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Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma

Abstract

Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy.

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Figure 1: Hepatic ELSs signify a poor prognosis in human HCC and are associated with NF-κB activation.
Figure 2: Persistent activation of IKK in the liver induces ELSs.
Figure 3: Persistent activation of IKK in hepatocytes induces aggressive HCC.
Figure 4: HCC progenitor cells appear in ELSs and progressively egress.
Figure 5: Cells of the adaptive immune system are required for ELS-dependent promotion of HCC.
Figure 6: Anti-Thy-1.2 immunoablative treatment during ELS development attenuates liver tumorigenesis.
Figure 7: ELS microniches provide a rich cytokine milieu.
Figure 8: Blockade of lymphotoxin signaling abolishes egress from microniches and tumorigenesis.

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Gene Expression Omnibus

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Acknowledgements

We thank K. Kohno (Nara Institute of Science and Technology) for plasmids pBstN and p2335A-1; M.-A. Buendia (Institut Pasteur, France) for Myc-Trp53−/− liver tumors; V. Factor and S. Thorgeirsson (US National Institutes of Health) for anti-A6; and E. Cinnamon, M.-A. Buendia, K. Kohno, M. Ringelhan, R. Hillermann, D. Kull, I. Gat-Viks, Y. Steuerman, S. Itzkovitz and K. Bahar-Halpern for help and advice. Supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (E.P. and Y.B.-N.), the European Research Council (LIVERMICROENV to E.P.; PICHO to Y.B.-N.; and LiverCancerMechanism to M.H.), the Israel Science Foundation (E.P., Y.B.-N., I.S. and O.P.), the Israel Cancer Research Fund (Y.B.-N.), the Helmholtz alliance “preclinical comprehensive cancer center” Graduiertenkolleg (GRK482 to M.H.), Krebsliga Schweiz (Oncosuisse) (A.W.), Promedica Stiftung (A.W.), the US National Institutes of Health (CA118165, SRP ES010337 and AI0043477 to M.K.; and DK099558 to Y.H.), the Hildyard chair for Mitochondrial and Metabolic Diseases (M.K.), the Japan Society for the Promotion of Science (K.T.), Irma T Hirschl Trust (Y.H.), the FLAGS foundation (N.G.) and the Uehara Memorial Foundation (S.N.).

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Authors

Contributions

K.R., M.Ka., M.H., Y.B.-N. and E.P. conceived of the study; S.F., D.Y. and I.S. designed and carried out most experiments and data analysis; K.T., A.W., K.U., N.G., S.N., G.G., M.E.S., M.Ko., H.K., M.B. and O.P. carried out additional experiments, contributed samples and performed data analysis; J.L.B. and K.R. supplied reagents; and S.F., D.Y., I.S., Y.H., M.Ka., M.H., Y.B.-N. and E.P. wrote the manuscript.

Corresponding authors

Correspondence to Mathias Heikenwalder, Yinon Ben-Neriah or Eli Pikarsky.

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The authors have filed a provisional patent related to this manuscript.

Integrated supplementary information

Supplementary Figure 1 Hepatic ELSs signify a poor prognosis in human HCC and are associated with NF-κB activation.

(a) Intrahepatic ELSs in human livers were classified as vague follicular aggregates (Agg), definite round-shaped clusters of small lymphocytes without germinal center (Fol), and follicles with definite germinal centers composed of large lymphocytes with clear cytoplasm (GC) according to published criteria (see Methods). (b-d) Kaplan-Meier curves for probability of early (b) or late (c) recurrence or of overall survival (d) after resection of HCC, in patients with high (red) and low (blue) ELS histological score in the liver parenchyma [66 patients with H&E staining out of 82 patients (14 high score, 52 low score); *p=0.04, n.s.- not significant, p=0.78, 0.18 for b, d, respectively, log-rank test]. (e) Kaplan Meier curves for probability of early recurrence after resection of HCC, in patients with high (red) and low (blue) ELS scores in the liver parenchyma [n=82 patients (15 high score, 67 low score); n.s.- not significant, p=0.34, Log-rank test]. (f) Gene set enrichment index assessing correlation between enrichment of 3 different published sets of NF-κB targets (X axis) and histological ELS score in human livers (Y axis) [see more details in Methods; n=66 patients (14 high score, 52 low score); p as indicated, two-tailed Students t-test]. See also Gene Set Enrichment Analysis (GSEA analysis), Supplementary Table 1.

