(αSMA-TKneg, n=13 mice, αSMA-TKpos: n=11 mice) (b). c-d, HSC depletion via LratCre × DTR in a non-fibrotic HCC model, induced by hydrodynamic tail vein injection of sleeping beauty-mediated expression MET + CTNNB1-Myc-tag, was highly efficient, as determined by TdT Tom fluorescence (c) but did not affect liver/body weight ratio, tumour size, tumour number or tumour area assessed by Myc-tag staining (DTRneg: n=11 mice, DTRpos: n=7 mice) (d). e-f analysis of cell-cell interactions by CellPhoneDB in snRNA-seq data from cirrhotic liver patients (n=4 cases) revealed HSC as a major cell type interacting with hepatocytes (e) or between different hepatocytes clusters and liver cells populations in the mouse fibrotic liver induced by 8xCCl₄ (n=3 mice) (f) as well as UMAP visualization of the proliferation marker *Mki67* (n=3 mice) (f). g-h, FACS analysis of total CD45+ leukocytes (g), and lymphocytes and myeloid cells (h) in the PME and TME after αSMA+ cell depletion during HCC development induced by DEN+17xCCl₄ (NT: n=8 mice per group, Tu: αSMA-TKneg, n=4 mice, αSMA-TKpos: n=5 mice - related to experiments in Extended Data Fig. 2 j-k) shows increased neutrophil and Ly6Chigh macrophage infiltration into non-tumour areas in αSMA-TK pos mice. i-j, CD45 IHC staining of the non-tumour and tumour tissue in DEN+CCl₄ treated *Yap*fl/fl (n=10 mice) and *Yap*AHSC mice (n=12 mice) (i), and in HSC-depleted DTRpos mice compared to DTRneg mice (NT: DTR neg: n=10 mice, DTRpos: n=12 mice; Tu: DTR neg: n=9 mice, DTRpos: n=11 mice) during HCC development induced by TAZ+FPC (j). k-l, endothelial cell analysis after HSC depletion induced by diphtheria toxin in DTRpos and DTRneg mice during HCC development induced by DEN+CCl₄ or TAZ+FPC diet evaluated by endomucin staining (k) and qPCR for the endothelial cell markers *Pecam1* (encoding for CD31) and *Kdr* (DEN+CCl₄: NT: DTR neg: n=15 mice, DTR pos: n=14 mice; Tu: 16 mice per group; *Kdr* mRNA: NT: 14 mice per group; Tu: 16 mice per group; *Pecam* and *Kdr* mRNA: TAZ+FPC: NT: DTRneg: n=10 mice, DTRpos: n=14 mice; Tu: DTRneg: n=10 mice, DTRpos: n=13 mice) (l). Data are shown as mean ± SEM, each data point represents one individual (a-d and g-l), scale bars: 100 µm (a-j) or 200 µm (k). GCV: ganciclovir; LBR: liver/body weight ratio; NK: Natural Killer, KC: Kupffer Cell, DC: Dendritic cell, NT: Non-tumour, Tu: Tumour. *Statistics*: all data in a besides *Acta2* in Tu, data in b besides LBR, d, g, all data in h besides B-cells, CD4+, CD8+, KC and Ly6ClowLy6Gneg in NT, CD45 in tumour in i, all data in l besides *Kdr* mRNA in DEN+CCl₄ and TAZ FPC in tumour were analysed by two-tailed Student's t-test. Data in a: *Acta2* mRNA in Tu, LBR in b, data in c, B-cells, CD4+, CD8+, KC and Ly6ClowLy6Gneg in NT, CD45 in tumour in h, CD45 in NT in l, data in j and *Kdr* mRNA in tumour in l were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data and gating strategy for h in Supplementary Information 2.
<figure><img src="image_5.png" /><figcaption>Extended Data Figure 5. Characterization of genetic mutations, inflammation, immune cell and tumor markers in mice with genetic HSC depletion, activation of inhibition.</figcaption></figure>

a, Mutations detected by PCR and sequencing for *Hras* Q61K/R/L, *Braf* V584E and *Egfr* F254I in *Lhx2*fl/fl (1 tumour/n; n=19 mice) vs *Lhx2*ΔHSC (1 tumour/n; n=7 mice) and *Lhx2*del (1 tumour/n; n=19 mice) mice treated with DEN; *Yap*fl/fl (2 tumours/n; n=14 mice) vs *Yap*ΔHSC (2 tumours/n; n=15 mice) mice and DTRneg (1 tumour/n; n=14 mice) vs DTRpos (1 tumour/n; n=15 mice) mice treated with DEN+CCl₄. b, Tumour grading represents average of 3 tumours per mouse in *Lhx2*fl/fl (n=8 mice) and *Lhx2*ΔHSC (n=7 mice) mice subjected to the DEN model; in *Yap*fl/fl (n=14 mice) and *Yap*ΔHSC (n=16 mice) mice subjected to the DEN+CCl₄ model and DTRneg (n=9 mice) and DTRpos (n=14 mice) mice
<figure><img src="image_6.png" /><figcaption>Extended Data Figure 5. Characterization of genetic mutations, inflammation, immune cell and tumor markers in mice with genetic HSC depletion, activation of inhibition.</figcaption></figure>

a, Mutations detected by PCR and sequencing for *Hras* Q61K/R/L, *Braf* V584E and *Egfr* F254I in *Lhx2*fl/fl (1 tumour/n; n=19 mice) vs *Lhx2*ΔHSC (1 tumour/n; n=7 mice) and *Lhx2*del (1 tumour/n; n=19 mice) mice treated with DEN; *Yap*fl/fl (2 tumours/n; n=14 mice) vs *Yap*ΔHSC (2 tumours/n; n=15 mice) mice and DTRneg (1 tumour/n; n=14 mice) vs DTRpos (1 tumour/n; n=15 mice) mice treated with DEN+CCl₄. b, Tumour grading represents average of 3 tumours per mouse in *Lhx2*fl/fl (n=8 mice) and *Lhx2*ΔHSC (n=7 mice) mice subjected to the DEN model; in *Yap*fl/fl (n=14 mice) and *Yap*ΔHSC (n=16 mice) mice subjected to the DEN+CCl₄ model and DTRneg (n=9 mice) and DTRpos (n=14 mice) mice
subjected to the TAZ+FPC HCC model. c-e, Characterization of inflammation, immune cells and tumors severity by qPCR for T cell markers, inflammatory genes and tumor-relevant marker genes in tumors from Lhx2<sup>AHSC</sup> mice with HSC activation in comparison to mice to Lhx2<sup>f/f</sup> littermates (n=7 mice per group) (c); Yap<sup>AHSC</sup> mice with HSC inhibition compared to Yap<sup>f/f</sup> mice (Cd3e, Cd4, Cd8, Ccl2, Ccl5, Tnfa, Ifng, Krt19, and Epcam mRNA: n=15 mice per group; Afp, Gpc3, Prom1, and Sox9 mRNA: YAP<sup>f/f</sup>: n=15 mice, YAP<sup>AHSC</sup>: n=14 mice). (d); and DTR<sup>pos</sup> (n=16 mice) mice with HSC depletion compared to DTR<sup>neg</sup> (n=16 mice) mice (e). Data are shown as mean ± SEM, each data point represents one individual. Data are shown as mean ± SEM, each data point represents one individual. <u>Statistics: a:</u> Mutation frequency were analysed by Chi-square test. <u>b:</u> tumour grading data were analysed by two-tailed Student's *t*-test. All data in <u>c</u> besides Afp and *Sox9* mRNA, Afp, Gpc3 and Prom1 mRNA in <u>d</u>, and Prom1 and *Sox9* mRNA in <u>e</u> were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data.
subjected to the TAZ+FPC HCC model. c-e, Characterization of inflammation, immune cells and tumors severity by qPCR for T cell markers, inflammatory genes and tumor-relevant marker genes in tumors from Lhx2<sup>AHSC</sup> mice with HSC activation in comparison to mice to Lhx2<sup>f/f</sup> littermates (n=7 mice per group) (c); Yap<sup>AHSC</sup> mice with HSC inhibition compared to Yap<sup>f/f</sup> mice (Cd3e, Cd4, Cd8, Ccl2, Ccl5, Tnfa, Ifng, Krt19, and Epcam mRNA: n=15 mice per group; Afp, Gpc3, Prom1, and Sox9 mRNA: YAP<sup>f/f</sup>: n=15 mice, YAP<sup>AHSC</sup>: n=14 mice). (d); and DTR<sup>pos</sup> (n=16 mice) mice with HSC depletion compared to DTR<sup>neg</sup> (n=16 mice) mice (e). Data are shown as mean ± SEM, each data point represents one individual. Data are shown as mean ± SEM, each data point represents one individual. <u>Statistics: a:</u> Mutation frequency were analysed by Chi-square test. <u>b:</u> tumour grading data were analysed by two-tailed Student's *t*-test. All data in <u>c</u> besides Afp and *Sox9* mRNA, Afp, Gpc3 and Prom1 mRNA in <u>d</u>, and Prom1 and *Sox9* mRNA in <u>e</u> were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data.
a

NASH (HF-CDAA diet)

<figure><img src="image_6.png" /></figure>

Mdr2<sup>KO</sup>

<figure><img src="image_7.png" /></figure>

TAZ + FPC

<figure><img src="image_8.png" /></figure>

b

myHSC in healthy and cirrhotic livers

<figure><img src="image_9.png" /></figure>

cyHSC in healthy and cirrhotic livers

<figure><img src="image_10.png" /></figure>

c

Genetic HSC activation (Lhx2 deletion)

<figure><img src="image_11.png" /></figure>

Genetic HSC inhibition (Yap deletion)

<figure><img src="image_12.png" /></figure>

d

myHSC/cyHSC

<figure><img src="image_13.png" /></figure>

Trajectories

<figure><img src="image_14.png" /></figure>

Col1a1

<figure><img src="image_15.png" /></figure>

Hgf

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

Start

<figure><img src="image_18.png" /></figure>

Expression level

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

cyHSC

<figure><img src="image_21.png" /></figure>

Expression level

<figure><img src="image_22.png" /></figure>

e

<figure><img src="image_23.png" /></figure>

f

<figure><img src="image_24.png" /></figure>

g

<figure><img src="image_25.png" /></figure>

Extended Data Figure 6]. Analysis of the myHSC and cyHSC in mouse and human fibrotic livers.

a, UMAPs of myHSC and cyHSC by scRNAseq, each visualized specific signatures as well as the correlation of cyHSC and myHSC signature score in HSC isolated from mice fed with HF-CDAA NASH diet for 12 weeks (n=1 mouse), from a 3 month old *Mdr2*<sup>KO</sup> mice (n=1 mouse) or a mouse treated with TAZ+FPC diet (n=1 mouse). b, visualization and quantification of myHSC and cyHSC populations in HSC from healthy (n=4 individuals) and cirrhotic (n=4 individuals) human livers by snRNAseq. c, analysis of microarray data from isolated HSC shows that genetic HSC activation via Mx1Cre-mediated *Lhx2* deletion (n=4 *Lhx2*<sup>fl/fl</sup>, n=4 *Lhx2*<sup>del</sup>) resulted in enriched myHSC signature and decreased cyHSC signature; and that genetic inhibition induced by LratCre-mediated *Yap1* deletion (n=5 *Yap1*<sup>fl/fl</sup>, n=4 *Yap1*<sup>ΔHSC</sup>) exerted the opposite effect with enriched cyHSC and decreased
a

NASH (HF-CDAA diet)

<figure><img src="image_7.png" /></figure>

Mdr2<sup>KO</sup>

<figure><img src="image_8.png" /></figure>

TAZ + FPC

<figure><img src="image_9.png" /></figure>

b

myHSC in healthy and cirrhotic livers

<figure><img src="image_10.png" /></figure>

cyHSC in healthy and cirrhotic livers

<figure><img src="image_11.png" /></figure>

c

Genetic HSC activation (Lhx2 deletion)

<figure><img src="image_12.png" /></figure>

Genetic HSC inhibition (Yap deletion)

<figure><img src="image_13.png" /></figure>

d

myHSC/cyHSC

<figure><img src="image_14.png" /></figure>

Trajectories

<figure><img src="image_15.png" /></figure>

Col1a1

<figure><img src="image_16.png" /></figure>

Hgf

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

Start

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

e

<figure><img src="image_24.png" /></figure>

f

<figure><img src="image_25.png" /></figure>

g

<figure><img src="image_26.png" /></figure>

Extended Data Figure 6]. Analysis of the myHSC and cyHSC in mouse and human fibrotic livers.

