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Chronic liver disease enables gut Enterococcus faecalis colonization to promote liver carcinogenesis

Nature Cancer volume 2, pages 1039–1054 (2021)Cite this article

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Abstract

Gut dysbiosis is observed in chronic hepatobiliary diseases and is frequently associated with liver carcinogenesis; however, the extent and specific mechanisms triggered by alterations in the microbiota mediating tumorigenesis in these patients remain unclear. Here we show that Enterococcus faecalis is abundant in the microbiota of patients with hepatitis C virus-related chronic liver disease. Xenotransplantation of gut microbiota from these patients increased the number of spontaneous liver tumors in mice and enhanced susceptibility to liver carcinogens. Hepatic colonization by gelE-positive E. faecalis increased liver expression of proliferative genes in a TLR4–Myd88-dependent manner, leading to liver tumorigenesis. Moreover, decreased fecal deoxycholic acid levels were associated with colonization by E. faecalis. Overall, these data identify E. faecalis as a key promoter of liver carcinogenesis.

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Fig. 1: Gut microbiota of patients with chronic liver disease are taxonomically and functionally different from those of HD subjects.
Fig. 2: Fecal microbiota of patients with chronic liver disease promote liver carcinogenesis in mice.
Fig. 3: E. faecalis in the gut promotes liver carcinogenesis.
Fig. 4: GelE-positive E. faecalis promotes liver carcinogenesis.
Fig. 5: Feces from patients with chronic liver disease exhibit high levels of gelatinase activity, which is associated with increased gut permeability.
Fig. 6: Fecal microbiota from patients with chronic liver disease promote carcinogenesis in a NASH model.
Fig. 7: Chronic liver disease results in dysbiosis of microbiota and decreased production of DCA in the gut.
Fig. 8: Specific microbiota inhibit colonization of E. faecalis and the subsequent promotion of carcinogenesis.

Data availability

All sequencing data and metadata have been archived in NCBI SRA as BioProject nos. PRJNA517801 and PRJNA517981. The KEGG database v.56 utilized in HUMAnN2 is available at HUMAnN2: http://huttenhower.sph.harvard.edu/humann2. Greengene database v.13_8_99, utilized in MacQIIME, is available from MacQIIME: http://www.wernerlab.org/software/macqiime. Source data for all numerical data are provided as Source data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

HUMAnN2 v.0.1.9 (http://huttenhower.sph.harvard.edu/humann2), MetaPhlAn2 v.2.0.0 (http://huttenhower.sph.harvard.edu/metaphlan2), MacQIIME v..9.1 (http://www.wernerlab.org/software/macqiime), PRINSEQ v.0.20.4 (http://prinseq.sourceforge.net/manual.html), bowtie2 v.2.2.4 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), SAMtools v.1.2 (http://www.htslib.org/), DIAMOND v.0.7.5 (https://github.com/bbuchfink/diamond) and the LEfSe web version (https://huttenhower.sph.harvard.edu/lefse/) are available for analysis of metagenomic data.

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Acknowledgements

We thank M. Wakabayashi and M. Nagashimada for assistance with the animal experiments; A. Katayama, N. Nishiyama and K. Hara for assistance with histology experiments; and M. Hattori, K. Ohshima, K. Hosomochi, A. Tajima and S. Hashimoto for advice on sequencing analysis. M.S.G. and F.L. were supported by grants from the US National Institutes of Health (AI072360) and the Harvard-wide Program on Antibiotic Resistance (AI083214). This work was supported by JSPS KAKENHI grant JP25893081.

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Authors and Affiliations

  1. Department of Gastroenterology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan

    Noriho Iida, Eishiro Mizukoshi, Tatsuya Yamashita, Jun Seishima, Ziyu Wang, Kuniaki Arai, Hikari Okada, Taro Yamashita, Yoshio Sakai, Masao Honda & Shuichi Kaneko

  2. Department of Bacteriology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan

    Masahiro Yutani & Yukako Fujinaga

  3. Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan

    Yusuke Masuo, Rina Agustina & Yukio Kato

  4. Division of Genetics, Innovative Cancer Model Research Center, Kanazawa University, Kanazawa, Japan

    Masanobu Oshima

  5. Department of Ophthalmology; Department of Microbiology, Harvard Medical School, Massachusetts Eye and Ear, Boston, MA, USA

    François Lebreton & Michael S. Gilmore

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Contributions

N.I. and E.M. designed the entire study. N.I. performed experiments and data analysis and wrote the manuscript. M.Y. and Y.F. performed bacterial cultivation. J.S., H.O., M.H., H.O. and Z.W. performed mouse and bacterial experiments. Tatsuya Yamashita and K.A. performed acquisition and analysis of clinical data. Y.M., R.A. and Y.K. performed liquid chromatography–mass spectrometry experiments. F.L. and M.S.G. provided the gelE-deleted E. faecalis strain and interpreted the data. Taro Yamashita, Y.S. and M.S.G edited the manuscript. E.M. and S.K. supervised the study and edited the manuscript.