Supplementary Figure 2 Constitutive activation of the NF-κB pathway in hepatocytes induces mild liver inflammation.

(a) NF-κB DNA binding was analyzed by EMSA in nuclear extracts from Alb-cre control, IKKβ(EE)Hep, TNF-treated Alb-cre for 30 or 60 minutes and Mdr2-/- mice. To examine the composition of NF-κB dimers, Alb-cre-TNF−treated nuclear extract was supershifted with RelA/p65 antibody. One of two Mdr2-/- lanes was removed from the figure for esthetic reasons. (b) Quantification of EMSA. Results are representative of three independent experiments (*p<0.05, two-tailed Students t-test, bars - mean ± SEM). (c) qPCR analysis for TNF and KC of mice described in a (control, IKKβ(EE)Hep, Mdr2-/-, TNF-treated Alb-cre 30'/60': n=7,5,6,4,4, respectively; *p=0.01, **p=0.0002; bars - mean ± SEM). (d) Representative immunohistochemical stains for GFP and RelA/p65 of livers from 6 months old Alb-cre control and IKKβ(EE)Hep mice (scale bars- 25 μm). (e) Representative H&E stains of liver tissue from 3 months-old Alb-cre control and IKKβ(EE)Hep mice (scale bars: upper panels- 200 mm, lower panels- 50 mm). (f) Representative photomicrographs of F4/80-stained liver sections from 7 months old Alb-cre control and IKKβ(EE)Hep mice (scale bars: 50 μm upper panels, 25 μm lower panels). (g) Quantification of F4/80+ cells (expressed as % of total) shown in f (control: n=11, IKKβ(EE)Hep: n=6, *p=0.002, two-tailed Students t-test, bars - mean ± SEM). (h,i) Alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were measured in sera of 7 months old Alb-cre control and IKKβ(EE)Hep mice (control: n=8, IKKβ(EE)Hep: n=5, *p<0.01, two-tailed Students t-test, bars- mean ± SEM). Normal range of ALT and AST: 17-77 and 54-191 U/L, respectively. Note that while AST levels in IKKβ(EE)Hep mice are significantly higher than in controls they are still within the normal range. (j) Representative Ki67 immunostains of Alb-cre control and IKKβ(EE)Hep mice liver parenchyma at the indicated ages (scale bars- 50 μm). (k) Quantification of Ki67 immunostains described in j (n=6,5,7,7 for 1,4,7,20 months control mice, respectively; n=5,4,7,7 for 1,4,7,20 months IKKβ(EE)Hep mice, respectively; n.s - non significant, *p=0.03, **p=1E-07, two-tailed Students t-test, bars - mean ± SEM; hpf - high power field). (l,m) Splenocytes of 5-months-old Alb-cre mice (l, upper panels) or from microscopically isolated ELSs from 5 months old DEN treated IKKβ(EE)Hep mice livers (l lower panels and m) were analyzed by flow cytometry for the following cell surface markers: CD4, CD8, CD44 and CD62L in l; CD45, CD11b+, MHCII+ (as a marker for activation) and F4/80+ in m. For both l and m results are representative of ELSs isolated from 6 IKKβ(EE)Hep mice (bars - mean ± SEM). (n) Representative co-immunofluorescence stains for B and T lymphocytes in IKKβ(EE)Hep mice (top) and human (bottom) ELSs, showing clear compartmentalization. CD3 labels T cells and B220/CD20 labels B cells. DAPI (blue) marks the nuclei (scale bars-100 µm). (o) mRNA qPCR analysis for the ELS gene signature in liver parenchyma from Alb-cre control and IKKβ(EE)Hep mice at the indicated ages (n=12,7,11 for control, 14 and 20 months old IKKβ(EE)Hep, respectively; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed Students t-test, bars - mean ± SEM. See Supplementary Table 2 for further details). Data are representative of one experiment except for (d), (f), (j), (n) and (o) which are representative of two independent experiments.