a, UMAPs of myHSC and cyHSC by scRNAseq, each visualized specific signatures as well as the correlation of cyHSC and myHSC signature score in HSC isolated from mice fed with HF-CDAA NASH diet for 12 weeks (n=1 mouse), from a 3 month old *Mdr2*<sup>KO</sup> mice (n=1 mouse) or a mouse treated with TAZ+FPC diet (n=1 mouse). b, visualization and quantification of myHSC and cyHSC populations in HSC from healthy (n=4 individuals) and cirrhotic (n=4 individuals) human livers by snRNAseq. c, analysis of microarray data from isolated HSC shows that genetic HSC activation via Mx1Cre-mediated *Lhx2* deletion (n=4 *Lhx2*<sup>fl/fl</sup>, n=4 *Lhx2*<sup>del</sup>) resulted in enriched myHSC signature and decreased cyHSC signature; and that genetic inhibition induced by LratCre-mediated *Yap1* deletion (n=5 *Yap1*<sup>fl/fl</sup>, n=4 *Yap1*<sup>ΔHSC</sup>) exerted the opposite effect with enriched cyHSC and decreased
myHSC signature in isolated HSC after treatment with 6xCCl₄. **d**, pseudotime analysis (of HSC from TAZ+FPC and *Mdr2*<sup>KO</sup>) as well as cyHSC, myHSC, *Collal* and *Hgf* mRNA expression in HSC using the same dataset as in a. **f**, *In situ* analysis of *Hgf* mRNA, visualized by RNAscope, *Collal*-GFP and LratCre-induced TdTom and subsequent quantification shows separate populations of *Collal*-GFP high *Hgf* low myHSC, and *Collal*-GFP low *Hgf* high cyHSC in *Mdr2*<sup>KO</sup> (n=3 mice) and TAZ+FPC-treated livers (n=1 mouse). **f-g**, analysis of *Hgf* and *Collal* expression by bulk RNA-seq (Ctrl and CCl₄: n=4 mice, FPC: n=5 mice) (**f**) or scRNA-seq (n=1 per condition) (**g**) of FACS-sorted HSC from control mice (Ctrl), or from CCl₄-treated or TAZ-FPC-treated mice. Dot plot bar graphsare shown as mean ± SEM. In the violin plots, box plots represent the interquartile range (IQR), Q1, median and Q3, whiskers as minimum (Q1-1.5×IQR) and maximum (Q3+1.5×IQR), and outlier data as individual dots. Each data point represents one cell (a,b,d) individual (f and *Mdr2*<sup>KO</sup> model in e) or one field of the same liver (TAZ+FPC model in e). Scale bar 50 µm. Statistics: a: P-values, coefficient of determination (R²) and statistical significance (P value) were determined using Pearson's. Data in d were analysed by two-tailed Mann-Whitney test. Data in b, d and g were analysed by two-tailed Mann-Whitney test. Data in f were analysed by two-tailed Student's t-test. Raw data are given in Source Data.
myHSC signature in isolated HSC after treatment with 6xCCl₄. **d**, pseudotime analysis (of HSC from TAZ+FPC and *Mdr2*<sup>KO</sup>) as well as cyHSC, myHSC, *Collal* and *Hgf* mRNA expression in HSC using the same dataset as in a. **f**, *In situ* analysis of *Hgf* mRNA, visualized by RNAscope, *Collal*-GFP and LratCre-induced TdTom and subsequent quantification shows separate populations of *Collal*-GFP high *Hgf* low myHSC, and *Collal*-GFP low *Hgf* high cyHSC in *Mdr2*<sup>KO</sup> (n=3 mice) and TAZ+FPC-treated livers (n=1 mouse). **f-g**, analysis of *Hgf* and *Collal* expression by bulk RNA-seq (Ctrl and CCl₄: n=4 mice, FPC: n=5 mice) (**f**) or scRNA-seq (n=1 per condition) (**g**) of FACS-sorted HSC from control mice (Ctrl), or from CCl₄-treated or TAZ-FPC-treated mice. Dot plot bar graphsare shown as mean ± SEM. In the violin plots, box plots represent the interquartile range (IQR), Q1, median and Q3, whiskers as minimum (Q1-1.5×IQR) and maximum (Q3+1.5×IQR), and outlier data as individual dots. Each data point represents one cell (a,b,d) individual (f and *Mdr2*<sup>KO</sup> model in e) or one field of the same liver (TAZ+FPC model in e). Scale bar 50 µm. Statistics: a: P-values, coefficient of determination (R²) and statistical significance (P value) were determined using Pearson's. Data in d were analysed by two-tailed Mann-Whitney test. Data in b, d and g were analysed by two-tailed Mann-Whitney test. Data in f were analysed by two-tailed Student's t-test. Raw data are given in Source Data.
<figure><img src="image_26.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>Extended Data Figure 7 | Determination of the role of HSC in hepatocarcinogenesis at different time points.</figcaption></figure>

a, The long-term effects of LratCre × DTR-mediated HSC depletion in the first half of DEN+CCl₄-induced hepatocarcinogenesis was tested by sacrificing mice 1.5 days (n=3 mice per group), 28 days (DTRneg: n=5 mice, DTRpos: n=4 mice per group) and 42 days (DTRneg: n=6 mice, DTRpos: n=4 mice) after the last DT injection and determining LratCre-induced TdTom expression. b, LratCrepos DTRpos (n=9 mice) with HSC depletion in the first half of DEN+CCl₄-induced hepatocarcinogenesis retained significant HSC depletion at time of sacrifice, as determined by qPCR for HSC markers *Lrat* and *Des*, but did not show significant changes in liver/body weight ratio (LBR), tumour number and tumour size compared to LratCrepos DTRneg littermates. c-d, Effects of poly I:C-induced
<figure><img src="image_27.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_30.png" /></figure>

Extended Data Figure 7 | Determination of the role of HSC in hepatocarcinogenesis at different time points.

a, The long-term effects of LratCre × DTR-mediated HSC depletion in the first half of DEN+CCl₄-induced hepatocarcinogenesis was tested by sacrificing mice 1.5 days (n=3 mice per group), 28 days (DTRneg: n=5 mice, DTRpos: n=4 mice per group) and 42 days (DTRneg: n=6 mice, DTRpos: n=4 mice) after the last DT injection and determining LratCre-induced TdTom expression. b, LratCrepos DTRpos (n=9 mice) with HSC depletion in the first half of DEN+CCl₄-induced hepatocarcinogenesis retained significant HSC depletion at time of sacrifice, as determined by qPCR for HSC markers *Lrat* and *Des*, but did not show significant changes in liver/body weight ratio (LBR), tumour number and tumour size compared to LratCrepos DTRneg littermates. c-d, Effects of poly I:C-induced
Lhx2 deletion at early time points (c, n=9 mice per group) and late time points (d, Lhx2<sup>del</sup>: n=10 mice, Lhx2<sup>del</sup>: n=12 mice) of DEN-induced HCC, as shown by representative images, liver/body ratio (LBR), tumour number and tumour size. Data are shown as mean ± SEM, each data point represents one individual scale bars: 200 µm. **Statistics**: Data in **a**: one way ANOVA (p<0.001) followed by Bonferroni's multiple comparison (comparison between samples sac at the same time point). Data in **b**, LBR in **c**, tumour number and tumour size in **d**, were analysed by two-tailed Student's t-test. Tumour number and tumour size in **c** and LBR in **d**: were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data.
Lhx2 deletion at early time points (c, n=9 mice per group) and late time points (d, Lhx2<sup>del</sup>: n=10 mice, Lhx2<sup>del</sup>: n=12 mice) of DEN-induced HCC, as shown by representative images, liver/body ratio (LBR), tumour number and tumour size. Data are shown as mean ± SEM, each data point represents one individual scale bars: 200 µm. **Statistics**: Data in **a**: one way ANOVA (p<0.001) followed by Bonferroni's multiple comparison (comparison between samples sac at the same time point). Data in **b**, LBR in **c**, tumour number and tumour size in **d**, were analysed by two-tailed Student's t-test. Tumour number and tumour size in **c** and LBR in **d**: were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data.
<figure><img src="image_30.png" /><figcaption>Extended Data Figure 8. Collagen type 1 deletion reduces liver stiffness and HCC development in DEN+CCl₄-, NASH diet- and *Mdr2*<sup>KO</sup>-induced HCC.</figcaption></figure>

a-b, UMAP visualization showing that *Col1a1* are mainly expressed in the HSC population in the 8xCCl₄ injured mouse liver by scRNAseq (n=3 mice) (a) and in the HSC/fibroblast cluster in the human normal (n=4 individuals), cirrhotic (n=4 individuals) and HCC (n=2 individuals) livers by snRNAseq (b). c, *Col1a1* mRNA expression, measured by qPCR, in untreated liver (n=5 mice, left panel) and the non-tumour tissue (n=8 mice) from DEN+14xCCl₄ treated mice; *COL1A1* mRNA expression in normal (n=84 individuals)
<figure><img src="image_31.png" /><figcaption>Extended Data Figure 8. Collagen type 1 deletion reduces liver stiffness and HCC development in DEN+CCl₄-, NASH diet- and *Mdr2*<sup>KO</sup>-induced HCC.</figcaption></figure>

a-b, UMAP visualization showing that *Col1a1* are mainly expressed in the HSC population in the 8xCCl₄ injured mouse liver by scRNAseq (n=3 mice) (a) and in the HSC/fibroblast cluster in the human normal (n=4 individuals), cirrhotic (n=4 individuals) and HCC (n=2 individuals) livers by snRNAseq (b). c, *Col1a1* mRNA expression, measured by qPCR, in untreated liver (n=5 mice, left panel) and the non-tumour tissue (n=8 mice) from DEN+14xCCl₄ treated mice; *COL1A1* mRNA expression in normal (n=84 individuals)
and adjacent non-tumour tissue from patients with F0-F1 (n=143 individuals), F2-F3 (n=59 individuals) and F4 (n=76 individuals) fibrosis (right panel). **d-e**, Mx1Cre<sup>pos</sup> *Colla1<sup>f/f</sup>* (*Colla1<sup>del</sup>* ) showed significant reduction of fibrosis in non-tumour tissue, determined by Sirius Red staining and *Colla1* mRNA, compared to *Colla1<sup>f/f</sup>* littermates in a profound fibrosis DEN+44xCCl₄ model (*Colla1<sup>f/f</sup>*: n=13 mice, *Colla1<sup>del</sup>*: n=14 mice) (d), and reduction of liver stiffness, determined by rheometry (control: n=1 mouse; DEN+19xCCl₄: n=4 mice/group) (e). f, Sirius red staining shows a strong increase of fibrosis, in the non-tumour tissue, induced by the profound fibrosis DEN+19xCCl₄ regimen (0.5 to 1.5 µl of CCl₄ per gram 2-3 injections/week; n=5 mice) compared to a moderate fibrosis model induced by DEN+10xCCl₄ (0.5 µl of CCl₄ per gram, 1 injection/week; n=5 mice; untreated controls n=3 mice). g, *Colla1<sup>del</sup>* mice treated with the moderate fibrosis DEN+15xCCl₄ (0.5 µl of CCl₄ per gram, 1injection/week as in f) reduced liver fibrosis (*Colla1<sup>f/f</sup>*: n=10 mice, *Colla1<sup>del</sup>*: n=14 mice) and *Colla1* mRNA (*Colla1<sup>f/f</sup>*: n=11 mice, *Colla1<sup>del</sup>*: n=8 mice) (g), but did not alter HCC development in comparison to *Colla1<sup>f/f</sup>* mice (*Colla1<sup>f/f</sup>*: n=17 mice, *Colla1<sup>del</sup>*: n=14 mice) (h) or i, liver stiffness assessed by rheometry in the non-tumour tissue from mice treated with DEN+10xCCl₄ (one injection per week as in f-h, n=3 mice for untreated and n=5 mice/group for DEN+CCl₄). j-k, Mx1Cre-mediated *Colla1* deletion in *Mdr2<sup>KO</sup>* female mice (*Mdr2<sup>KO</sup> *Colla1<sup>del</sup>* , n=11 mice and n=7 mice for *Colla1* mRNA) efficiently reduced liver fibrosis (j) and HCC development (k), compared to Mx1Cre<sup>neg</sup> *Mdr2<sup>KO</sup>* littermates (*Mdr2<sup>KO</sup> *Colla1<sup>f/f</sup>* , n=18 and n=7 mice for *Colla1* mRNA). l, Liver stiffness assessed by rheometry was reduced in 22 week old *Mdr2<sup>KO</sup> *Colla1<sup>del</sup>* male mice (n=2 mice) compared to their. *Colla1<sup>f/f</sup>* *Mdr2<sup>KO</sup>* (n=3 mice) littermates, livers from 8 week old untreated mice were used as control (n=3). m-o, HSC-selective ablation of *Colla1* in LratCre<sup>pos</sup> *Colla1<sup>f/f</sup>* (*Colla1<sup>ΔHSC</sup>* ) efficiently reduced liver fibrosis (Sirius Red: *Colla1<sup>f/f</sup>*: n=16 mice, *Colla1<sup>ΔHSC</sup>*: n=14 mice; *Colla1* mRNA: *Colla1<sup>f/f</sup>*: n=15 mice, *Colla1<sup>ΔHSC</sup>*: n=13 mice) (m), HCC development (*Colla1<sup>f/f</sup>*: n=16 mice, *Colla1<sup>ΔHSC</sup>*: n=14 mice), (n) as well as hepatocyte proliferation in the non-tumour liver, as determined by Ki67 IHC and quantification of proliferating hepatocytes (*Colla1<sup>f/f</sup>*: n=13 mice, *Colla1<sup>ΔHSC</sup>*: n=14 mice; Ki67+ tumour cells: n=9 mice) (o), compared to Lrat<sup>neg</sup> littermate controls (*Colla1<sup>f/f</sup>* ) in the DEN + CCl₄ profound fibrosis model (as in d). p-r, *Colla1<sup>ΔHSC</sup>* mice displayed strong reductions of liver fibrosis (p), HCC development (q), but not in liver triglycerides and cholesterol content (r) compared to *Colla1<sup>f/f</sup>* control mice in a model of NASH-associated HCC induced by 8 months of HF-CDAA diet (*Colla1<sup>f/f</sup>*: n=4 mice for Sirius red and triglycerides, n=11 mice for *Colla1* mRNA and HCC, *Colla1<sup>ΔHSC</sup>*: n=7 mice for Sirius red and triglycerides, n=10 mice for *Colla1* mRNA and HCC). Data are shown as mean ± SEM, box plots represent the interquartile Range (IQR), Q1, median and Q3, each data point represents one individual (c-r), scale bars: 200 µm (d, f, g, j, m and p), 50 µm (o), Red arrows show the Ki67+ proliferating hepatocytes or tumour cells. Statistics: *Colla1* mRNA in mouse liver in c, data in d, f, g, LBR and tumour number in h, j, LBR and tumour size in k, m, LBR and tumour size in n, o, p, q and cholesterol in r were analysed by two-tailed Student's t-test. Tumour size in h, tumour number in k and in n, and triglycerides in r were analysed by two-tailed Mann-Whitney test. # P<0.05, **,## P<0.001, ### P<0.001. Data in c and l: *COL1A1* mRNA in human liver was analysed by one-way ANOVA (Kruskal-Wallis's test) followed by Dunn's multiple comparison test with normal liver group. Data in
and adjacent non-tumour tissue from patients with F0-F1 (n=143 individuals), F2-F3 (n=59 individuals) and F4 (n=76 individuals) fibrosis (right panel). **d-e**, Mx1Cre<sup>pos</sup> *Colla1<sup>f/f</sup>* (*Colla1<sup>del</sup>* ) showed significant reduction of fibrosis in non-tumour tissue, determined by Sirius Red staining and *Colla1* mRNA, compared to *Colla1<sup>f/f</sup>* littermates in a profound fibrosis DEN+44xCCl<sub>4</sub> model (*Colla1<sup>f/f</sup>*: n=13 mice, *Colla1<sup>del</sup>*: n=14 mice) (d), and reduction of liver stiffness, determined by rheometry (control: n=1 mouse; DEN+19xCCl<sub>4</sub>: n=4 mice/group) (e). **f**, Sirius red staining shows a strong increase of fibrosis, in the non-tumour tissue, induced by the profound fibrosis DEN+19xCCl<sub>4</sub> regimen (0.5 to 1.5 µl of CCl<sub>4</sub> per gram 2-3 injections/week; n=5 mice) compared to a moderate fibrosis model induced by DEN+10xCCl<sub>4</sub> (0.5 µl of CCl<sub>4</sub> per gram, 1 injection/week; n=5 mice; untreated controls n=3 mice). **g**, *Colla1<sup>del</sup>* mice treated with the moderate fibrosis DEN+15xCCl<sub>4</sub> (0.5 µl of CCl<sub>4</sub> per gram, 1injection/week as in f) reduced liver fibrosis (*Colla1<sup>f/f</sup>*: n=10 mice, *Colla1<sup>del</sup>*: n=14 mice) and *Colla1* mRNA (*Colla1<sup>f/f</sup>*: n=11 mice, *Colla1<sup>del</sup>*: n=8 mice) (g), but did not alter HCC development in comparison to *Colla1<sup>f/f</sup>* mice (*Colla1<sup>f/f</sup>*: n=17 mice, *Colla1<sup>del</sup>*: n=14 mice) (h) or **i**, liver stiffness assessed by rheometry in the non-tumour tissue from mice treated with DEN+10xCCl<sub>4</sub> (one injection per week as in f-h, n=3 mice for untreated and n=5 mice/group for DEN+CCl<sub>4</sub>). **j-k**, Mx1Cre-mediated *Colla1* deletion in *Mdr2<sup>KO</sup>* female mice (*Mdr2<sup>KO</sup> *Colla1<sup>del</sup>* , n=11 mice and n=7 mice for *Colla1* mRNA) efficiently reduced liver fibrosis (j) and HCC development (k), compared to Mx1Cre<sup>neg</sup> *Mdr2<sup>KO</sup>* littermates (*Mdr2<sup>KO</sup> *Colla1<sup>f/f</sup>* , n=18 and n=7 mice for *Colla1* mRNA). **l**, Liver stiffness assessed by rheometry was reduced in 22 week old *Mdr2<sup>KO</sup> *Colla1<sup>del</sup>* male mice (n=2 mice) compared to their. *Colla1<sup>f/f</sup>* *Mdr2<sup>KO</sup>* (n=3 mice) littermates, livers from 8 week old untreated mice were used as control (n=3). **m-o**, HSC-selective ablation of *Colla1* in *LratCre<sup>pos</sup> *Colla1<sup>f/f</sup>* (*Colla1<sup>ΔHSC</sup>* ) efficiently reduced liver fibrosis (Sirius Red: *Colla1<sup>f/f</sup>*: n=16 mice, *Colla1<sup>ΔHSC</sup>*: n=14 mice; *Colla1* mRNA: *Colla1<sup>f/f</sup>*: n=15 mice, *Colla1<sup>ΔHSC</sup>*: n=13 mice) (m), HCC development (*Colla1<sup>f/f</sup>*: n=16 mice, *Colla1<sup>ΔHSC</sup>*: n=14 mice), (n) as well as hepatocyte proliferation in the non-tumour liver, as determined by Ki67 IHC and quantification of proliferating hepatocytes (*Colla1<sup>f/f</sup>*: n=13 mice, *Colla1<sup>ΔHSC</sup>*: n=14 mice; Ki67+ tumour cells: n=9 mice) (o), compared to *Lrat<sup>neg</sup>* littermate controls (*Colla1<sup>f/f</sup>* ) in the DEN + CCl<sub>4</sub> profound fibrosis model (as in d). **p-r**, *Colla1<sup>ΔHSC</sup>* mice displayed strong reductions of liver fibrosis (p), HCC development (q), but not in liver triglycerides and cholesterol content (r) compared to *Colla1<sup>f/f</sup>* control mice in a model of NASH-associated HCC induced by 8 months of HF-CDAA diet (*Colla1<sup>f/f</sup>*: n=4 mice for Sirius red and triglycerides, n=11 mice for *Colla1* mRNA and HCC, *Colla1<sup>ΔHSC</sup>*: n=7 mice for Sirius red and triglycerides, n=10 mice for *Colla1* mRNA and HCC). Data are shown as mean ± SEM, box plots represent the interquartile Range (IQR), Q1, median and Q3, each data point represents one individual (c-r), scale bars: 200 µm (d, f, g, j, m and p), 50 µm (o), Red arrows show the Ki67+ proliferating hepatocytes or tumour cells. **Statistics:** *Colla1* mRNA in mouse liver in c, data in d, f, g, LBR and tumour number in h, j, LBR and tumour size in k, m, LBR and tumour size in n, o, p, q and cholesterol in r were analysed by two-tailed Student's t-test. Tumour size in h, tumour number in k and in n, and triglycerides in r were analysed by two-tailed Mann-Whitney test. # P<0.05, **,## P<0.001, ### P<0.001. Data in c and l: *COL1A1* mRNA in human liver was analysed by one-way ANOVA (Kruskal-Wallis's test) followed by Dunn's multiple comparison test with normal liver group. Data in
e: was analysed by two-way ANOVA followed by Tukey's multiple comparison test. Raw data are given in Source Data.

