Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling

Abstract

Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from premalignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.

Figure 1. DEN-Induced Hepatocytic Aggregates Contain CD44⁺ HCC Progenitors

(A) Fifteen-day-old BL/6 males were given DEN or vehicle. After 3 or 5 months, their livers were removed and collagenase digested. Left: typical digest appearance (magnification: 400×; 3 months after DEN). Red arrow indicates a collagenase-resistant aggregate. Right: aggregates per liver (n = 5; ± SD for each point). (B) Livers were collagenase digested 5 months after DEN administration. Aggregates were separated from nonaggregated cells and mechanically dispersed into a single-cell suspension (left upper panels; 200×). 10⁴ viable aggregated or nonaggregated cells were i.s. injected into MUP-uPA mice whose livers and spleens were analyzed for tumors 5 months later (left lower panels). The number of HCC nodules per liver was determined (n = 5; ± SD). (C) Adult BL/6 mice were given retrorsine twice with a 2 week interval to inhibit hepatocyte proliferation. After 1 month, mice were i.s. transplanted with dispersed hepatocyte aggregates (10⁴ cells) from DEN-treated mice and, 2 weeks later, were given three weekly i.p. injections of CCl₄ or vehicle. Tumor multiplicity and size were evaluated 5 months later (n = 5; ± SD). (D) Hepatocyte aggregates were prepared as in (A), stained with CD44 antibody and DAPI, and examined by fluorescent microscopy (400×). (E) Hepatocyte aggregates were dispersed as above, and CD44⁺ cells were separated from CD44⁻ cells. The indicated cell numbers were injected into MUP-uPA mice, and HCC development was evaluated 5 months later. n values are in parentheses (n.d., not done). See also Figure S1.

Figure 2. Tak1^Δhep Livers Contain Collagenase-Resistant HcPC Aggregates

(A) Livers, free of tumors (upper panels), were removed from 1-month-old Tak1^F/F and Tak1^Δhep males and collagenase digested (lower panels; red arrow indicates collagenase-resistant aggregate). (B) 10⁴ nonaggregated or dispersed aggregated hepatocytes from (A) were i.s. injected into MUP-uPA mice that were analyzed 6 months later to identify mice with at least one liver tumor (n = 5–8 mice per genotype). (C) BL/6 males were injected with vehicle or CCl₄ twice weekly for 2 weeks. Hepatocytes were isolated by collagenase digestion and photographed (right panels; 400×). Liver sections were stained with Sirius red to reveal collagen deposits (left panels). (D) 8-week-old BL/6 males were subjected to 70% partial hepatectomy, pulsed with BrdU at 46 and 70 hr, and sacrificed 2 hr later. Isolated hepatocytes were photographed. Liver sections were analyzed for BrdU incorporation (400×). See also Figure S2 and Table S1.

Figure 3. Aggregated Hepatocytes Exhibit an Altered Transcriptome Similar to that of HCC Cells

Aggregated and matched nonaggregated hepatocytes were isolated 5 months after DEN treatment. HCC cells were isolated from DEN-induced tumors, and normal hepatocytes were from age- and gender-matched control mice. RNA was extracted and subjected to microarray analysis (n = 3 for each sample). (A) Scatterplot representing fold changes (log 2 of expression ratio) in gene expression for HCC versus normal (y axis) and aggregated versus nonaggregated (x axis) pairwise transcriptome comparisons. The plot is displayed twice: in the left panel, genes with an FDR < 0.01 in the aggregated versus nonaggregated comparison are highlighted in red, and in the right panel, genes with an FDR < 0.01 in the HCC versus normal comparison are highlighted in cyan. DE, differentially expressed. (B) Venn diagram showing overlap between genes that are differentially expressed between aggregated and nonaggregated hepatocytes and between HCC cells and normal hepatocytes with an FDR < 0.01 (cyan and red dots from A). The probability to find 583 overlapping genes is <7.13 × 10⁻²⁴³. From these 583 common genes, only 4 behaved differently. (C) The ten most enriched biological processes (upper table) and cellular compartments (lower panel) represented by genes that are significantly upregulated (left panel) or downregulated (right panel) in HCC relative to normal hepatocytes (HCC) or in aggregated relative to nonaggregated hepatocytes (aggregated). (D) Heatmap displaying positive fold changes (FC) in expression of genes of interest in aggregated versus nonaggregated HcPCs (left) and in HCC versus normal hepatocytes (right). (E) Expression of selected genes was examined by real-time PCR and is depicted as fold change relative to normal hepatocytes given an arbitrary value of 1.0 (n = 3; ± SD). (1) Normal hepatocytes; (2) nonaggregated hepatocytes from DEN-treated liver; (3) HcPC aggregates from DEN-treated liver; and (4) DEN-induced HCCs. See also Figure S3.