Supplementary Figure 3 IKKβ(EE) expression in hepatocytes induces HCC and metastases.

(a) Representative immunostains for A6, glutamine synthetase (GS) and Ki67 in tumors of IKKβ(EE)Hep mice (scale bars- 50 μm). (b) Immunostains for collagen IV of liver tissue from untreated Alb-cre or tumors from 9-months old DEN-treated Alb-cre control mouse and 20-months old IKKβ(EE)Hep mouse. Parenchyma (P) and Tumor (T) areas are indicated; Red dashed line depicts P/T border (scale bars- 50 μm). (c) Representative H&E stained sections showing lymph node and lung metastasis in 20 months old IKKβ(EE)Hep mice (scale bars: left panels 500 μm, right panels 50 μm). (d,e) ELS number (d) and diameter (e) in livers of Alb-cre control, IKKβ(+/E)Hep hemizygotes and IKKβ(EE)Hep homozygotes mice at the indicated ages. Control Alb-cre mice do not develop ELSs (n=10,8,6,5,4 for control, 4,7 and 14 months old IKKβ(EE)Hep or IKKβ(+/E)Hep mice, respectively; *p≤0.002, **p≤0.0003, two-tailed Students t-test, bars- mean±SEM). (f,g) Tumor number (f, ≥0.5 cm) and volume (g) in livers of 20-month-old Alb-cre control, IKKβ(+/E)Hep hemizygotes and IKKβ(EE)Hep homozygotes mice (n=13,9,11 for control, IKKβ(+/E)Hep and IKKβ(EE)Hep mice, respectively; n.s.=not significant, *p<0.05, **p<0.0001, two-tailed Students t-test, red cross line signifies mean). Data regarding control and IKKβ(EE)Hep homozygotes mice are the same as in Fig. 3a-b and are shown here as a reference. (h) Representative photomicrographs of F4/80-stained liver sections from 9 months-old control and Alb-IKKβ(EE) mice (scale bars, upper panel- 50 μm, lower panel- 25 μm). (i) H&E stains of liver tissue from 9 months-old Alb-IKKβ(EE) mice reveal the presence of ELSs (scale bars-50 mm). (j) Liver and H&E stained section of liver tissue from 12-month-old Alb-IKKβ(EE) mouse. Arrows indicate tumors on the liver surface (scale bar- 50 μm). (k) Representative H&E stained sections of livers from DEN-treated IKKβ(EE)Hep mice at the indicated ages (scale bars - upper panels: 200 μm, lower panels: 50 mm). (l,m) Quantification of ELSs number/cm2 and diameter (μm) in untreated and DEN-treated IKKβ(EE)Hep mice. Alb-cre control mice, either untreated or DEN-treated, do not develop ELSs (n=5/5,8/9,8/9,6/10 for control, 3,6 and 9 months old untreated or DEN-treated IKKβ(EE)Hep mice, respectively; *p=0.001, **p<0.0001 for (l) and *p<0.0001 for (m), two-tailed Students t-test, mean ± SEM). (n,o) Tumor (≥0.5 cm) number and total volume in livers of 9-month-old DEN-treated Alb-cre control and IKKβ(EE)Hep mice (n=12, 8, 11 for DEN-treated control, untreated IKKβ(EE)Hep and DEN-treated IKKβ(EE)Hep mice, respectively; *p<0.001 for (n) and *p<0.01 for (o), two-tailed Students t-test, red cross line signifies mean). (p) Representative livers and H&E stained sections of liver tissue from 9-month-old DEN-treated Alb-cre control and IKKβ(EE)Hep mice. Arrows and dashed lines indicate tumors (scale bars-200 mm). (q) Representative Ki-67 immunostains of DEN-treated Alb-cre control and IKKβ(EE)Hep mice liver parenchyma or tumor (mon-months, scale bars-50 μm). (r) Quantification of Ki67+ hepatocytes in liver parenchyma (par) and tumors of DEN-treated Alb-cre control and IKKβ(EE)Hep mice (n=5,6,5,5,7,8,4,4,7,8 for the indicated mice, respectively; *p<0.01, **p<0.001, two-tailed Students t-test, bars- mean ± SEM). (s) Aberration scores for each of the tumors analyzed by aCGH [Data are stored and available from ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) accession number E-MTAB-3848]. Score (0-3) was determined based on size and number of aberrations in each tumor. Well differentiated tumors (WD-HCC) were compared to mixed (HCC-CCC) tumors in each group (n=6,7,7 for control, IKKβ(EE)Hep+DEN and IKKβ(EE)Hep mice, respectively; *p<0.05, two-tailed Students t-test, bars- mean ± SEM). (t) Genomic DNA was extracted from parenchyma of Alb-cre control mice or from tumors of IKKβ(EE)Hep mice and subjected to copy number variation analysis by digital PCR. Rgs2 and Gab2 are genes located at the centers of two chromosomal regions found to be amplified in aCGH analysis of HCCs from IKKβ(EE)Hep mice (see s). Red and purple dashed lines depict average plus 2 standard deviations of the control group for Rgs2 and Gab2, respectively. Tert - a reference for unamplified DNA (n=11,13 for control and IKKβ(EE)Hep, respectively). Data are representative of one experiment except (l), (m), (n) and (q), which are representative of two independent experiments.