<figure><img src="image_31.png" /><figcaption>Extended Data Figure 9. Characterization of Col1a1 on hepatocyte death, HSC activation, inflammation and immune cell infiltration and YAP activation and effects of hepatocyte-specific Yap or Itgb1 deletion on hepatocarcinogenesis.</figcaption></figure>

a-b, Col1a1<sup>f/f</sup> and Col1a1<sup>del</sup> mice were treated with the profound fibrosis DEN+44xCCL₄ regimen. Hepatocyte death was determined by TUNEL staining (n=6 mice per group) in non-tumour tissue at the end of the DEN+44xCCL₄ regimen 1 week after the last CCL₄
e: was analysed by two-way ANOVA followed by Tukey's multiple comparison test. Raw data are given in Source Data.

<figure><img src="image_32.png" /><figcaption>Extended Data Figure 9. Characterization of *Col1a1* on hepatocyte death, HSC activation, inflammation and immune cell infiltration and YAP activation and effects of hepatocyte-specific Yap or *Itgb1* deletion on hepatocarcinogenesis.</figcaption></figure>

a-b, *Col1a1*<sup>fl/fl</sup> and *Col1a1*<sup>del</sup> mice were treated with the profound fibrosis DEN+44xCCl<sub>4</sub> regimen. Hepatocyte death was determined by TUNEL staining (n=6 mice per group) in non-tumour tissue at the end of the DEN+44xCCl<sub>4</sub> regimen 1 week after the last CCl<sub>4</sub>
injection, as well as by ALT measured 2 days after 20xCCl₄ (n=6 per group) (a). HSC activation and inflammation were assessed by αSMA staining (n=8 per group) and qPCR for HSC activation and inflammatory markers in non-tumour tissue two days after 44xCCl₄ (n=8 mice per group) (b). c-d, HSC activation and *Il1b* mRNA were assessed in *Colla*<sup>f/f</sup> (n=11 mice) and *Colla*<sup>ΔHSC</sup> (n=1 mice) mice fed eight months with HF-CDAA diet (c) and in 14 months-old *Mdr2*<sup>KO</sup> *x* *Colla*<sup>f/f</sup> and *Mdr2*<sup>KO</sup> *x* *Colla*<sup>del</sup> mice (n=7 mice per group) (d). e-g, Determination of CD45+ lymphocytes (e), myeloid subpopulations) (f) and lymphoid (g) subpopulations by FACS in tumour and non-tumour areas of LratCre<sup>neg</sup> *Colla*<sup>f/f</sup> mice (n=5 mice) and LratCre<sup>pos</sup> *Colla*<sup>f/f</sup> mice (*Colla*<sup>ΔHSC</sup>, n=5 mice) with the profound fibrosis DEN+44xCCl₄ regimen. h, YAP staining in *Colla*<sup>f/f</sup> and *Colla*<sup>del</sup> mice treated with the profound fibrosis DEN+19xCCl₄ showed YAP expression mostly in non-parenchymal cells rather than in hepatocytes. i, *Yap* was efficiently delete by injection of AAV8-TBG-Cre (*Yap*<sup>ΔHep</sup>, n=10 mice) compared to AAV-TGB-empty (*Yap*<sup>f/f</sup>, n=14 mice) but did not affect HCC development were in the DEN+CCl₄ profound fibrosis HCC model determined by liver/body weight ratio (LBR), tumour number and tumour size. j, *Itgb1*<sup>f/f</sup> mice were injected with either AAV8-TGB-empty (*Itgb1*<sup>f/f</sup>, n=13) or AAV8-TBG-Cre (*Itgb1*<sup>ΔHep</sup>, n=11) and effects of hepatocyte-specific *Itgb1* deletion on HCC development were determined in the DEN+CCl₄ profound fibrosis HCC model by the liver/body weight ratio (LBR), tumour number and tumour size. Data are shown as mean ± SEM, each data point represents one individual. NT: non-tumour, Tum: tumour NK: Natural killer, Treg: T regulatory cells, GZB: Granzyme B, DC: Dendritic cells, KC: Kupffer cells, LBR: liver/body weight ratio. Scale bars: 100 µm (a and h), 200 µm (b). Statistics: TUNEL in a, data in b and d besides *Il1b* mRNA, *Colla2* mRNA in c, f, data in g besides B cells, NKT cells, CD8 and Tregs in non-tumour area, i, and data in j besides LBR were analysed by two-tailed Student's t-test. ALT in a, *Il1b* mRNA in b and d, all data in c besides *Colla2*, e, B cells, NKT cells, CD8 and Tregs in non-tumour area in g and LBR in j were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data and gating strategy for e-g in Supplementary Information 4.
injection, as well as by ALT measured 2 days after 20xCCl₄ (n=6 per group) (a). HSC activation and inflammation were assessed by αSMA staining (n=8 per group) and qPCR for HSC activation and inflammatory markers in non-tumour tissue two days after 44xCCl₄ (n=8 mice per group) (b). c-d, HSC activation and *Il1b* mRNA were assessed in *Colla1f/f* (n=11 mice) and *Colla1*<sup>ΔHSC</sup> (n=1 mice) mice fed eight months with HF-CDAA diet (c) and in 14 months-old *Mdr2<sup>KO</sup> x Colla1f/f* and *Mdr2<sup>KO</sup> x Colla1del* mice (n=7 mice per group) (d). e-g, Determination of CD45+ lymphocytes (e), myeloid subpopulations) (f) and lymphoid (g) subpopulations by FACS in tumour and non-tumour areas of LratCre<sup>neg</sup> *Colla1f/f* mice (n=5 mice) and LratCre<sup>pos</sup> *Colla1f/f* mice (*Colla1*<sup>ΔHSC</sup>, n=5 mice) with the profound fibrosis DEN+44xCCl₄ regimen. h, YAP staining in *Colla1f/f* and *Colla1del* mice treated with the profound fibrosis DEN+19xCCl₄ showed YAP expression mostly in non-parenchymal cells rather than in hepatocytes. i, *Yap* was efficiently delete by injection of AAV8-TBG-Cre (*Yap*<sup>ΔHep</sup>, n=10 mice) compared to AAV-TGB-empty (*Yap*<sup>fl/fl</sup>, n=14 mice) but did not affect HCC development were in the DEN+CCl₄ profound fibrosis HCC model determined by liver/body weight ratio (LBR), tumour number and tumour size. j, *Itgb1*<sup>fl/fl</sup> mice were injected with either AAV8-TGB-empty (*Itgb1*<sup>fl/fl</sup>, n=13) or AAV8-TBG-Cre (*Itgb1*<sup>ΔHep</sup>, n=11) and effects of hepatocyte-specific *Itgb1* deletion on HCC development were determined in the DEN+CCl₄ profound fibrosis HCC model by the liver/body weight ratio (LBR), tumour number and tumour size. Data are shown as mean ± SEM, each data point represents one individual. NT: non-tumour, Tum: tumour NK: Natural killer, Treg: T regulatory cells, GZB: Granzyme B, DC: Dendritic cells, KC: Kupffer cells, LBR: liver/body weight ratio. Scale bars: 100 µm (a and h), 200 µm (b). Statistics: TUNEL in a, data in b and d besides *Il1b* mRNA, *Colla2* mRNA in c, f, data in g besides B cells, NKT cells, CD8 and Tregs in non-tumour area, i, and data in j besides LBR were analysed by two-tailed Student's t-test. ALT in a, *Il1b* mRNA in b and d, all data in c besides *Colla2*, e, B cells, NKT cells, CD8 and Tregs in non-tumour area in g and LBR in j were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data and gating strategy for e-g in Supplementary Information 4.
a

Ddr1

Normal liver

8xCCl₄ liver

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

DDR1

Normal

Cirrhotic

HCC

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

b

Huh7

DDR1

GAPDH

<figure><img src="image_37.png" /></figure>

Hepa1-6

DDR1

GAPDH

<figure><img src="image_38.png" /></figure>

Primary hepatocytes

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

c

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

d

Regular DEN+CCl₄

<figure><img src="image_43.png" /></figure>

Tumour

<figure><img src="image_44.png" /></figure>

αSMA

Extensive fibrosis DEN+CCl₄

<figure><img src="image_45.png" /></figure>

Tumour

<figure><img src="image_46.png" /></figure>

αSMA

<figure><img src="image_47.png" /></figure>

e

ECM

<figure><img src="image_48.png" /></figure>

f

<figure><img src="image_49.png" /></figure>

g

<figure><img src="image_50.png" /></figure>

h

Mmp1a/1b

<figure><img src="image_51.png" /></figure>

Mmp2

<figure><img src="image_52.png" /></figure>

Mmp8

<figure><img src="image_53.png" /></figure>

i

<figure><img src="image_54.png" /></figure>

j

<figure><img src="image_55.png" /></figure>

Mmp9

<figure><img src="image_56.png" /></figure>

Mmp13

<figure><img src="image_57.png" /></figure>

Mmp14

<figure><img src="image_58.png" /></figure>

k

CD3 IHC non-tumour

<figure><img src="image_59.png" /></figure>

CD3 IHC tumour

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

Pptprc

<figure><img src="image_63.png" /><figcaption>Extended Data Figure 10 | Regulation and role of DDR1 in the fibrotic liver and HCC development.</figcaption></figure>

a, UMAPs of scRNA-seq data showing hepatocyte *Ddr1* expression from normal (n=1 mouse) and 8xCCl₄ fibrotic mouse livers (n=3 mice – top panel) and of snRNA-seq from patients showing *DDR1* expression in hepatocytes HCC from healthy patients (n=4 individuals), non-tumor (n=2 individuals) and cirrhotic (n=2 individuals) or HCC tumour (n=2 individuals) liver tissues (bottom panel). **b**, Huh7, Hepa1-6, HepG2 HCC cells and mouse primary hepatocytes were treated with different cytokines for 24h, followed by immunoblotting for DDR1 and GAPDH (each n=1 well, n=1 experiment). **c**, Confirmation of *Ddr1* deletion by pPCR in non-tumour liver and tumour liver from AAV8-TGB-empty treated *Ddr1*<sup>f/f</sup> and AAV8-TGB-Cre treated *Ddr1*<sup>ΔHep</sup> mice treated with DEN+44xCCl₄ (in NT: *Ddr1*<sup>f/f</sup>: n=9 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice; in Tu: *Ddr1*<sup>f/f</sup>: n=10 mice, *Ddr1*<sup>ΔHep</sup> n=8
a