Corresponding author

Correspondence to Eishiro Mizukoshi.

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The authors declare no competing interests.

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Peer review information Nature Cancer thanks Tim Greten, Florencia McAllister and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Comparison of gut microbiota based on subjects-related variates.

(a) Chao1 was calculated from metagenomic taxonomy data. Boxes represent 25th-75th percentile of the distribution. The median is shown in the middle of the boxes. Whiskers represent min to max. The number of samples were 21–24 and 10 iterations were performed in the analysis. (b–h) Unweighted UniFrac distances were calculated with taxonomic data for fecal samples based on subjects-related variates including age (b), use of PPI/H2RA (c), BCAA (d), obesity (e), daily alcohol consumption (f), smoking (g) and cirrhosis (h). Distances were visualized by principal coordinates analysis. Significant differences between groups were determined by PERMANOVA, and P values and q value are shown at the bottom of the plots. (a-b) LDA was performed with relative abundance of taxa in the feces. (i) HD subjects (n=24) and CLD/HCC subjects (n=23) without treatment of PPI, H2RA or BCAA were compared by LDA. (j) Ages were matched between HD subjects (n=13) and CLD/HCC subjects (n=36) at over 55 years old and compared by LDA. LDA scores with P value < 0.05 are shown in the graphs. q, adjusted P value by false discovery rate method; PPI, proton pump inhibitors; H2RA, H2 receptor antagonists; BCAA, branched-chain amino acids. (a), Kruskal Wallis test;. *, P < 0.05; **, P < 0.01. Exact P-values were as follows: (a) HD vs. CLD, P>0.999; HD vs. HCC, P=0.0045; CLD vs. HCC, P=0.0143.

Source data

Extended Data Fig. 2 ß-diversity analysis of the mouse feces after the fecal transplantation from the human subjects.

(a) Microbiota-depleted mice received the fecal microbiota from the subjects HD2, CLD1, CLD6, HCC8 or HCC14. Unweighted UniFrac distances were calculated with taxonomy data in feces of the mice at various time points after the fecal transplantation. The results are visualized by principal coordinate analysis. d, day; M, months. (b) Chao1 was calculated from taxonomy data. Boxes represent 25th-75th percentile of the distribution. The median is shown in the middle of the boxes. Whiskers represent min to max. The number of samples were 21–24 and 10 iterations were performed in the analysis. (c to f) Germ-free or antibiotics-pretreated mice received feces of thee subjects HD50, CLD9 and HCC11 (c), HD55, CLD13 and HCC12 (d), and HD59, CLD10 and HCC13 (e). Unweighted UniFrac distances were calculated with taxonomy data in feces of the mice and visualized. (f) UniFrac distances between the subjects and feces-transplanted germ-free mice, and between those and feces-transplanted antibiotics-pretreated mice were compared. ABX, antibiotics; FT, feces-transplanted. n=9. (b), Kruskal Wallis test; (f), two-sided Mann-Whitney U test. *, P < 0.05; ***, P < 0.001. Exact P-values were as follows: (b) HD vs. CLD, P<0.0001; HD vs. HCC, P<0.0001; CLD vs. HCC, P=0.0139: (f) P=0.0244.

Source data

Extended Data Fig. 3 The number of liver tumors and gene expression in the liver of in individual mouse that received fecal transplantation from the human subjects.

Fecal transplantation from the HD, CLD, or HCC subjects was performed in microbiota-depleted mice and followed by injection of DEN and CCl4 to induce liver carcinogenesis. (a) The mice were euthanized in 8 months after the DEN injection. The number of liver tumors per mouse is shown. (b) The mice were euthanized after injection of DEN once and CCl4 twice. mRNA expression of indicated genes was shown. These figures a and b correspond to individual plots of Fig. 2c, d, respectively.

Source data

Extended Data Fig. 4 Liver gene expression after transplantation of the subjects’ feces to germ-free mice.