Supplementary Figure 4 Tumor progenitor cells gradually egress from their supportive microniche.

(a-c) Consecutive liver sections were immune-stained for the HCC progenitor markers CD44v6, Sox9 and CK19 as indicated. The percent of positive cells (out of total hepatocytes) within ELS and in liver parenchyma is shown (n=10, *P≤1.6E-10, two-tailed Students t-test, bars- mean ± SEM). (d) Representative immunostains for GFP (scale bars-50 µm). (e) Representative H&E stained sections of IKKβ(EE)Hep livers depicting ELS to HCC progression (arrows point to small ELSs; scale bars, upper panels-200 µm, lower panels- 50 µm). Lower panels are the same ones shown in Fig. 4c. (f,g) Representative whole slide scans of H&E stained sections from 20 months old IKKβ(EE)Hep liver (f, see also high resolution scan: http://viro2.helmholtz-muenchen.de/dih/webViewer.php?snapshotId=14313306898875) and 9 months old IKKβ(EE)Hep DEN-treated liver (g, see also high resolution scan: http://viro2.helmholtz-muenchen.de/dih/webViewer.php?snapshotId=14313306213780). Note that in each liver multiple ELSs and tumors at various stages of progression are present (arrows indicate ELSs, scale bars-1 mm). (h) Genomic DNA, extracted from parenchyma or from tumor progenitors cells isolated by laser capture micro-dissection from ELSs of 5 months old IKKβ(EE)Hep livers, was subjected to copy number variation analysis by digital PCR. Rgs2 and Gab2 are genes located at the centers of two chromosomal regions found to be amplified in aCGH analysis of HCCs from IKKβ(EE)Hep mice (see Supplementary Fig. 3t). Red and purple dashed lines depict average plus 2 standard deviations of the control group for Rgs2 and Gab2, respectively. Tert used as a reference for unamplified region (n=10,11 for parenchymal and ELS derived hepatocytes, respectively). (i) Representative co-immunofluorescence stains depicting the ELS egression process. Lymphocytes (CD3+B220) highlight ELS border. GFP labels all hepatocytes, Sox9 labels malignant hepatocytes. Hoechst 33342 (blue) marks the nuclei. Arrows points to egressing cluster (scale bars-100 µm). (j) Quantification of ELS zonation in livers of 14-20-months-old untreated IKKβ(EE)Hep mice and 6 months old DEN treated IKKβ(EE)Hep mice (n=9, *p=2.1E-09, two-tailed Students t-test, bars- mean ± SEM). Data are representative of one experiment except for (d) and (j) which are representative of two independent experiments.