Ddr1

Normal liver

8xCCl₄ liver

<figure><img src="image_33.png" /></figure>

DDR1

Normal

Cirrhotic

HCC

<figure><img src="image_34.png" /></figure>

b

Huh7

DDR1

GAPDH

<figure><img src="image_35.png" /></figure>

Hepa1-6

DDR1

GAPDH

<figure><img src="image_36.png" /></figure>

HepG2

<figure><img src="image_37.png" /></figure>

Primary hepatocytes

<figure><img src="image_38.png" /></figure>

c

<figure><img src="image_39.png" /></figure>

d

Regular DEN+CCl₄

<figure><img src="image_40.png" /></figure>

Tumour

<figure><img src="image_41.png" /></figure>

αSMA

Extensive fibrosis DEN+CCl₄

<figure><img src="image_42.png" /></figure>

Tumour

<figure><img src="image_43.png" /></figure>

αSMA

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

αSMA+ area (%)

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

e

<figure><img src="image_48.png" /></figure>

f

<figure><img src="image_49.png" /></figure>

g

<figure><img src="image_50.png" /></figure>

h

Mmp1a/1b

<figure><img src="image_51.png" /></figure>

Mmp2

<figure><img src="image_52.png" /></figure>

Mmp8

<figure><img src="image_53.png" /></figure>

i

<figure><img src="image_54.png" /></figure>

j

<figure><img src="image_55.png" /></figure>

Mmp9

<figure><img src="image_56.png" /></figure>

Mmp13

<figure><img src="image_57.png" /></figure>

Mmp14

<figure><img src="image_58.png" /></figure>

k

CD3 IHC non-tumour

<figure><img src="image_59.png" /></figure>

CD3 IHC tumour

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

Pptprc

<figure><img src="image_62.png" /><figcaption>Extended Data Figure 10 | Regulation and role of DDR1 in the fibrotic liver and HCC development.</figcaption></figure>

a, UMAPs of scRNA-seq data showing hepatocyte *Ddr1* expression from normal (n=1 mouse) and 8xCCl₄ fibrotic mouse livers (n=3 mice – top panel) and of snRNA-seq from patients showing *DDR1* expression in hepatocytes HCC from healthy patients (n=4 individuals), non-tumor (n=2 individuals) and cirrhotic (n=2 individuals) or HCC tumour (n=2 individuals) liver tissues (bottom panel). **b**, Huh7, Hepa1-6, HepG2 HCC cells and mouse primary hepatocytes were treated with different cytokines for 24h, followed by immunoblotting for DDR1 and GAPDH (each n=1 well, n=1 experiment). **c**, Confirmation of *Ddr1* deletion by pPCR in non-tumour liver and tumour liver from AAV8-TGB-empty treated *Ddr1*<sup>f/f</sup> and AAV8-TGB-Cre treated *Ddr1*<sup>ΔHep</sup> mice treated with DEN+44xCCl₄ (in NT: *Ddr1*<sup>f/f</sup>: n=9 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice; in Tu: *Ddr1*<sup>f/f</sup>: n=10 mice, *Ddr1*<sup>ΔHep</sup> n=8
mice - related to Fig. 5c). d, Fibrosis, αSMA+ myofibroblast accumulation and *Ddr1* mRNA expression within tumours were quantified by Sirius Red staining, IHC and qPCR, respectively, in the "moderate fibrosis" DEN + 14xCCl₄ HCC model (αSMA and Sirius Red: n=10 mice; *Ddr1* mRNA n=11 mice) and in the "profound fibrosis" DEN+44xCCl₄ HCC model (αSMA and Sirius Red: n=9 mice; *Ddr1* mRNA n=10 mice). e-f, Huh7 HCC cells (n=3 biological replicate; n=2 experiments) (e) and primary mouse hepatocyte (n=1 well; n=1 experiment) (f) were plated on either plastic or fibroblast ECM, samples were harvested at 24h or at the indicated times after plating, followed by western blot for pDDR1, DDR1, pAKT, AKT and b-actin or GAPDH. g, Huh7 cells were plated on ECM from wild-type fibroblasts or from MMP-resistant Col I (RR) fibroblasts, followed by western blot for pDDR1, DDR1, pAKT, AKT and b-actin (n=1 well, n=3 experiments). h, expression of collagen-degrading MMPs, determined by qPCR, in the profound fibrosis DEN+CCl₄ model (n=4 normal liver controls [Ctrl], n=9 mice for non-tumour [NT] and tumour [Tu] samples). i, survival analysis in the TCGA dataset based on the expression of a collagen-degrading MMP signature (*MMP1*, *MMP2*, *MMP8*, *MMP9*, *MMP13* and *MMP14*). j, YAP, TAZ, GAPDH, pAKT and AKT expression were determined by western blot in non-tumor tissue from *Ddr1*<sup>fl/fl</sup> and *Ddr1*<sup>ΔHep</sup> mice (n=6 mice per group). k, Infiltration of CD3+ T cells was determined by IHC and quantified in both non-tumor and tumor tissue (CD3 IHC, *Cd3e* and *Ptprc* mRNA in non-tumour area: *Ddr1*<sup>fl/fl</sup>: n=9 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice; CD3 IHC, *Cd3e* and *Ptprc* mRNA in non-tumour area: *Ddr1*<sup>fl/fl</sup>: n=10 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice); expression of *Cd3e* and *Ptprc* (encoding CD45) was determined by qPCR. Data are shown as mean ± SEM, each data point represents one well (b,e,f,g) or one individual (c,h,k); scale bars: 200 µm (d), 50 µm (k). nd: non-detected. Immunoblots in j were performed on two different gels using the same samples: one gel for YAP/TAZ and GAPDH and a second gel for pAKT and AKT. Data are shown as mean ± SEM. Statistics: *Ddr1* mRNA in NT in c, data in d, CD3+ IHC and *Cd3e* mRNA in non-tumour and *Ptprc* mRNA in tumour in g were analysed by two-tailed Student's t-test. CD3+ IHC and *Cd3e* mRNA in tumour and *Ptprc* mRNA in non-tumour in g were analysed by two-tailed Mann-Whitney test. Data in h were analysed by one way ANOVA (Kruskal Wallis) for *Mmp2* (p<0.001), *Mmp8* (p=0.004) and *Mmp9* mRNA (p=0.004) followed by Dunn's multiple comparison or for *Mmp13* (p=0.001) and *Mmp14* mRNA one-way one-way ANOVA (p<0.001) followed by Tukey's multiple comparison. Raw data are given in Source Data and raw western blot gels in Supplementary Information.
mice - related to Fig. 5c). d, Fibrosis, αSMA+ myofibroblast accumulation and *Ddr1* mRNA expression within tumours were quantified by Sirius Red staining, IHC and qPCR, respectively, in the "moderate fibrosis" DEN + 14xCCl₄ HCC model (αSMA and Sirius Red: n=10 mice; *Ddr1* mRNA n=11 mice) and in the "profound fibrosis" DEN+44xCCl₄ HCC model (αSMA and Sirius Red: n=9 mice; *Ddr1* mRNA n=10 mice). e-f, Huh7 HCC cells (n=3 biological replicate; n=2 experiments) (e) and primary mouse hepatocyte (n=1 well; n=1 experiment) (f) were plated on either plastic or fibroblast ECM, samples were harvested at 24h or at the indicated times after plating, followed by western blot for pDDR1, DDR1, pAKT, AKT and b-actin or GAPDH. g, Huh7 cells were plated on ECM from wild-type fibroblasts or from MMP-resistant Col I (RR) fibroblasts, followed by western blot for pDDR1, DDR1, pAKT, AKT and b-actin (n=1 well, n=3 experiments). h, expression of collagen-degrading MMPs, determined by qPCR, in the profound fibrosis DEN+CCl₄ model (n=4 normal liver controls [Ctrl], n=9 mice for non-tumour [NT] and tumour [Tu] samples). i, survival analysis in the TCGA dataset based on the expression of a collagen-degrading MMP signature (*MMP1*, *MMP2*, *MMP8*, *MMP9*, *MMP13* and *MMP14*). j, YAP, TAZ, GAPDH, pAKT and AKT expression were determined by western blot in non-tumor tissue from *Ddr1*<sup>fl/fl</sup> and *Ddr1*<sup>ΔHep</sup> mice (n=6 mice per group). k, Infiltration of CD3+ T cells was determined by IHC and quantified in both non-tumor and tumor tissue (CD3 IHC, *Cd3e* and *Ptprc* mRNA in non-tumour area: *Ddr1*<sup>fl/fl</sup>: n=9 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice; CD3 IHC, *Cd3e* and *Ptprc* mRNA in non-tumour area: *Ddr1*<sup>fl/fl</sup>: n=10 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice); expression of *Cd3e* and *Ptprc* (encoding CD45) was determined by qPCR. Data are shown as mean ± SEM, each data point represents one well (b,e,f,g) or one individual (c,h,k); scale bars: 200 µm (d), 50 µm (k). nd: non-detected. Immunoblots in j were performed on two different gels using the same samples: one gel for YAP/TAZ and GAPDH and a second gel for pAKT and AKT. Data are shown as mean ± SEM. Statistics: *Ddr1* mRNA in NT in c, data in d, CD3+ IHC and *Cd3e* mRNA in non-tumour and *Ptprc* mRNA in tumour in g were analysed by two-tailed Student's t-test. CD3+ IHC and *Cd3e* mRNA in tumour and *Ptprc* mRNA in non-tumour in g were analysed by two-tailed Mann-Whitney test. Data in h were analysed by one way ANOVA (Kruskal Wallis) for *Mmp2* (p<0.001), *Mmp8* (p=0.004) and *Mmp9* mRNA (p=0.004) followed by Dunn's multiple comparison or for *Mmp13* (p=0.001) and *Mmp14* mRNA one-way one-way ANOVA (p<0.001) followed by Tukey's multiple comparison. Raw data are given in Source Data and raw western blot gels in Supplementary Information.
<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

<figure><img src="image_72.png" /></figure>

Extended Data Figure 11 | Expression and role of myHSC-enriched hyaluronan and cyHSC-expressed HHIP and CXCL12 in hepatocarcinogenesis.

a, UMAP visualization and dotplot show predominant expression of *Has2* in myHSC in the profound fibrosis regimen, induced by 19xCCL₄ (n=1 mouse). b, bulk RNA sequencing in FACS-sorted HSC showed upregulation of *Has2* in HSC from 12xCCL₄-injured liver (n=4 mice) compared to quiescent HSC (n=4 mice). c-d, HCC was induced by DEN+14xCCL₄ and hyaluronan (HA) staining showed strong reduction of HA deposition in *Has2*<sup>ΔHSC</sup> mice compared to *Has2*<sup>Δf</sup> littermates (n=13 mice per group) (c). HCC evaluation showed a slight reduction of HCC development in *Has2*<sup>ΔHSC</sup> (n=20 mice) mice compared to *Has2*<sup>Δf</sup> littermates (n=21 mice) (d). e, UMAPs of *Hhip* and *Cxcl12* mRNA in normal (n=1 mouse) and in 8xCCL₄-treated (n=3 mice) mouse livers. f, Mice with LratCre-mediated conditional deletion of *Hhip* (*Hhip*<sup>ΔHSC</sup>) show efficient deletion in healthy control liver
<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

Extended Data Figure 11 | Expression and role of myHSC-enriched hyaluronan and cyHSC-expressed HHIP and CXCL12 in hepatocarcinogenesis.

a, UMAP visualization and dotplot show predominant expression of *Has2* in myHSC in the profound fibrosis regimen, induced by 19xCCL₄ (n=1 mouse). b, bulk RNA sequencing in FACS-sorted HSC showed upregulation of *Has2* in HSC from 12xCCL₄-injured liver (n=4 mice) compared to quiescent HSC (n=4 mice). c-d, HCC was induced by DEN+14xCCL₄ and hyaluronan (HA) staining showed strong reduction of HA deposition in *Has2*<sup>ΔHSC</sup> mice compared to *Has2*<sup>Δf</sup> littermates (n=13 mice per group) (c). HCC evaluation showed a slight reduction of HCC development in *Has2*<sup>ΔHSC</sup> (n=20 mice) mice compared to *Has2*<sup>Δf</sup> littermates (n=21 mice) (d). e, UMAPs of *Hhip* and *Cxcl12* mRNA in normal (n=1 mouse) and in 8xCCL₄-treated (n=3 mice) mouse livers. f, Mice with LratCre-mediated conditional deletion of *Hhip* (*Hhip*<sup>ΔHSC</sup>) show efficient deletion in healthy control liver
(n=4 mice), non-tumor tissue and tumor tissue (Hhip<sup>f/f</sup>: n=17 mice, Hhip<sup>ΔHSC</sup> n=10 mice) as well as upregulation of Hedgehog target gene *Gli1* in comparison to Hhip<sup>f/f</sup> controls.
**g, Hhip<sup>ΔHSC</sup> (n=11 mice) and Hhip<sup>fl/fl</sup> (n=17 mice) mice were subjected to DEN-induced hepatocarcinogenesis, followed by evaluation of the liver/body weight ratio (LBR), tumour size and tumour number.** **h, Deletion of *Cxcl12* was determined by qPCR in HSC isolated from mice with LratCre-mediated deletion (Cxcl12<sup>ΔHSC</sup>) in comparison to Cxcl12<sup>fl/fl</sup> HSC (n=2 mice per group).** **i, Cxcl12<sup>ΔHSC</sup> (n=12 mice) and Cxcl12<sup>fl/fl</sup> (n=14 mice) mice were subjected to DEN+CCl<sub>4</sub> induced HCC followed by evaluation of the liver/body weight ratio, tumour size and tumour number.** Data are shown as mean ± SEM. In the dotplot graphs, each data point represent one cell (a) or one individual (b,c,d,g,h,i), scale bars: 400 µm.

**Statistics:** Data in **a**, LBR in **d**, *Gli1* mRNA in non-tumour in **f**, LBR in **g** and **i** were analysed by two-tailed Mann-Whitney test. Data in **b**, **c**, all data in **d** besides *Gli1* mRNA, LBR in **g** and **i**, were analysed by two-tailed Student's *t*-test. Raw data are given in Source Data and Supplementary Information.
(n=4 mice), non-tumor tissue and tumor tissue (Hhip<sup>f/f</sup>: n=17 mice, Hhip<sup>ΔHSC</sup> n=10 mice) as well as upregulation of Hedgehog target gene *Gli1* in comparison to Hhip<sup>f/f</sup> controls.
**g, Hhip<sup>ΔHSC</sup> (n=11 mice) and Hhip<sup>fl/fl</sup> (n=17 mice) mice were subjected to DEN-induced hepatocarcinogenesis, followed by evaluation of the liver/body weight ratio (LBR), tumour size and tumour number.** **h, Deletion of *Cxcl12* was determined by qPCR in HSC isolated from mice with LratCre-mediated deletion (Cxcl12<sup>ΔHSC</sup>) in comparison to Cxcl12<sup>fl/fl</sup> HSC (n=2 mice per group).** **i, Cxcl12<sup>ΔHSC</sup> (n=12 mice) and Cxcl12<sup>fl/fl</sup> (n=14 mice) mice were subjected to DEN+CCl<sub>4</sub> induced HCC followed by evaluation of the liver/body weight ratio, tumour size and tumour number.** Data are shown as mean ± SEM. In the dotplot graphs, each data point represent one cell (a) or one individual (b,c,d,g,h,i), scale bars: 400 µm.