Germ-free mice received fecal transplantation from the selected subjects, HD50, CLD9 and HCC11 (a and d), HD55, CLD13, HCC12 (b), HD59, CLD10, and HCC13 (c). (a–c) Then, after DEN injection once followed by CCl4 injection twice, the mice were euthanized and mRNA expression in the liver was analyzed by real-time PCR. n=4–5. (d) The mice received DEN injection twice followed by CCl4 injection and were euthanized in 8 months after the DEN injection. n=5. Means and SE are shown. GF, germ-free. (a–c), one-way ANOVA followed by Tukey’s test; (d), Kruskal-Wallis test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Exact P-values were as follows: (a) mki67 HD50 vs. CLD9, P=0.0073; HD50 vs. HCC11, P=0.0072; HD50 vs. GF, P=0.998; CLD9 vs. HCC11, P>0.999; CLD9 vs.GF, P=0.0054; HCC11 vs. GF, P=0.0053: ccnb1 HD50 vs. CLD9, P=0.0314; HD50 vs. HCC11, P=0.0209; HD50 vs. GF, P=0.999; CLD9 vs. HCC11, P=0.996; CLD9 vs.GF, P=0.0378; HCC11 vs. GF, P=0.0252: col1a1 HD50 vs. CLD9, P=0.433; HD50 vs. HCC11, P=0.181; HD50 vs. GF, P=0.303; CLD9 vs. HCC11, P=0.932; CLD9 vs.GF, P=0.0186; HCC11 vs. GF, P=0.0056: tnf HD50 vs. CLD9, P=0.151; HD50 vs. HCC11, P=0.0219; HD50 vs. GF, P=0.0432; CLD9 vs. HCC11, P=0.736; CLD9 vs.GF, P=0.0005; HCC11 vs. GF, P<0.0001: birc5 HD50 vs. CLD9, P=0.0053; HD50 vs. HCC11, P=0.010; HD50 vs. GF, P=0.998; CLD9 vs. HCC11, P=0.989; CLD9 vs.GF, P=0.0040; HCC11 vs. GF, P=0.0075: (b) mki67 HD55 vs. CLD13, P=0.0011; HD55 vs. HCC12, P=0.0003; CLD13 vs. HCC12, P=0.507: ccnb1 HD55 vs. CLD13, P=0.0031; HD55 vs. HCC12, P<0.0001; CLD13 vs. HCC12, P=0.045: col1a1 HD55 vs. CLD13, P=0.146; HD55 vs. HCC12, P=0.248; CLD13 vs. HCC12, P=0.929: tnf HD55 vs. CLD13, P=0.122; HD55 vs. HCC12, P=0.757; CLD13 vs. HCC12, P=0.339: birc5 HD55 vs. CLD13, P=0.0174; HD55 vs. HCC12, P<0.0001; CLD13 vs. HCC12, P=0.0047: (c) mki67 HD59 vs. CLD10, P=0.455; HD59 vs. HCC13, P=0.0205; CLD10 vs. HCC13, P=0.172: ccnb1 HD59 vs. CLD10, P=0.692; HD59 vs. HCC13, P=0.100; CLD10 vs. HCC13, P=0.354: col1a1 HD59 vs. CLD10, P=0.305; HD59 vs. HCC13, P=0.177; CLD10 vs. HCC13, P=0.929: tnf HD59 vs. CLD10, P=0.365; HD59 vs. HCC13, P=0.0403; CLD10 vs. HCC13, P=0.383: birc5 HD59 vs. CLD10, P=0.383; HD59 vs. HCC13, P=0.0467; CLD10 vs. HCC13, P=0.405: (d) HD50 vs. CLD9, P=0.303; HD50 vs. HCC11, P=0.019; CLD9 vs. HCC11, P=0.839.

Source data

Extended Data Fig. 5 Abundance of Enterococcus species in feces of the subjects.

Copy number of E. faecalis (a, d, e), E. faecium (b), and E. casseliflavus (c) in feces of the subjects was determined by quantitative PCR. PPI-untreated subjects or subjects over 55 years old were analyzed in (d, e), respectively. n=21–24 (a-c), n=13–23 (d–f) Number of liver tumors in E. faecalis-bearing mice is shown according to presence of fecal gelE. n=13–50. (g–j) Relative abundance (%) of Bacillus (g), Staphylococcus (h), Pseudomonas (i), and Serratia (j) in feces of the mice transplanted with human feces was determined by 16SrRNA-based sequencing. n=21–24. (a–j) Means and SE are shown. (a–e and g–j) Kruskal-Wallis test. (f) two-sided Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Exact P-values were as follows: (a) HD vs. CLD, P=0.0007; HD vs. HCC, P=0.0002; CLD vs. HCC, P>0.999: (b) HD vs. CLD, P=0.206; HD vs. HCC, P=0.229; CLD vs. HCC, P>0.999: (c) HD vs. CLD, P>0.999; HD vs. HCC, P=0.238; CLD vs. HCC, P>0.999 (d) HD vs. CLD, P=0.0068; HD vs. HCC, P=0.0031; CLD vs. HCC, P>0.999: (e) HD vs. CLD, P=0.0014; HD vs. HCC, P=0.0019; CLD vs. HCC, P>0.999: (f) P=0.049: (g) HD vs. CLD, P>0.999; HD vs. HCC, P=0.0587; CLD vs. HCC, P=0.151: (h) HD vs. CLD, P>0.999; HD vs. HCC, P>0.999; CLD vs. HCC, P>0.999: (i) HD vs. CLD, P=0.100; HD vs. HCC, P=0.318; CLD vs. HCC, P>0.999: (j) HD vs. CLD, P>0.999; HD vs. HCC, P=0.657; CLD vs. HCC, P=0.704.