Figure 4. DEN-Induced HcPC Aggregates Express Pathways and Markers Characteristic of HCC and Hepatobiliary Stem Cells

(A) Cytospin preps of collagenase-resistant aggregates from 5-month-old DEN-injected mice were stained with antibodies to CK19, AFP, EpCAM, A6, phospho-Y-STAT3 (Tyr705), albumin, phospho-c-Jun, Sox9, and phospho-c-Met. Black arrows indicate aggregates, and yellow arrows indicatenonaggregated cells(magnification: 400×). (B) 5-month-old DEN-treated mice were injected with BrdU, and 2 hr later, collagenase-resistant aggregates were isolated and analyzed for BrdU incorporation (400×). See also Figure S4.

Figure 5. HcPC-Containing Aggregates May Originate from Liver Premalignant Lesions

(A and B) Male and female mice were injected with PBS or DEN at 15 days. At the indicated time points, BrdU was administrated, and livers were collected 2 hr later and stained with H&E (A) or a BrdU-specific antibody (B). Arrows indicate borders of FAH (magnification: 200×). (C) Sections of male livers treated as above were subjected to IHC with the indicated antibodies (400×).

Figure 6. Liver Premalignant Lesions and HcPCs Exhibit Elevated IL-6 and LIN28 Expression

(A and B) Livers of 5-month-old DEN injected mice were analyzed for IL-6 expression by IHC (magnification: 400×) (A) and ISH (magnification: 100×, top; 400×, bottom) (B). (C and D) Quantification of IL-6 (C) and LIN28 (D) mRNA in aggregated versus nonaggregated hepatocytes from 5-month-old DEN-treated livers and in normal versus tumor-bearing livers (n = 6; ± SEM) (ND, not detected). (E) Immunoblot analyses of LIN28A in normal (NL) and tumor-bearing (TU) livers. (F) DEN-treated livers were subjected to IHC with a LIN28A antibody. Broken lines indicate borders of FAH (400×). (G) LIN28B was silenced with shRNA in HCC (dih) cells and cultured HcPCs, and LIN28B and IL-6 mRNAs were quantitated by qRT-PCR (n = 3; ± SEM). See also Figure S5 and Table S2.

Figure 7. HCC Growth Depends on Autocrine IL-6 Production

(A) HCC cells (dih10) were transduced with lenti-viruses containing scrambled or IL-6-specific shRNA. IL-6 mRNA was analyzed by qRT-PCR. (B) Dih10 cells (1.2 × 10⁵) transduced as above were i.s. injected into MUP-uPA mice that were analyzed 6 months later for HCC development (n =3;± SEM). (C) HcPCs from WT and Il6^−/− mice were injected (1 × 10⁴ cells/mice) into MUP-uPA mice and analyzed 5 months later for HCC development (n =5;± SEM). (D) HcPCs isolated from DEN-treated WT mice were transduced with shRNA against IL-6 or scrambled shRNA, cultured for 3 to 4 days, i.s. transplanted (1 × 10⁴ cells/mice) into MUP-uPA mice, and analyzed 6 months later (n = 3; ± SEM). (E) Livers of MUP-uPA mice from (D) were immunostained with GFP antibody 6 months after transplantation (200×). The bicistronic lentivirus in this experiment expresses GFP along with control or IL-6 shRNA, allowing tracking of the infected cells. (F) DEN-treated Il6^Δhep and Il6^F/F mice were sacrificed after 9 months to evaluate tumor multiplicity and size (n = 6–10, ± SEM). (G) IHC analysis of autocrine IL-6 signaling in human premalignant lesions in HCV-infected livers. Expression of LIN28, p-STAT3, and IL-6 was analyzed in 25 needle biopsies of dysplastic nodules, and representative positive specimens (n = 4) are shown. The dysplastic nodules and paired nontumor tissue were obtained from the same HCV-infected patient (n = 25). Nontumor tissue of metastatic liver cancer was used as normal control. See also Figures S6 and S7.