**Statistics:** Data in **a**, LBR in **d**, *Gli1* mRNA in non-tumour in **f**, LBR in **g** and **i** were analysed by two-tailed Mann-Whitney test. Data in **b**, **c**, all data in **d** besides *Gli1* mRNA, LBR in **g** and **i**, were analysed by two-tailed Student's *t*-test. Raw data are given in Source Data and Supplementary Information.
a

<figure><img src="image_73.png" /></figure>

Fibrotic mouse liver (8xCCl₄)

Cell identity

Hgf

Met

b

<figure><img src="image_74.png" /></figure>

Normal and cirrhotic human

Cell identity

HGF

c

<figure><img src="image_75.png" /></figure>

Normal mouse liver

Cell identity

Met

d

<figure><img src="image_76.png" /></figure>

e

<figure><img src="image_77.png" /></figure>

f

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g

<figure><img src="image_79.png" /></figure>

h

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i

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Extended Data Figure 12. HGF deletion in HSC promotes fibrosis and inflammation in CCl₄-induced liver injury.

a-c, UMAP visualization of *Hgf* and/or *Met* in the 8xCCl₄ injured (n=3 mice) (a) or normal mouse livers (n=1) by scRNAseq (c) and in the human normal (n=4 cases), cirrhotic (n=4 cases) and HCC (n=2 cases) livers by snRNAseq (b). d, significant reduction of *Hgf* mRNA in normal (n=4 mice/group) and CCl₄ liver (UT: n=3 mice, *Hgf*<sup>fl/fl</sup>: n=8 mice, *Hgf*<sup>ΔHSC</sup>: n=7 mice) as well as of HGF protein in liver (UT: n=4 mice, CCl₄: n=5 mice/group) e, Fibrosis was assessed by Sirius Red staining in the non-tumour tissue from *Hgf*<sup>ΔHSC</sup> mice
<figure><img src="image_72.png" /><figcaption>a</figcaption></figure>

Fibrotic mouse liver (8xCCl₄)

Cell identity

Hgf

Met

<figure><img src="image_73.png" /><figcaption>c</figcaption></figure>

Normal mouse liver

Cell identity

Met

<figure><img src="image_74.png" /><figcaption>e</figcaption></figure>

Hgf fl/fl

Sirius red

Sirius red

<figure><img src="image_75.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_76.png" /></figure>

Normal and cirrhotic human

Cell identity

HGF

<figure><img src="image_77.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>g</figcaption></figure>

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<figure><img src="image_80.png" /><figcaption>h</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>i</figcaption></figure>

<figure><img src="image_82.png" /></figure>

Extended Data Figure 12. HGF deletion in HSC promotes fibrosis and inflammation in CCl₄-induced liver injury.

a-c, UMAP visualization of *Hgf* and/or *Met* in the 8xCCl₄ injured (n=3 mice) (a) or normal mouse livers (n=1) by scRNAseq (c) and in the human normal (n=4 cases), cirrhotic (n=4 cases) and HCC (n=2 cases) livers by snRNAseq (b). d, significant reduction of *Hgf* mRNA in normal (n=4 mice/group) and CCl₄ liver (UT: n=3 mice, *Hgf* fl/fl: n=8 mice, *Hgf* ΔHSC: n=7 mice) as well as of HGF protein in liver (UT: n=4 mice, CCl₄: n=5 mice/group) e, Fibrosis was assessed by Sirius Red staining in the non-tumour tissue from *Hgf* ΔHSC mice
(n=10 mice) compared to *Hgf*<sup>f/f</sup> littermates (n=11 mice) during HCC development induced by DEN+12xCCL₄. **f**, HSC activation was determined by qPCR for the fibrogenic genes *Colla1* and *Lox* in livers from *Hgf*<sup>ΔHSC</sup> (n=4 mice) and *Hgf*<sup>f/f</sup> mice (n=7 mice) treated with injections of 6xCCL₄. **g**, flow cytometric analysis of CD45+ cells as well as myeloid and lymphocyte populations in livers from *Hgf*<sup>ΔHSC</sup> (n=7 mice) compared to *Hgf*<sup>f/f</sup> mice (n=8 mice) treated with injections of 6xCCL₄. **h**, qPCR for inflammatory genes from *Hgf*<sup>ΔHSC</sup> (n=7 mice) and *Hgf*<sup>f/f</sup> (n=8 mice) livers after treatment with 6xCCL₄ injections. **i**, Liver/body ratio (LBR) in *Hgf*<sup>f/f</sup> (Ctrl: n=10 mice, pHx: n=6 mice) and *Hgf*<sup>ΔHSC</sup> (Ctrl: n=8 mice, pHx: n=7 mice) mice in untreated mice or after 48h partial hepatectomy (pHx) (left panel). Hepatocyte proliferation assessed by Ki67 staining 48h after pHx (right panel) - *Hgf*<sup>f/f</sup>: n=6 mice, *Hgf*<sup>ΔHSC</sup>: n=7 mice). Data are shown as mean ± SEM, each data point represents one individual, scale bars: 200 µm. GZB, granzyme B. **Statistics**: Data in **d-g** and **i** were analysed by two-tailed Student's *t*-test. Data in **h** were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data and gating strategy for **g** in Supplementary Information 5.

<figure><img src="image_82.png" /><figcaption>**b**</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>**c**</figcaption></figure>

<figure><img src="image_84.png" /></figure>

**Extended Data Figure 13.** Dysbalance between myHSC and cyHSC occurs in advanced liver disease and elevates HCC risk in patients.

a, UMAP visualization showing that the genes from the myHSC and cyHSC signatures, encoding secreted mediators, are strongly enriched in myHSC or cyHSC, and weakly or not expressed in other liver cell populations in 8xCCL₄ injured mouse liver (n=3 mice) analysed by scRNAseq. **b**, myHSC and cyHSC secreted gene signatures were applied to
(n=10 mice) compared to *Hgf*<sup>f/f</sup> littermates (n=11 mice) during HCC development induced by DEN+12xCCL₄. **f**, HSC activation was determined by qPCR for the fibrogenic genes *Col1a1* and *Lox* in livers from *Hgf*<sup>ΔHSC</sup> (n=4 mice) and *Hgf*<sup>f/f</sup> mice (n=7 mice) treated with injections of 6xCCL₄. **g**, flow cytometric analysis of CD45+ cells as well as myeloid and lymphocyte populations in livers from *Hgf*<sup>ΔHSC</sup> (n=7 mice) compared to *Hgf*<sup>f/f</sup> mice (n=8 mice) treated with injections of 6xCCL₄. **h**, qPCR for inflammatory genes from *Hgf*<sup>ΔHSC</sup> (n=7 mice) and *Hgf*<sup>f/f</sup> (n=8 mice) livers after treatment with 6xCCL₄ injections. **i**, Liver/body ratio (LBR) in *Hgf*<sup>f/f</sup> (Ctrl: n=10 mice, pHx: n=6 mice) and *Hgf*<sup>ΔHSC</sup> (Ctrl: n=8 mice, pHx: n=7 mice) mice in untreated mice or after 48h partial hepatectomy (pHx) (left panel). Hepatocyte proliferation assessed by Ki67 staining 48h after PHx (right panel) - *Hgf*<sup>f/f</sup>: n=6 mice, *Hgf*<sup>ΔHSC</sup>: n=7 mice). Data are shown as mean ± SEM, each data point represents one individual, scale bars: 200 µm. GZB, granzyme B. **Statistics**: Data in **d-g** and **i** were analysed by two-tailed Student's *t*-test. Data in **h** were analysed by two-tailed Mann-Whitney test. Raw data are given in Source Data and gating strategy for **g** in Supplementary Information 5.

<figure><img src="image_83.png" /><figcaption>**b**</figcaption></figure>

<figure><img src="image_84.png" /><figcaption>**c**</figcaption></figure>

<figure><img src="image_85.png" /></figure>

**Extended Data Figure 13.** Dysbalance between myHSC and cyHSC occurs in advanced liver disease and elevates HCC risk in patients.

a, UMAP visualization showing that the genes from the myHSC and cyHSC signatures, encoding secreted mediators, are strongly enriched in myHSC or cyHSC, and weakly or not expressed in other liver cell populations in 8xCCL₄ injured mouse liver (n=3 mice) analysed by scRNAseq. **b**, myHSC and cyHSC secreted gene signatures were applied to
bulk RNA-seq data from cohorts of patients with HCV-induced liver disease (GSE10140) or NAFLD/NASH-induced liver disease (GSE49541), and the proportion of patients with high cyHSC/myHSC dysbalance was determined as described in the methods. c, HCC incidence was compared between patients with high cyHSC/myHSC dysbalance (i.e. a status with higher myHSC and lower cyHSC) and low cyHSC/myHSC dysbalance (i.e. a status lower myHSC and higher cyHSC) in a HCV-induced liver disease cohort (GSE15654). *Statistics*: data in b were analysed by two-sided Fisher's exact test. Survival curves in c were represented using the Kaplan-Meier method and compared with log-rank statistics.

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgements

This work was supported by grants R01CA190844 and R01CA228483 (to RFS) and R01DK116620 (to RFS and IT) and the Columbia University Digestive and Liver Disease Research Center (1P30DK132710) and its Bioinformatics and Single Cell Analysis Core. J.Z.-R was supported by Ligue Nationale contre le Cancer (Equipe Labellisée) and Labex OncoImmunology (Investissement d'avenir). N.C.H. is supported by a Wellcome Trust Senior Research Fellowship in Clinical Science (ref. 219542/Z/19/Z), Medical Research Council and a Chan Zuckerberg Initiative Seed Network Grant. Y.H. was supported by NIH grant CA233794 and Cancer Prevention and Research Institute of Texas grant RR180016; BI was supported by NIH grants R37CA258829 and R21CA263381. These studies used the resources of the Herbert Irving Comprehensive Cancer Center at Columbia University: The Flow Core Facility funded in part through Center Grant P30CA013696 and S10OD020056, the Molecular Pathology and the Confocal and Specialized Microscopy, supported by NIH grant #P30 CA013696. A.F. was funded by a Foundation pour la Recherche Medicale postdoctoral fellowship (SPE20170336778), an American Liver Foundation Postdoctoral Research Award, an International Liver Cancer Association's Fellowship and the Mandl Connective Tissue Research Fellowship. Y.S. by Uehara Memorial Foundation and Naomi Berrie Diabetes Center Russell Berrie Foundation. D.D. by F31 DK091980, S.B. by Deutsche Forschungsgemeinschaft grant GZ:BH 155/1-1; S.A. was funded by an American Liver Foundation Postdoctoral Research Fellowship Award, a Cholangiocarcinoma Foundation's Innovation Award, and a Research Scholar Award from the American Gastroenterological Association. We thank Dr. Edwin Monuki (University of California, Irvine) for the Lhx2 floxed mice and Dr. Matthias Mack (University of Regensburg, Germany) for the Collal floxed mice, Dr. Raghu Kalluri for the αSMA-TK mice, and Dr. Yu Yamaguchi (Stamford Burnham Prebys Medical Discovery Institute, La Jolla) for the Has2 floxed mice. We thank Dr Ekihiro Seki (University of California, Los Angeles), Dr. Céline Hernandez (The University of Birmingham, United Kingdom) and Dr. Christian Kuntzen (Columbia University) for scientific support and discussions.

## ADDITIONAL INFORMATION

### Sample sizes, biological replicates and statistical tests

**Additional information for Figure 1 | Promotion of HCC development by hepatic stellate cells.** a: Sirius Red: Lhx2<sup>fl/fl</sup>: n=8 mice, Lhx2<sup>ΔHSC</sup>: n=7 mice; Collal mRNA: Lhx2<sup>fl/fl</sup>: n=8 mice, Lhx2<sup>ΔHSC</sup>: n=6 mice. b: LBR, tumour number and tumour size: Lhx2<sup>fl/fl</sup>: n=8 mice, Lhx2<sup>ΔHSC</sup>: n=7 mice. c: αSMA area: YAP<sup>fl/fl</sup>: n=14 mice, YAP<sup>ΔHSC</sup>: n=16 mice; Collal mRNA: YAP<sup>fl/fl</sup>: n=15 mice, YAP<sup>ΔHSC</sup>: n=14 mice. d: LBR, tumour number and tumour size: YAP<sup>fl/fl</sup>: n=15 mice, YAP<sup>ΔHSC</sup>: n=16 mice. e: TdTomato: DTR<sup>neg</sup>: n=12 mice, DTR<sup>pos</sup>: n=7 mice; Collal mRNA: DTR<sup>neg</sup>: n=15 mice, DTR<sup>pos</sup>: n=16 mice. f: LBR, tumour number and tumour size: DTR<sup>neg</sup>: n=15 mice, DTR<sup>pos</sup>: n=17 mice. *Statistics*: all data in a, b, d, e, αSMA in c and all data in f besides LBR were analysed by two-tailed Student's t-test; Collal in c and LBR in f were analysed by two-tailed Mann-Whitney test.
bulk RNA-seq data from cohorts of patients with HCV-induced liver disease (GSE10140) or NAFLD/NASH-induced liver disease (GSE49541), and the proportion of patients with high cyHSC/myHSC dysbalance was determined as described in the methods. c, HCC incidence was compared between patients with high cyHSC/myHSC dysbalance (i.e. a status with higher myHSC and lower cyHSC) and low cyHSC/myHSC dysbalance (i.e. a status lower myHSC and higher cyHSC) in a HCV-induced liver disease cohort (GSE15654). *Statistics*: data in **b** were analysed by two-sided Fisher's exact test. Survival curves in **c** were represented using the Kaplan-Meier method and compared with log-rank statistics.

