure 3) Expression of Jag1 and Hey1 proteins in human Burkitt s lymphoma tissues (A) Jag1 expression in lymphoma tissues of Burkitt s lymphoma patients and normal lymph node. Three representative staining images of Jag1, EC-specific marker VE-cadherin, and Hey1 are shown in Figure 3A, and staining of 4 th Burkitt s lymphoma patient is shown here. Scale bar= 50 µm. (B, C) Proliferation of (B) and Hey1 expression (C) in human Burkitt s LCs harboring MYC chromosomal translocation under immunoglobulin heavy chain enhancer. Human LCs co-cultured with ECs (LC + EC group) were compared with those cultured in serum free medium (LC group); n= 4. (D-F) Efficiency of shrna-mediated knockdown of HEY1 (D), NOTCH1 (E), and NOTCH2 (F) in human Burkitt s LCs. Protein level was detected by immunoblot and quantified by densitometry. Each target gene was silenced by at least three individual shrna clones; n=3. (G-H) After knockdown of HEY1 (G) and NOTCH2 (H), expansion of human LCs after EC co-culture was determined; n=3. (I-J) HEY1 (I) and NOTCH2 (J) were silenced in human LCs with three individual shrna constructs and intrasplenically injected into mice. Hepatic lymphoma load was then compared by H&E staining. 5x10 5 human LCs were injected into the spleen of NSG mice. Scale bar= 50 µm. 6
Table S2 (related to Figure 3) Expression of Jagged1 and Hey1 in human Burkitt's lymphoma tissue Patient Number Age Gender Tumor site Jag1 expression in lymphoma ECs 1 Hey1 + cell percentage 2 1 8 M Nasopharynx +++ ++ 2 7 M Right Tonsil ++ +++ 3 8 M Left Tonsil ++ ++ 4 5 M Abdominal wall +++ +++ 1 Jag1 + ECs in lymphoma per high power field. +++, over 60%; ++, between 40%-60%; + lower than 40%.. 2 Hey1 + lymphoma cells in lymphoma per high power field. +++, over 40%; ++, between 20%-40%; + lower than 20%.. 7
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Figure S4 (related to Figure 4) Regulation of Jag1 expression in human ECs by FGF4 from Burkitt s LCs and inducible EC-specific deletion of Fgfr1 (Fgfr1 iδec/iδec ) in adult mice (A) FGF4 expression in two Burkitt s lymphoma patients and normal lymph node was examined by immunostaining. FGFR1 activation in lymphoma ECs was examined by immunostaining of phosphorylation/activation of downstream effector FRS2 (pfrs2). Note that pfrs2 is predominantly localized in VE-cadherin-expressing ECs (white arrow head). Scale bar= 50 µm. (B, C) FGFR1 activation in ECs co-cultured with human Burkitt s LCs was tested.fgf4 expression was knocked down in human LCs by four individual clones (B). After 2,000 LCs cells were incubated with 1x10 5 human ECs, FGFR1 activation in co-cultured human ECs was examined by immunoblot of the phosphorylation/activation of downstream effector FRS-2 (C). Scrambled (Srb) transduced human LCs were used as control. (D) Jag1 transcriptional level in human ECs maintained in serum-free medium containing 20 ng/ml VEGF- A. Mouse LCs were transduced with Srb or Fgf4 shrna and co-cultured with human ECs; n= 3. (E, F) Injection of tamoxifen into VE-Cad-Cre ERT+ Fgfr1 loxp/loxp and VE-Cad-Cre ERT+ Fgfr1 loxp/+ mice caused EC-specific deletion of Fgfr1 in adult mice (Fgfr1 iδec/iδec ). Deletion of Fgfr1 in ECs was verified by qpcr (E) and immunoblot (F) analyses. 9
Figure S5 (related to Figure 6) In vivo conversion of indolent LC subset to aggressive LCs by host ECs (A) 2x10 6 Myc + mouse LC Serum were subcutaneously injected to mice with EC-specific deletion of Jag1 (Jag1 iδec/iδec ) and control mice. Tumor volume was monitored; n= 4-6. (B) Individual LC clones were derived from both CD44 - IGF1R - CSF1R - and CD44 + IGF1R + CSF1R + subpopulations and subcutaneously injected to wild type mice. At day 28 after injection, LCs were isolated from tumor mass and analyzed for expression of CD44, IGF1R, and CSF1R. CD44 + IGF1R + CSF1R + LC clones were also transplanted as control. Representative flow cytometry graph of injected clones is shown. Each derived clone was injected into 3 mice. 10
Figure S6 (related to Figure 8) Vascular perfusion function in Eµ-Myc mice with EC-specific deletion of Jag1 (Myc + Jag1 iδec/iδec ) (A) Representative image of lymph node (arrow) in Myc + Jag1 iδec/iδec and control mice. (B, C) Vascular perfusion of Myc + Jag1 iδec/iδec and control mice was tested by intravenously injecting 50 µg isolectinb4 that binds to perfused and patent vasculature. Representative fluorescent image of isolectin and VE-cadherin staining is presented in (B), and quantification of patent ECs (isolectin + VE-cadherin + ) is shown in (C). Scale bar = 50 µm and 10 µm in inset. (D)Hypoxia in the lymphoma mass in Myc + Jag1 iδec/iδec and control mice was tested by probe Pimonidazole staining (Hypoxyprobe -1 Plus Kit, Hypoxyprobe, Inc). As control, Myc + mice were injected with VEGFR2 inhibitor SU1498 to induce hypoxia in enlarging tumor mass. Sale bar = 50 µm. 11