Supplementary Figure 5 Proliferation, apoptosis and NF-κB activation in well-differentiated HCCs from IKKβ(EE)Hep mice do not differ from that in those from Rag1–/– IKKβ(EE)Hep mice.

Representative immunostains for the proliferation marker Ki67 (a), apoptosis marker Cleaved Caspase 3 (b) and RelA/p65 (c) in livers of 6 months old IKKβ(EE)Hep and Rag1-/- -IKKβ(EE)Hep mice (scale bars-50 μm). Graphs on the right (d,e,f, respectively) depict quantification of the corresponding immunostain. Ki-67 and cleaved Caspase 3 positive hepatocytes were counted in 10, arbitrary chosen, high power fields; RelA/p65 stain was quantified using an arbitrary subjective scoring scale (0-4) of nuclear staining (n=5, n.s - not significant, two-tailed Students t-test, bars - mean ± SEM).

Supplementary Figure 6 Anti-Thy-1.2 treatment during ELS development attenuates liver tumorigenesis.

(a) Blood samples were taken from TLF-2 (isotype control antibody) injected or from anti-Thy1.2 injected 6 months old DEN-treated IKKβ(EE)Hep mice and analyzed by flow cytometry for two T cell markers, Thy1 and TCRβ. Numbers above the bars represent percentage of positively stained cells. (b) Liver to body ratio of control or anti-Thy1.2 treated 6 months old DEN-injected IKKβ(EE)Hep mice (n=6,10 for control and anti-Thy1.2 treated mice, respectively; *p<0.01, two-tailed Students t-test, bars- mean ± SEM). (c,d) AST and ALT levels were measured in sera of control or anti-Thy1.2 treated 6 months old DEN-injected IKKβ(EE)Hep mice (n=5, *p<0.01, two-tailed Students t-test, bars- mean ± SEM).

Supplementary Figure 7 Activation of the lymphotoxin pathway in IKKβ(EE)Hep mice and hepatitis C virus–infected patients.

(a,b) qPCR analysis of mRNA levels of NF-κB2 and Bcl3, respectively, in DEN-treated Alb-cre control and IKKβ(EE)Hep mice at the indicated ages (n=5,5,6, respectively; *p<0.001, two-tailed Students t-test, bars - mean ± SEM). (c) Immunoblot analysis of protein extracts from either liver parenchyma or HCCs of 20-months old Alb-cre control and IKKβ(EE)Hep mice for p100, RelB and p52. Actin - loading control. (d) Immunoblot analysis of protein extracts from livers of DEN-treated Alb-cre control, IKKβ(+/E)Hep and IKKβ(EE)Hep mice at the indicated ages for p100 and p52. Tubulin – loading control. (e,f) Spearman correlation plots of mRNA expression levels of LTβ vs. CCL17 and LTβ vs. CCL20, respectively, in ELSs dissected from 6 months old DEN-treated IKKβ(EE)Hep mice (n=9). (g,h) Pearson correlation plots of mRNA expression levels of LTβ vs. CCL17 and LTβ vs. CCL20, respectively, in human HCV infected livers (n=43). (i) Representative LTβ-mRNA in-situ hybridization in mouse livers (scale bars-100 µm upper panels, 50 µm lower panels). (j) Cells from microscopically isolated ELSs from IKKβ(EE)Hep mice livers were FACS-sorted for the shown cell types and then analyzed for LTβ expression by real time PCR. Hep (par)=parenchymal hepatocytes from IKKβ(EE)Hep mice livers. Results are representative of ELSs isolated from 3 IKKβ(EE)Hep mice. (k) Quantification of the percentage of LTβ positive progenitor malignant hepatocytes in parenchyma, small (200 μm) and large (>200 μm) ELSs (10 sections containing ELSs from IKKβ(EE)Hep mice were quantified, *p<0.0001, two-tailed Students t-test, red cross line signifies mean). (l) Quantification of tumor progenitor LTβ variability. LTβ mRNA expression in ELS associated hepatocytes was assessed in 10 individual IKKβ(EE)Hep mice (labeled #1 to #10). For each mouse, all ELSs in a single liver section were scored. Each ELS was scored as either “inner>outer” where stronger staining was noted in hepatocytes in the ELS core compared with the periphery (yellow); “no variability” indicating no apparent difference in LTβ hepatocyte expression (blue); “outer>inner” – higher expression in hepatocytes in the ELS periphery (red). Total: cumulative score of all assessed ELSs: 26 out of 54 ELSs were scored as "outer>inner" vs. 1 out of 54 that was scored as "inner>outer", p=0.0001, Fisher exact test. (m,n) Representative images (m) and quantification (n) of LTβ-mRNA in-situ hybridization in liver tumors of 6-month-old DEN-treated IKKβ(EE)Hep mice or IKKβ(EE)Hep-Rag1-/- (IKK-Rag) mice (scale bars-50 µm, n=5, *p<0.0001, two-tailed Students t-test, bars- mean ± SEM). (o,p) Representative images (o) and quantification (p) of LTβ-mRNA in-situ hybridization in liver ELSs and tumors of 6-month-old DEN-injected IKKβ(EE)Hep mice treated with either isotype control antibody (LTF-2) or anti-Thy1.2 antibody (scale bars-50 µm, n=5,4, respectively; *p<0.0001, two-tailed Students t-test, bars- mean ± SEM).