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgements

This work was supported by grants R01CA190844 and R01CA228483 (to RFS) and R01DK116620 (to RFS and IT) and the Columbia University Digestive and Liver Disease Research Center (1P30DK132710) and its Bioinformatics and Single Cell Analysis Core. J.Z.-R was supported by Ligue Nationale contre le Cancer (Equipe Labellisée) and Labex OncoImmunology (Investissement d'avenir). N.C.H. is supported by a Wellcome Trust Senior Research Fellowship in Clinical Science (ref. 219542/Z/19/Z), Medical Research Council and a Chan Zuckerberg Initiative Seed Network Grant. Y.H. was supported by NIH grant CA233794 and Cancer Prevention and Research Institute of Texas grant RR180016; BI was supported by NIH grants R37CA258829 and R21CA263381. These studies used the resources of the Herbert Irving Comprehensive Cancer Center at Columbia University: The Flow Core Facility funded in part through Center Grant P30CA013696 and S10OD020056, the Molecular Pathology and the Confocal and Specialized Microscopy, supported by NIH grant #P30 CA013696. A.F. was funded by a Foundation pour la Recherche Medicale postdoctoral fellowship (SPE20170336778), an American Liver Foundation Postdoctoral Research Award, an International Liver Cancer Association's Fellowship and the Mandl Connective Tissue Research Fellowship. Y.S. by Uehara Memorial Foundation and Naomi Berrie Diabetes Center Russell Berrie Foundation. D.D. by F31 DK091980, S.B. by Deutsche Forschungsgemeinschaft grant GZ:BH 155/1-1; S.A. was funded by an American Liver Foundation Postdoctoral Research Fellowship Award, a Cholangiocarcinoma Foundation's Innovation Award, and a Research Scholar Award from the American Gastroenterological Association. We thank Dr. Edwin Monuki (University of California, Irvine) for the Lhx2 floxed mice and Dr. Matthias Mack (University of Regensburg, Germany) for the Collal floxed mice, Dr. Raghu Kalluri for the αSMA-TK mice, and Dr. Yu Yamaguchi (Stamford Burnham Prebys Medical Discovery Institute, La Jolla) for the Has2 floxed mice. We thank Dr Ekihiro Seki (University of California, Los Angeles), Dr. Céline Hernandez (The University of Birmingham, United Kingdom) and Dr. Christian Kuntzen (Columbia University) for scientific support and discussions.

## ADDITIONAL INFORMATION

### Sample sizes, biological replicates and statistical tests

**Additional information for Figure 1 | Promotion of HCC development by hepatic stellate cells.**
a: Sirius Red: Lhx2<sup>fl/fl</sup>: n=8 mice, Lhx2<sup>ΔHSC</sup>: n=7 mice; Collal mRNA: Lhx2<sup>fl/fl</sup>: n=8 mice, Lhx2<sup>ΔHSC</sup>: n=6 mice. b: LBR, tumour number and tumour size: Lhx2<sup>fl/fl</sup>: n=8 mice, Lhx2<sup>ΔHSC</sup>: n=7 mice. c: αSMA area: YAP<sup>fl/fl</sup>: n=14 mice, YAP<sup>ΔHSC</sup>: n=16 mice; Collal mRNA: YAP<sup>fl/fl</sup>: n=15 mice, YAP<sup>ΔHSC</sup>: n=14 mice. d: LBR, tumour number and tumour size: YAP<sup>fl/fl</sup>: n=15 mice, YAP<sup>ΔHSC</sup>: n=16 mice. e: TdTomato: DTR<sup>neg</sup>: n=12 mice, DTR<sup>pos</sup>: n=7 mice; Collal mRNA: DTR<sup>neg</sup>: n=15 mice, DTR<sup>pos</sup>: n=16 mice. f: LBR, tumour number and tumour size: DTR<sup>neg</sup>: n=15 mice, DTR<sup>pos</sup>: n=17 mice. *Statistics*: all data in **a, b, d, e, αSMA** in **c** and all data in **f** besides LBR were analysed by two-tailed Student's *t*-test; Collal in **c** and LBR in **f** were analysed by two-tailed Mann-Whitney test.
**Additional information for Figure 2 | Hepatic stellate cell accumulation and activation occurs predominantly in the non-tumour environment.** **a:** Col-GFP/TdTom double+ area: DEN+CCl₄: n=3 mice per group, TAZ+FPC: n=2 mice per group, KRAS/sgp19: n=3 mice per group; Sirius Red: DEN+CCl₄: n=3 mice per group, TAZ+FPC: n=3 mice per group, KRAS/sgp19: n=3 mice per group. **b:** αSMA: Human HCC: n=45 cases per group, ICC: n=5 cases per group, Sirius Red: Human HCC: n=45 cases per group, ICC: n=4 cases per group. **c:** Ki67+hepatocytes: *YAP*<sup>f/f</sup>: n=10 mice, *YAP*<sup>ΔHSC</sup>: n=11 mice; *DTR*<sup>neg</sup>: n=10 mice, *DTR*<sup>pos</sup>: n=10 mice. **d:** CellPhoneDB in scRNAseq: n=3 mice. *Statistics:* all data in **a** and **c** were analysed by two-tailed Student's *t*-test. Data in **b** were analysed by one-way ANOVA (Kruskal-Wallis's test) followed by Dunn's multiple comparison test.

**Additional information for Figure 3 | Single cell RNA-seq analysis reveals subsets of HSC with differential expression of HGF and COL1A1 in the fibrotic liver.** Fig a,e:
scRNAseq on n=1 mouse, **b,f:** snRNAseq on normal and cirrhotic human livers n= 4 cases per group. **c:** scRNAseq on n=1 mouse per group. **d:** CellPhoneDB in scRNAseq: n=3 mice. **g:** normal liver: n=1 mouse and quantification on 4 areas per group; profound fibrosis: n=1 mouse and quantification on 4 areas per group. *Statistics:* P-values in **a-b**, coefficient of determination (R²) and statistical significance (P value) were determined using Pearson's. Data in **c** were analysed by one-way ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison test. Data in **e-f** were analysed by two-tailed Mann-Whitney test.

**Additional information for Figure 4 | Promotion of HCC by type I collagen and stiffness-associated accumulation of TAZ in hepatocytes.** **a:** LBR and tumour number: n=21 mice per group; tumour size: *Colla*<sup>f/f</sup>: n=20 mice, *Colla*<sup>del</sup>: n=21 mice. **b:** Ki67+hepatocytes: n=13 mice per group; Ki67+tumour cells: *Colla*<sup>f/f</sup>: n=11 mice, *Colla*<sup>del</sup>: n=10 mice. **c:** Youngs modulus: untreated: 1 mouse, 19xCCl₄: n=4 mice per group. **d:** TAZ/GAPDH and *YAP*/GAPDH: n=6 mice per group. **e:** nuclear TAZ+ hepatocytes: n= 9 mice per group. **f:** LBR, tumour number and tumour size: n= 10 mice per group. **g:** Ki67+hepatocytes: n=10 mice per group; Ki67+tumour cells: *Wwtr*<sup>f/f</sup>: n=10 mice, *Wwtr*<sup>ΔHep</sup>: n=8 mice. *Statistics:* tumour size in **a**, Ki67+ tumour cells in **b**, and all data in **d,e,f** and **g** were analysed by two-tailed Student's *t*-test. LBR and tumour number in **a** and Ki67+ hepatocytes in **b** were analysed by two-tailed Mann-Whitney test. Data in **e** were analysed by two-way ANOVA followed by Tukey's multiple comparison test. * Comparison between *Colla*<sup>f/f</sup> and *Colla*<sup>del</sup> mice, *p*<0.05

**Additional information for Figure 5 | Promotion of hepatocarcinogenesis by DDR1.** **b.** DDR1 IHC: normal: n=6, non-tumour and tumour: n=64 per group; *DDR1* mRNA: normal: n=4, non-tumour and tumour: n=16 per group. **c:** LBR and tumour size: *Ddr1*<sup>f/f</sup>: n=10 mice, *Ddr1*<sup>ΔHep</sup> n=9 mice, tumour number: n=9 mice per group. **d:** Ki67+ hepatocytes and Ki67+ tumours cells: *Ddr1*<sup>f/f</sup>: n=9 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice. **e:** (n=3 biological replication/group; n=1 experiment). **f,** (n=1 biological replicate; n=2 experiments). *Statistics:* all data in **b** and Ki67+ tumour cells in **d** were analysed by two-tailed Mann-Whitney test. All data in **c** and Ki67+ hepatocytes in **d** were analysed by two-tailed Student's *t*-test.

**Additional information for Figure 6 | CyHSC-enriched HGF protects hepatocytes from death and reduces HCC development.** **a:** LBR and tumour number: n= 10 mice per group;
**Additional information for Figure 2 | Hepatic stellate cell accumulation and activation occurs predominantly in the non-tumour environment.** **a:** Col-GFP/TdTom double+ area: DEN+CCl₄: n=3 mice per group, TAZ+FPC: n=2 mice per group, KRAS/sgp19: n=3 mice per group; Sirius Red: DEN+CCl₄: n=3 mice per group, TAZ+FPC: n=3 mice per group, KRAS/sgp19: n=3 mice per group. **b:** αSMA: Human HCC: n=45 cases per group, ICC: n=5 cases per group, Sirius Red: Human HCC: n=45 cases per group, ICC: n=4 cases per group. **c:** Ki67+hepatocytes: *YAP*<sup>f/f</sup>: n=10 mice, *YAP*<sup>ΔHSC</sup>: n=11 mice; *DTR*<sup>neg</sup>: n=10 mice, *DTR*<sup>pos</sup>: n=10 mice. **d:** CellPhoneDB in scRNAseq: n=3 mice. *Statistics:* all data in **a** and **c** were analysed by two-tailed Student's *t*-test. Data in **b** were analysed by one-way ANOVA (Kruskal-Wallis's test) followed by Dunn's multiple comparison test.

**Additional information for Figure 3 | Single cell RNA-seq analysis reveals subsets of HSC with differential expression of HGF and COL1A1 in the fibrotic liver.** Fig a,e:
scRNAseq on n=1 mouse, **b,f:** snRNAseq on normal and cirrhotic human livers n= 4 cases per group. **c:** scRNAseq on n=1 mouse per group. **d:** CellPhoneDB in scRNAseq: n=3 mice. **g:** normal liver: n=1 mouse and quantification on 4 areas per group; profound fibrosis: n=1 mouse and quantification on 4 areas per group. *Statistics:* P-values in **a-b**, coefficient of determination (R²) and statistical significance (P value) were determined using Pearson's. Data in **c** were analysed by one-way ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison test. Data in **e-f** were analysed by two-tailed Mann-Whitney test.

**Additional information for Figure 4 | Promotion of HCC by type I collagen and stiffness-associated accumulation of TAZ in hepatocytes.** **a:** LBR and tumour number: n=21 mice per group; tumour size: *Colla*<sup>f/f</sup>: n=20 mice, *Colla*<sup>del</sup>: n=21 mice. **b:** Ki67+hepatocytes: n=13 mice per group; Ki67+tumour cells: *Colla*<sup>f/f</sup>: n=11 mice, *Colla*<sup>del</sup>: n=10 mice. **c:** Youngs modulus: untreated: 1 mouse, 19xCCl₄: n=4 mice per group. **d:** TAZ/GAPDH and *YAP*/GAPDH: n=6 mice per group. **e:** nuclear TAZ+ hepatocytes: n= 9 mice per group. **f:** LBR, tumour number and tumour size: n= 10 mice per group. **g:** Ki67+hepatocytes: n=10 mice per group; Ki67+tumour cells: *Wwtr*<sup>f/f</sup>: n=10 mice, *Wwtr*<sup>ΔHep</sup>: n=8 mice. *Statistics:* tumour size in **a**, Ki67+ tumour cells in **b**, and all data in **d,e,f** and **g** were analysed by two-tailed Student's *t*-test. LBR and tumour number in **a** and Ki67+ hepatocytes in **b** were analysed by two-tailed Mann-Whitney test. Data in **e** were analysed by two-way ANOVA followed by Tukey's multiple comparison test. * Comparison between *Colla*<sup>f/f</sup> and *Colla*<sup>del</sup> mice, *p*<0.05

**Additional information for Figure 5 | Promotion of hepatocarcinogenesis by DDR1.** **b.** DDR1 IHC: normal: n=6, non-tumour and tumour: n=64 per group; *DDR1* mRNA: normal: n=4, non-tumour and tumour: n=16 per group. **c:** LBR and tumour size: *Ddr1*<sup>f/f</sup>: n=10 mice, *Ddr1*<sup>ΔHep</sup> n=9 mice, tumour number: n=9 mice per group. **d:** Ki67+ hepatocytes and Ki67+ tumours cells: *Ddr1*<sup>f/f</sup>: n=9 mice, *Ddr1*<sup>ΔHep</sup> n=8 mice. **e:** (n=3 biological replication/group; n=1 experiment). **f,** (n=1 biological replicate; n=2 experiments). *Statistics:* all data in **b** and Ki67+ tumour cells in **d** were analysed by two-tailed Mann-Whitney test. All data in **c** and Ki67+ hepatocytes in **d** were analysed by two-tailed Student's *t*-test.

**Additional information for Figure 6 | CyHSC-enriched HGF protects hepatocytes from death and reduces HCC development.** **a:** LBR and tumour number: n= 10 mice per group;
tumour size: Hgf<sup>f/f</sup>: n=10 mice, Hgf<sup>ΔHSC</sup>: n=9 mice. **b**: TUNEL and Ki67+ hepatocytes: Hgf<sup>f/f</sup>: n=8 mice, Hgf<sup>ΔHSC</sup>: n=4 mice; ALT (CCl<sub>4</sub>): Hgf<sup>f/f</sup>: n=7 mice, Hgf<sup>ΔHSC</sup>: n=4 mice; Mki67 mRNA: Hgf<sup>f/f</sup>: n=6 mice, Hgf<sup>ΔHSC</sup>: n=4 mice; ALT (NASH): n= 11 mice per group. **c**: PI+ area: control: n= 3 wells, treatment: n= 5 wells per group. **d**: Hgf mRNA: untreated: n= 5 mice, NT and HCC: n= 8 mice per group. **e**: HGF mRNA: normal: n=124 cases, F0-F1 non-tumour: n=261 cases, F2-F3: n=115 cases, F4: n=182 cases, DMN: n=31 cases, HCC: n=687 cases. **Statistics**: all data in **a**, **b**, **c** and **d** were analysed by two-tailed Student's t-test. Data in **e** between normal, F0-F1, F2-F3 and F4 were analysed by one-way ANOVA (Kruskal-Wallis's test) followed by Dunn's multiple comparison test with normal liver group. Dysplastic macronodules (DMN) and HCC comparison in **e** was analysed by two-tailed Mann-Whitney test. Kaplan-Meier survival curves in **f** were compared with log-rank statistics.