Supplemental Experimental Procedures: Animals Fgfr1 loxp/loxp or Jag1 loxp/loxp mice were bred with VE-cadherin-Cre ERT2 (cdh5-pac-cre ERT2 ) transgenic mice to establish VE-cadherin-Cre ERT2 Fgfr1 loxp/loxp or VE-cadherin-Cre ERT2 Jag1 loxp/loxp mice. Eµ-Myc mice were crossed with VE-cadherin-Cre ERT2 Fgfr1 loxp/loxp or VE-cadherin-Cre ERT2 Jag1 loxp/loxp mice to generate Myc + VE-cadherin-Cre ERT2 Fgfr1 loxp/loxp or Myc + VE-cadherin-Cre ERT2 Jag1 loxp/loxp mice.ecspecific deletion of Fgfr1 or Jag1 was induced by treating mice with tamoxifen at a dose of 250 mg/kg i.p. for 6 days interrupted for 3 days after the third dose, resulting in EC-specific gene deletion in lymphoma mice (Myc + Fgfr1 iδec/iδec or Myc + Jag1 iδec/iδec ).Transgenic Notch Reporter (TNR) mice were crossed with Myc + Jag1 loxp/loxp mice to analyze Notch pathway activity upon EC-specific deletion of Jag1. Deletion of target genes in tumor ECs was corroborated by quantitative PCR and immunoblot analysis. All animal experiments were carried out under the guidelines set by Institutional Animal Care and Use Committee at Weill Cornell Medical College. Co-culture of lymphoma cells (LCs) with VeraVec endothelial cells in serum/growth factor-free condition For serum/growth factor-free co-culture experiments, wild-type (WT) endothelial cells (ECs) or E4ORF1 transduced human umbilical endothelial cells (VeraVec ECs) in which Jag1 was knocked down by shrna (EC shjag1 ) were plated in 6 well plate at 1x10 5 cells/well. VeraVec ECs are cultivated without the confounding factors associated with the use of serum or exogenous recombinant angiogenic factors, including VEGF-A and FGF-2 as well as without supplementation with bovine brain extracts or xenobiotic factors. As such, VeraVec ECs sustain their native vascular niche attributes and supply the physiological levels of angiocrine factors necessary for the unbiased growth of the tumor cells. LCs isolated from Eµ- 12
Myc mice were seeded on top of the monolayer formed by WT ECs or EC shjag1 at 1000 cells/well. After 8 or 12 days after co-culture, the number of LCs was quantified by flow cytometric analysis. VeraVec ECs are referred here for simplicity as ECs. Inhibition of Notch pathway in LCs and Notch ligand Jag1 in ECs: shrna-mediated gene knockdown was utilized to selectively knock down Notch ligand (Jag1) and receptors (Notch1, 2) in VeraVec ECs and LCs, respectively. Human Notch1 was silenced by clones TRCN0000003358, TRCN0000003359, TRCN0000003360, and Notch2 was knocked down with clones TRCN0000004894, TRCN0000004895, TRCN0000004896, TRCN0000004897. shrna clones targeting human Hey1, TRCN0000020214, TRCN0000020215, and TRCN0000020216 were utilized. For mouse Hey1 knock down, we utilized shrna Clones: TRCN0000086480, TRCN0000086482, TRCN0000086481, TRCN0000086479. Mouse Notch1 was knocked down with shrna Clones: TRCN0000025895, TRCN0000025902, TRCN0000025908. Mouse Notch2 was silenced by clones: TRCN0000025887, TRCN0000025896, TRCN0000025898, and TRCN0000025924.Lentiviruses were generated by cotransfecting 15 μg of shuttle lentiviral vector containing target gene or scrambled shrna, 3 μg of penv/vsv-g, 5 μg of prre, and 2.5 μg of prsv-rev in 293T cells by the calcium precipitation method. Viral supernatants were concentrated by ultracentrifugation and used to transduce HUVECs or LCs. γ- secretase inhibitor compound E was utilized to abolish Notch pathway activation in LCs. LCs were incubated with 1 µm compound E before injection into mice or co-culture with ECs. Compound E was injected to mice at 2 mg/kg. Detection of Notch2 activation in LCs Cultured LCs were retrieved from either serum containing medium or co-culture with serum/growth factorfree VeraVec ECs. Notch activation was detected by translocation of ICD of Notch 1 and 2. Notch 1 and 2 ICDs were detected by both immunostaining and immunoblot. Antibody detecting Notch1 ICD was from 13
Cell signaling, Notch 2 ICD was examined by antibody from Abcam. For ICD staining, retrieved LCs were cytospun onto glass slides and fixed with 4% paraformaldehyde. After rehydratation for 10 min at room temperature in PBS containing 10% bovine serum albumin, cells were incubated with primary antibodies and secondary antibody conjugated with fluorescent dyes. After nuclear countstaining with DAPI, cells were washed, mounted under glass and analyzed by confocal microscopy. Chromatin immunoprecipitation (ChIP) analysis was utilized to test the activation of RBPJ. ChIP was performed with antibody recognizing RBPJ (Cell Signaling, Cat. No. 5313). Cells were fixed in 1% formaldehyde for 9 min, washed in PBS and quenched with ph 8 Glycine solution (125 mm final concentration). ChIP was performed with Chromatin IP kit (Cell signaling). Different groups of LCs with knockdown of Notch1, Notch2, and inhibition of γ-secretase inhibitor compound E were compared. Flow cytometry Labeled LCs were measured by LSRII flow cytometer (Beckton Dickenson); compensation for multivariate experiments was carried out with FACS Diva software (Becton Dickinson Immunocytometry Systems). Flow cytometry analysis was performed using various controls, including isotype antibodies and unstained LCs for determining gates and compensations in flow cytometry. 14