Supplementary Figure 8 Blockade of lymphotoxin signaling abolishes egress from the microniche and tumorigenesis.

(a) Schematic representation of long term LTβR-Ig treatment. IKKβ(EE)Hep mice were given a single injection of DEN at 15 days of age, followed by 3 different regimens of 10 weeks LTβR-Ig or control-Ig administration (100 mg per week), as indicated. All mice were sacrificed at 33 weeks of age. (b) Representative immunostains for FDC-M1 of spleen and liver sections from DEN-treated IKKβ(EE)Hep mice injected for 10 consecutive weeks (13-22 weeks, see a for details) with control-Ig or LTβR-Ig and sacrificed three days after the last injection (scale bars-50 μm). (c,d) Quantification (c) and representative high power confocal microscopy images (d) for p65 (red) staining in control or LTβR-Ig treated IKKβ(EE)Hep mice. GFP (green) marks hepatocytes, DAPI (blue) marks the nuclei (*p= 0.002, arrows point to nuclear p65 in intra-ELS HCC progenitors, scale bars-50 μm). (e) Tumor volume of livers of 33 weeks old IKKβ(EE)Hep mice treated with either control-Ig or LTβR-Ig for the indicated periods (n=12,11,10,11 for control-Ig, 3-12W, 13-23W, 23-32W, respectively; *p=0.02, **p<0.0007, two-tailed Students t-test, red cross line signifies mean). (f) Representative images of whole livers and H&E stained sections. Arrows and dashed lines indicate visible tumors on the liver surface and H&E stained sections, respectively (scale bars-200 μm). (g) High power confocal microscopy images: GFP (green) marks all hepatocytes, Sox9 (red) marks progenitor hepatocytes, B220+CD3 (white) mark lymphocytes and Hoechst 33342 (blue) marks the nuclei. Arrows point to egressing malignant hepatocytes (scale bars-upper panels- 100 mm, lower panels- 50 mm).

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Supplementary Figures 1–8 and Supplementary Tables 1–7 (PDF 2987 kb)

Progenitor cells egressing out of an ELS

3D reconstruction of an ELS from DEN-treated IKKβ(EE)Hep mouse. Note green CD44v6+ progenitor cells egressing out of the ELS at multiple points. (AVI 43556 kb)

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Finkin, S., Yuan, D., Stein, I. et al. Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma. Nat Immunol 16, 1235–1244 (2015). https://doi.org/10.1038/ni.3290

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