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19. Hoshida Y et al. Gene expression in fixed tissues and outcome in hepatocellular carcinoma. N Engl J Med 359, 1995–2004, doi:10.1056/NEJMoa0804525 (2008). [PubMed: 18923165]

20. Ji J et al. Hepatic stellate cell and monocyte interaction contributes to poor prognosis in hepatocellular carcinoma. Hepatology 62, 481–495, doi:10.1002/hep.27822 (2015). [PubMed: 25833323]

21. Mu X et al. Hepatocellular carcinoma originates from hepatocytes and not from the progenitor/ biliary compartment. J Clin Invest 125, 3891–3903, doi:10.1172/JCI177995 (2015). [PubMed: 26348897]

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36. Suzuki A et al. Hepatocyte growth factor promotes cell survival from fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death-inducing signaling complex suppression. Hepatology 32, 796-802, doi:10.1053/jhep.2000.17738 (2000). [PubMed: 11003625]

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38. Alkasalias T et al. Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent. Proc Natl Acad Sci U S A 111, 17188-17193, doi:10.1073/pnas.1419554111 (2014). [PubMed: 25404301]

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## References (Methods)

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42. Krempen K et al. Far upstream regulatory elements enhance position-independent and uterus-specific expression of the murine alpha1(I) collagen promoter in transgenic mice. Gene Expr 8, 151-163 (1999). [PubMed: 10634317]

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44. Geisler F, Algul H, Paxian S & Schmid RM Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro. Gastroenterology 132, 2489-2503, doi:10.1053/j.gastro.2007.03.033 (2007). [PubMed: 17570221]

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46. Affo S et al. Promotion of Cholangiocarcinoma Growth by Diverse Cancer-associated Fibroblast Subpopulations. Cancer Cell In Press (2021).

47. Phaneuf D, Moscioni AD, LeClair C, Raper SE & Wilson JM Generation of a mouse expressing a conditional knockout of the hepatocyte growth factor gene: demonstration of impaired liver regeneration. DNA Cell Biol 23, 592-603, doi:10.1089/dna.2004.23.592 (2004). [PubMed: 15383179]

48. Matsumoto K et al. Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb. Development 136, 2825-2835, doi:10.1242/dev.038505 (2009). [PubMed: 19633173]

49. Murtaugh LC, Stanger BZ, Kwan KM & Melton DA Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A 100, 14920-14925, doi:10.1073/pnas.2436557100 (2003). [PubMed: 14657333]

50. Ozdemir BC et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25, 719-734, doi:10.1016/j.ccr.2014.04.005 (2014). [PubMed: 24856586]

51. Mitchell C & Willenbring H A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nature protocols 3, 1167-1170, doi:10.1038/nprot.2008.80 (2008). [PubMed: 18600221]

52. Zhang S et al. The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic beta-Catenin to Induce Hepatoblastoma Development in Mice and Humans. Am J Pathol 190, 1397-1413, doi:10.1016/j.ajpath.2020.03.011 (2020). [PubMed: 32283103]
34. Ueki T et al. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med 5, 226-230, doi:10.1038/5593 (1999). [PubMed: 9930873]

35. Santoni-Rugiu E et al. Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model. Proc Natl Acad Sci U S A 93, 9577-9582, doi:10.1073/pnas.93.18.9577 (1996). [PubMed: 8790372]

36. Suzuki A et al. Hepatocyte growth factor promotes cell survival from fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death-inducing signaling complex suppression. Hepatology 32, 796-802, doi:10.1053/jhep.2000.17738 (2000). [PubMed: 11003625]

37. Stoker MG, Shearer M & O'Neill C Growth inhibition of polyoma-transformed cells by contact with static normal fibroblasts. J Cell Sci 1, 297-310 (1966). [PubMed: 4291022]

38. Alkasalias T et al. Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent. Proc Natl Acad Sci U S A 111, 17188-17193, doi:10.1073/pnas.1419554111 (2014). [PubMed: 25404301]

39. Sun X et al. Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion. Nature 599, 673-678, doi:10.1038/s41586-021-04057-2 (2021). [PubMed: 34732895]

40. Chen Y et al. Type I collagen deletion in alphaSMA(+) myofibroblasts augments immune suppression and accelerates progression of pancreatic cancer. Cancer Cell 39, 548-565 e546, doi:10.1016/j.ccell.2021.02.007 (2021). [PubMed: 33667385]

## References (Methods)

41. Nault JC et al. Clinical Impact of Genomic Diversity From Early to Advanced Hepatocellular Carcinoma. Hepatology 71, 164-182, doi:10.1002/hep.30811 (2020). [PubMed: 31206197]

42. Krempen K et al. Far upstream regulatory elements enhance position-independent and uterus-specific expression of the murine alpha1(I) collagen promoter in transgenic mice. Gene Expr 8, 151-163 (1999). [PubMed: 10634317]

43. Mangale VS et al. Lhx2 selector activity specifies cortical identity and suppresses hippocampal organizer fate. Science 319, 304-309, doi:10.1126/science.1151695 (2008). [PubMed: 18202285]

44. Geisler F, Algul H, Paxian S & Schmid RM Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro. Gastroenterology 132, 2489-2503, doi:10.1053/j.gastro.2007.03.033 (2007). [PubMed: 17570221]

45. Buchtler S et al. Cellular Origin and Functional Relevance of Collagen I Production in the Kidney. J Am Soc Nephrol 29, 1859-1873, doi:10.1681/ASN.2018020138 (2018). [PubMed: 29777019]

46. Affo S et al. Promotion of Cholangiocarcinoma Growth by Diverse Cancer-associated Fibroblast Subpopulations. Cancer Cell In Press (2021).

47. Phaneuf D, Moscioni AD, LeClair C, Raper SE & Wilson JM Generation of a mouse expressing a conditional knockout of the hepatocyte growth factor gene: demonstration of impaired liver regeneration. DNA Cell Biol 23, 592-603, doi:10.1089/dna.2004.23.592 (2004). [PubMed: 15383179]

48. Matsumoto K et al. Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb. Development 136, 2825-2835, doi:10.1242/dev.038505 (2009). [PubMed: 19633173]

49. Murtaugh LC, Stanger BZ, Kwan KM & Melton DA Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A 100, 14920-14925, doi:10.1073/pnas.2436557100 (2003). [PubMed: 14657333]

50. Ozdemir BC et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25, 719-734, doi:10.1016/j.ccr.2014.04.005 (2014). [PubMed: 24856586]

51. Mitchell C & Willenbring H A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nature protocols 3, 1167-1170, doi:10.1038/nprot.2008.80 (2008). [PubMed: 18600221]

52. Zhang S et al. The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic beta-Catenin to Induce Hepatoblastoma Development in Mice and Humans. Am J Pathol 190, 1397-1413, doi:10.1016/j.ajpath.2020.03.011 (2020). [PubMed: 32283103]
53. Tao J et al. Modeling a human hepatocellular carcinoma subset in mice through coexpression of met and point-mutant beta-catenin. Hepatology 64, 1587-1605, doi:10.1002/hep.28601 (2016). [PubMed: 27097116]

54. Mederacke I, Dapito DH, Affo S, Uchinami H & Schwabe RF High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nature protocols 10, 305-315, doi:10.1038/nprot.2015.017 (2015). [PubMed: 25612230]

55. Pradere JP et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 58, 1461-1473, doi:10.1002/hep.26429 (2013). [PubMed: 23553591]

56. Chung AS et al. Immune Checkpoint Inhibition is Safe and Effective for Liver Cancer Prevention in a Mouse Model of Hepatocellular Carcinoma. Cancer Prev Res (Phila) 13, 911-922, doi:10.1158/1940-6207.CAPR-20-0200 (2020). [PubMed: 32839204]

57. Love MI, Huber W & Anders S Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550, doi:10.1186/s13059-014-0550-8 (2014). [PubMed: 25516281]

58. Zhou Y et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 10, 1523, doi:10.1038/s41467-019-09234-6 (2019). [PubMed: 30944313]

59. Hao Y et al. Integrated analysis of multimodal single-cell data. bioRxiv (2020).

60. Slyper M et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat Med 26, 792-802, doi:10.1038/s41591-020-0844-1 (2020). [PubMed: 32405060]

61. Andrews TS et al. Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatol Commun 6, 821-840, doi:10.1002/hep4.1854 (2022). [PubMed: 34792289]

62. Fleming SJ, Marioni JC & Babadi M CellBender remove-background: a deep generative model for unsupervised removal of background noise from scRNA-seq datasets. bioRxiv doi: 10.1101/791699 (2019).

63. Franzen O, Gan LM & Bjorkegren JLM PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database (Oxford) 2019, doi:10.1093/database/baz046 (2019).

64. Cao J et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496-502, doi:10.1038/s41586-019-0969-x (2019). [PubMed: 30787437]

65. Hoshida Y Nearest template prediction: a single-sample-based flexible class prediction with confidence assessment. PLoS One 5, e15543, doi:10.1371/journal.pone.0015543 (2010). [PubMed: 21124904]

66. Kuleshov MV et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44, W90-97, doi:10.1093/nar/gkw377 (2016). [PubMed: 27141961]

67. Vento-Tormo R et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature 563, 347-353, doi:10.1038/s41586-018-0698-6 (2018). [PubMed: 30429548]

68. Efremova M, Vento-Tormo M, Teichmann SA & Vento-Tormo R CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nature protocols 15, 1484-1506, doi:10.1038/s41596-020-0292-x (2020). [PubMed: 32103204]
53. Tao J et al. Modeling a human hepatocellular carcinoma subset in mice through coexpression of met and point-mutant beta-catenin. Hepatology 64, 1587-1605, doi:10.1002/hep.28601 (2016). [PubMed: 27097116]

54. Mederacke I, Dapito DH, Affo S, Uchinami H & Schwabe RF High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nature protocols 10, 305-315, doi:10.1038/nprot.2015.017 (2015). [PubMed: 25612230]

55. Pradere JP et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 58, 1461-1473, doi:10.1002/hep.26429 (2013). [PubMed: 23553591]

56. Chung AS et al. Immune Checkpoint Inhibition is Safe and Effective for Liver Cancer Prevention in a Mouse Model of Hepatocellular Carcinoma. Cancer Prev Res (Phila) 13, 911-922, doi:10.1158/1940-6207.CAPR-20-0200 (2020). [PubMed: 32839204]

57. Love MI, Huber W & Anders S Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550, doi:10.1186/s13059-014-0550-8 (2014). [PubMed: 25516281]

58. Zhou Y et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 10, 1523, doi:10.1038/s41467-019-09234-6 (2019). [PubMed: 30944313]

59. Hao Y et al. Integrated analysis of multimodal single-cell data. bioRxiv (2020).

60. Slyper M et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat Med 26, 792-802, doi:10.1038/s41591-020-0844-1 (2020). [PubMed: 32405060]

61. Andrews TS et al. Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatol Commun 6, 821-840, doi:10.1002/hep4.1854 (2022). [PubMed: 34792289]

62. Fleming SJ, Marioni JC & Babadi M CellBender remove-background: a deep generative model for unsupervised removal of background noise from scRNA-seq datasets. bioRxiv doi: 10.1101/791699 (2019).

63. Franzen O, Gan LM & Bjorkegren JLM PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database (Oxford) 2019, doi:10.1093/database/baz046 (2019).

64. Cao J et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496-502, doi:10.1038/s41586-019-0969-x (2019). [PubMed: 30787437]

65. Hoshida Y Nearest template prediction: a single-sample-based flexible class prediction with confidence assessment. PLoS One 5, e15543, doi:10.1371/journal.pone.0015543 (2010). [PubMed: 21124904]

66. Kuleshov MV et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44, W90-97, doi:10.1093/nar/gkw377 (2016). [PubMed: 27141961]

67. Vento-Tormo R et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature 563, 347-353, doi:10.1038/s41586-018-0698-6 (2018). [PubMed: 30429548]

68. Efremova M, Vento-Tormo M, Teichmann SA & Vento-Tormo R CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nature protocols 15, 1484-1506, doi:10.1038/s41596-020-0292-x (2020). [PubMed: 32103204]
<figure><img src="image_85.png" /><figcaption>**Figure 1** | Promotion of HCC development by hepatic stellate cells.</figcaption></figure>

a-b, Induction of liver fibrosis, shown by Sirius Red staining, and *Colla1* by HSC-selective *Lhx2* deletion via LratCre (*Lhx2*<sup>ΔHSC</sup>) compared to *Lhx2*<sup>fl/fl</sup> littermates (n=7-8 mice/group), (a) and increased DEN-induced HCC, shown by representative liver pictures, the liver-body ratio (LBR), tumour number and size (b). c-d, Decreased DEN+CCl₄-induced HSC activation in *Yap*<sup>ΔHSC</sup> compared to *Yap*<sup>fl/fl</sup> mice (n=15-16/group), shown by αSMA IHC and *Colla1* qPCR (c), as well as reduced HCC burden (d). e-f, Efficient HSC depletion in non-tumour tissue in the DEN+CCl₄ model in DTR<sup>pos</sup> compared DTR<sup>neg</sup> LratCre<sup>pos</sup>, TdTom<sup>pos</sup> littermates (n=16-17/group), determined by TdTom fluorescence and *Colla1* qPCR (e) as well as reduced tumour development (f). Data are shown as mean ± SEM, each data point represents one individual, Tu: Tumour, HTVI: hydrodynamic tail vein injection, all scale bars: 200 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_86.png" /><figcaption>Figure 1 | Promotion of HCC development by hepatic stellate cells.</figcaption></figure>

a-b, Induction of liver fibrosis, shown by Sirius Red staining, and *Colla1* by HSC-selective *Lhx2* deletion via LratCre (*Lhx2*<sup>ΔHSC</sup>) compared to *Lhx2*<sup>fl/fl</sup> littermates (n=7–8 mice/group), (a) and increased DEN-induced HCC, shown by representative liver pictures, the liver-body ratio (LBR), tumour number and size (b). c-d, Decreased DEN+CCl₄-induced HSC activation in *Yap*<sup>ΔHSC</sup> compared to *Yap*<sup>fl/fl</sup> mice (n=15–16/group), shown by αSMA IHC and *Colla1* qPCR (c), as well as reduced HCC burden (d). e-f, Efficient HSC depletion in non-tumour tissue in the DEN+CCl₄ model in DTR<sup>pos</sup> compared DTR<sup>neg</sup> LratCre<sup>pos</sup>, TdTom<sup>pos</sup> littermates (n=16–17/group), determined by TdTom fluorescence and *Colla1* qPCR (e) as well as reduced tumour development (f). Data are shown as mean ± SEM, each data point represents one individual, Tu: Tumour, HTVI: hydrodynamic tail vein injection, all scale bars: 200 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_86.png" /><figcaption>Figure 2 | Hepatic stellate cell accumulation and activation occurs predominantly in the non-tumour environment.</figcaption></figure>

a, HSC and collagen-expressing fibroblasts were visualized in non-tumour (NT) and tumour (Tu) areas of DEN+CCl₄ and TAZ+FPC diet HCC models or the KRAS/sgp19 ICC model by LratCre-induced TdT Tom and *Colla1*-GFP, respectively (n=2–3/group); liver fibrosis was determined by Sirius Red staining (n=3/group). b, myofibroblasts were visualized by αSMA IHC and liver fibrosis by Sirius Red staining in paired non-tumour and tumour sections from human HCC (n=45, displayed are HCCs arising in fibrotic livers) and from human ICC (n=4–5). c, proliferation determined by Ki67 IHC in non-tumour tissue from *Yap*fl/fl and *Yap*ΔHSC littermates or DTRpos and DTRneg mice in the DEN+CCl₄ HCC model. d, analysis of cell-cell interactions by CellPhoneDB in scRNA-seq data from 8xCCl₄-injured liver (n=3) revealed HSC as a major cell type interacting with hepatocytes. Data are shown as mean ± SEM, each data point (a,b,c) represents one individual; scale bars: 150 µm (a,b), 100 µm (c). Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_87.png" /><figcaption>Figure 2 | Hepatic stellate cell accumulation and activation occurs predominantly in the non-tumour environment.</figcaption></figure>

a, HSC and collagen-expressing fibroblasts were visualized in non-tumour (NT) and tumour (Tu) areas of DEN+CCl₄ and TAZ+FPC diet HCC models or the KRAS/sgp19 ICC model by LratCre-induced TdT Tom and Colla1-GFP, respectively (n=2–3/group); liver fibrosis was determined by Sirius Red staining (n=3/group). b, myofibroblasts were visualized by αSMA IHC and liver fibrosis by Sirius Red staining in paired non-tumour and tumour sections from human HCC (n=45, displayed are HCCs arising in fibrotic livers) and from human ICC (n=4–5). c, proliferation determined by Ki67 IHC in non-tumour tissue from Yapfl/fl and YapΔHSC littermates or DTRpos and DTRneg mice in the DEN+CCl₄ HCC model. d, analysis of cell-cell interactions by CellPhoneDB in scRNA-seq data from 8xCCl₄-injured liver (n=3) revealed HSC as a major cell type interacting with hepatocytes. Data are shown as mean ± SEM, each data point (a,b,c) represents one individual; scale bars: 150 µm (a,b), 100 µm (c). Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_87.png" /><figcaption>Figure 3 | Single cell RNA-seq analysis reveals subsets of HSC with differential expression of *HGF* and *COL1A1* in the fibrotic liver.</figcaption></figure>

a-b, UMAP visualization of myHSC and cyHSC signatures from scRNAseq of HSC from 19xCCl₄ fibrotic mouse liver (n=1) (a) or snRNAseq of HSC from normal and cirrhotic human livers (n=4 each) (b). c, Violin plots of scRNA-seq data show cyHSC and myHSC subsets in untreated (Ctrl) or injured (CCl₄) livers (n=1 each). d, Cell-cell interaction analysis of hepatocytes with other liver cells by CellPhoneDB from 8xCCl₄-injured liver (n=3). e-f, UMAP and violin plot aligning *Col1a1* and *Hgf* mRNA expression with myHSC
<figure><img src="image_88.png" /><figcaption>Figure 3 | Single cell RNA-seq analysis reveals subsets of HSC with differential expression of *HGF* and *COL1A1* in the fibrotic liver.</figcaption></figure>

a-b, UMAP visualization of myHSC and cyHSC signatures from scRNAseq of HSC from 19xCCl₄ fibrotic mouse liver (n=1) (a) or snRNAseq of HSC from normal and cirrhotic human livers (n=4 each) (b). c, Violin plots of scRNA-seq data show cyHSC and myHSC subsets in untreated (Ctrl) or injured (CCl₄) livers (n=1 each). d, Cell-cell interaction analysis of hepatocytes with other liver cells by CellPhoneDB from 8xCCl₄-injured liver (n=3). e-f, UMAP and violin plot aligning *Col1a1* and *Hgf* mRNA expression with myHSC
and cyHSC in scRNA-seq from 19xCCl₄ treated liver (e, n=1) or snRNA-seq of HSC from human normal and cirrhotic livers (n=4 each) (f). g, in situ analysis of *Hgf* mRNA by RNAscope with *Collal1*-GFP and LratCre-induced TdTm fluorescence, shows separate populations of *Collal1*-GFP^high* *Hgf*^low* myHSC, and *Collal1*-GFP^low* *Hgf*^high* cyHSC in normal (n=1) and 21xCCl₄-treated livers (n=1) (g). AU=arbitrary units. Data are shown as mean ± SEM. In c and e, box plots represent the interquartile range (IQR), Q1, median and Q3, whiskers (Q1–1.5×IQR and Q3+1.5×IQR) and outlier data as individual dots. each data point represents one cell (a,b,e,f), or one area (g). Scale bars: 50 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
and cyHSC in scRNA-seq from 19xCCl₄ treated liver (e, n=1) or snRNA-seq of HSC from human normal and cirrhotic livers (n=4 each) (f). g, *in situ* analysis of *Hgf* mRNA by RNAscope with *Collal1*-GFP and LratCre-induced TdTm fluorescence, shows separate populations of *Collal1*-GFP^high Hgf^low myHSC, and *Collal1*-GFP^low Hgf^high cyHSC in normal (n=1) and 21xCCl₄-treated livers (n=1) (g). AU=arbitrary units. Data are shown as mean ± SEM. In c and e, box plots represent the interquartile range (IQR), Q1, median and Q3, whiskers (Q1–1.5×IQR and Q3+1.5×IQR) and outlier data as individual dots. each data point represents one cell (a,b,e,f), or one area (g). Scale bars: 50 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_88.png" /><figcaption>**Figure 4** | Promotion of HCC by type I collagen and stiffness-associated accumulation of TAZ in hepatocytes.</figcaption></figure>

a, significantly reduced HCC, shown by representative images, liver body ratio (LBR), tumour size and number, in Mx1Cre<sup>Pos</sup> *Colla1<sup>f/f</sup>* (*Colla1<sup>del</sup>*) compared to *Colla1<sup>f/f</sup>* littermates (n=21 per group) in a profound fibrosis DEN+44xCCl<sub>4</sub> model. b, Ki67 staining in non-tumour and tumour tissue (n=10–13 mice per group) shows a reduction of hepatocyte proliferation and tumour cell proliferation in *Colla1<sup>del</sup>* compared to *Colla1<sup>f/f</sup>* mice. c, reduction of liver stiffness, shown by rheometry (untreated: n=1; DEN+19xCCl<sub>4</sub>: n=4/group). d-e, pAKT, pERK, YAP, TAZ and GAPDH, detected by western blot (d), and TAZ IHC and nuclear translocation (e), in livers from the DEN+19xCCl<sub>4</sub> profound fibrosis model (macroscopic tumors not yet present) from *Colla1<sup>del</sup>* and *Colla1<sup>f/f</sup>* mice (n=6/group for d, n=9/group for e). f-g, HCC development (f) and hepatocyte proliferation (g) in mice with
<figure><img src="image_89.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_90.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_91.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_92.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_93.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_94.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_95.png" /><figcaption>**Figure 4** | Promotion of HCC by type I collagen and stiffness-associated accumulation of TAZ in hepatocytes.</figcaption></figure>

a, significantly reduced HCC, shown by representative images, liver body ratio (LBR), tumour size and number, in Mx1Cre<sup>pos</sup> *Colla1<sup>f/f</sup>* (*Colla1<sup>del</sup>*) compared to *Colla1<sup>f/f</sup>* littermates (n=21 per group) in a profound fibrosis DEN+44xCCl<sub>4</sub> model. b, Ki67 staining in non-tumour and tumour tissue (n=10–13 mice per group) shows a reduction of hepatocyte proliferation and tumour cell proliferation in *Colla1<sup>del</sup>* compared to *Colla1<sup>f/f</sup>* mice. c, reduction of liver stiffness, shown by rheometry (untreated: n=1; DEN+19xCCl<sub>4</sub>: n=4/group). d-e, pAKT, pERK, YAP, TAZ and GAPDH, detected by western blot (d), and TAZ IHC and nuclear translocation (e), in livers from the DEN+19xCCl<sub>4</sub> profound fibrosis model (macroscopic tumors not yet present) from *Colla1<sup>del</sup>* and *Colla1<sup>f/f</sup>* mice (n=6/group for d, n=9/group for e). f-g, HCC development (f) and hepatocyte proliferation (g) in mice with
hepatocyte-specific TAZ deletion (*Wwtr1*ΔHep, n=8–10) compared to *Wwtr1*fl/fl littermates (n=10) in the profound fibrosis DEN+44xCCl₄ HCC model. Using the same samples, immunoblots in **d** were done using one gel for YAP/TAZ and GAPDH and a second gel for pAKT, AKT, pERK1/2 and ERK. Data are shown as mean ± SEM, each data point represents one individual. Scale bars: 50 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
hepatocyte-specific TAZ deletion (*Wwtr1*ΔHep, n=8–10) compared to *Wwtr1*fl/fl littermates (n=10) in the profound fibrosis DEN+44xCCl₄ HCC model. Using the same samples, immunoblots in **d** were done using one gel for YAP/TAZ and GAPDH and a second gel for pAKT, AKT, pERK1/2 and ERK. Data are shown as mean ± SEM, each data point represents one individual. Scale bars: 50 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_89.png" /><figcaption>**Figure 5** | Promotion of hepatocarcinogenesis by DDR1.</figcaption></figure>

a, Western blot for DDR1 in healthy, non-tumour and tumour liver from different mouse models of HCC. b, DDR1 IHC in healthy human control livers (n=6) and in non-tumour and tumour areas from patients (TMA containing n=64 each), as well as *DDR1* mRNA expression in healthy controls (n=4), and non-tumor and tumor areas from HCC patients (n=16). c-d, HCC development (c) and Ki67+ hepatocytes (d) were assessed in mice with hepatocyte-specific DDR1 deletion (*Ddr1fl/fl*, n=12 and *Ddr1ΔHep*, n=9) in the DEN+CCl₄ profound fibrosis HCC model. e, Western blot for pDDR1, pAKT, DDR1, AKT and b-actin in HepG2 cells, plated on plastic or ECM. f, western blot for pDDR1, pAKT, DDR1, AKT and b-actin in Huh7 cells plated on plastic, or ECM and treated with or without DDR1 inhibitor 7rh. Data are shown as mean ± SEM, each datapoint represents one individual (a-d) or one well (e-f). Scale bars: 500 µm (b), 50 µm (d). Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
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b
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<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

b
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<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

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<figure><img src="image_100.png" /></figure>

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<caption>**Figure 5** | Promotion of hepatocarcinogenesis by DDR1.</caption>

a, Western blot for DDR1 in healthy, non-tumour and tumour liver from different mouse models of HCC. b, DDR1 IHC in healthy human control livers (n=6) and in non-tumour and tumour areas from patients (TMA containing n=64 each), as well as *DDR1* mRNA expression in healthy controls (n=4), and non-tumor and tumor areas from HCC patients (n=16). c-d, HCC development (c) and Ki67+ hepatocytes (d) were assessed in mice with hepatocyte-specific DDR1 deletion (*Ddr1fl/fl*, n=12 and *Ddr1ΔHep*, n=9) in the DEN+CCl₄ profound fibrosis HCC model. e, Western blot for pDDR1, pAKT, DDR1, AKT and b-actin in HepG2 cells, plated on plastic or ECM. f, western blot for pDDR1, pAKT, DDR1, AKT and b-actin in Huh7 cells plated on plastic, or ECM and treated with or without DDR1 inhibitor 7rh. Data are shown as mean ± SEM, each datapoint represents one individual (a-d) or one well (e-f). Scale bars: 500 µm (b), 50 µm (d). Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_90.png" /><figcaption>Figure 6 | CyHSC-enriched HGF protects hepatocytes from death and reduces HCC development.</figcaption></figure>

a, *Hgf* deletion in HSC promoted DEN+12xCCl₄-induced HCC development (n=10/group).

b, hepatocyte death assessed by TUNEL staining and ALT measurement as well as hepatocyte Ki67 IHC and *mKi67* RNA in *Hgf*ΔHSC and to *Hgf*fl/fl mice in the 6xCCl₄ model (n=4–8/group) and ALT measurement in mice on HF-CDAA-diet for 1 month (n=9/group).

c, Propidium iodide staining (red) in cultured murine primary hepatocytes treated with ActD+Jo2-induced +/− murine recombinant HGF (50 ng/µL) for 6h (n=3–5 wells/group).

d, *Hgf* mRNA expression in non-tumour (NT) and HCC tumour tissue (n=8 each) from mice after DEN+14xCCl₄ treatment compared to untreated liver (Ctrl, n=5).

e, *HGF* mRNA expression in normal liver (n=124), F0-F1 non-tumour (n=261), F2-F3 (n=115), F4 (n=182), dyplastic macronodules (DMN, n=31) and HCC (n=687) from patients.

f, survival of HCC patients with low *HGF*(n=280) or high *HGF*(n=280) expression. Data are shown as mean ± SEM, box plots represent interquartile range (IQR), Q1, median and Q3, each data point represents one individual. Scale bars 100 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.
<figure><img src="image_101.png" /><figcaption>Figure 6 | CyHSC-enriched HGF protects hepatocytes from death and reduces HCC development.</figcaption></figure>

a, *Hgf* deletion in HSC promoted DEN+12xCCl₄-induced HCC development (n=10/group).

b, hepatocyte death assessed by TUNEL staining and ALT measurement as well as hepatocyte Ki67 IHC and *mKi67* RNA in *Hgf*ΔHSC and to *Hgf*fl/fl mice in the 6xCCl₄ model (n=4–8/group) and ALT measurement in mice on HF-CDAA-diet for 1 month (n=9/group).

c, Propidium iodide staining (red) in cultured murine primary hepatocytes treated with ActD+Jo2-induced +/− murine recombinant HGF (50 ng/µL) for 6h (n=3–5 wells/group).

d, *Hgf* mRNA expression in non-tumour (NT) and HCC tumour tissue (n=8 each) from mice after DEN+14xCCl₄ treatment compared to untreated liver (Ctrl, n=5).

e, *HGF* mRNA expression in normal liver (n=124), F0-F1 non-tumour (n=261), F2-F3 (n=115), F4 (n=182), dyplastic macronodules (DMN, n=31) and HCC (n=687) from patients. *f*, survival of HCC patients with low *HGF* (n=280) or high *HGF* (n=280) expression. Data are shown as mean ± SEM, box plots represent interquartile range (IQR), Q1, median and Q3, each data point represents one individual. Scale bars 100 µm. Details of sample sizes, biological replicates and statistical tests are given in the Additional Information and Source Data.