Source data

Extended Data Fig. 6 Deletion of gelE results in decreased colonization in the gut.

(a) Inoculation of E. faecalis V583 wild-type (WT) or ∆gelE strains was performed orally and rectally in germ-free or mice whose microbiota were depleted by antibiotics . CFU of V583 in feces was counted 8 days after the inoculation. Means and SE are shown. n=4. Four independent experiments were combined. One-way ANOVA followed by Tukey’s test. ***, P < 0.001. (b) The indicated E. faecalis strains were cultivated on gelatin-containing agar plates and gelatinase activity was evaluated. (c) After inoculation of E. faecalis V583 WT or ∆gelE strains in germ-free mice, the mice received DEN injection twice followed by CCl4 injection and were euthanized in 8 months after the DEN injection. Number of liver tumors is shown. Kruskal-Wallis test. n=5–7. (d) Homogenized liver harvested from V583 WT- or ∆gelE–inoculated microbiota-depleted mice was cultivated and CFU count was obtained. n=5. Representative of 2 independent repeats with similar results. Means and SE are shown. Mann-Whitney U test. Exact P-values were as follows: (a) GF + V583WT vs. GF + V583ΔgelE, P=0.998; GF + V583WT vs. microbiota-depleted + V583WT, P<0.0001; GF + V583WT vs. microbiota-depleted + V583ΔgelE, P<0.0001; GF + V583ΔgelE vs. microbiota-depleted + V583WT, P<0.0001; GF + V583ΔgelE vs. microbiota-depleted + V583ΔgelE, P<0.0001; microbiota-depleted + V583WT vs. microbiota-depleted + V583ΔgelE, P=0.0021.

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Extended Data Fig. 7 Effects of E. faecalis colonization on liver tumor in Myd88-/- and Tlr4-/- mice.

Inoculation of E. faecalis V583 wild-type (WT) strains was performed orally and rectally in microbiota-depleted WT or Myd88-/- mice (a, b) and Tlr4-/-, Tlr4floxLyz2cre mice (c, d). (a) the mice received DEN injection twice followed by CCl4 injection and were euthanized in 8 months later. Number of liver tumors is shown. n=7. (b, c) CFU of E. faecalis strain V583 in feces of WT, Myd88-/- (a), or Tlr4-/- (b) after inoculation was determined. n=4–6. Four independent experiments were combined. (d) After DEN injection once followed by CCl4 injection twice, the mice were euthanized and mRNA expression in the liver was analyzed by real-time PCR. n=8–9. Representative of 2 independent repeats with similar results. (a–d) Means and SE are shown. (a), two-sided Mann-Whitney U test; (b–d), one-way ANOVA followed by Tukey’s test. *, P < 0.05; **, P < 0.01. ***, P < 0.001. Exact P-values were as follows: (a) P=0.0017: (b) WT mice + V583WT vs. WT mice + V583ΔgelE, P=0.0003; WT mice + V583WT vs. Myd88-/- mice + V583WT, P=0.962; WT mice + V583WT vs. Myd88-/- mice + V583ΔgelE, P=0.002; WT mice + V583ΔgelE vs. Myd88-/- mice + V583WT, P=0.0001; WT mice + V583ΔgelE vs. Myd88-/- mice + V583ΔgelE, P=0.845; Myd88-/- mice + V583WT vs. Myd88-/- mice + V583ΔgelE, P=0.0007: (d) mki67 Tlr4fl/flLyz2wt/wt vs. Tlr4fl/flLyz2cre/wt, P=0.417; Tlr4fl/flLyz2wt/wt vs. Tlr4-/-, P=0.0091; Tlr4fl/flLyz2cre/wt vs. Tlr4-/-, P=0.151: ccnb1 Tlr4fl/flLyz2wt/wt vs. Tlr4fl/flLyz2cre/wt, P=0.836; Tlr4fl/flLyz2wt/wt vs. Tlr4-/-, P=0.0029; Tlr4fl/flLyz2cre/wt vs. Tlr4-/-, P=0.0135: col1a1 Tlr4fl/flLyz2wt/wt vs. Tlr4fl/flLyz2cre/wt, P=0.888; Tlr4fl/flLyz2wt/wt vs. Tlr4-/-, P=0.389; Tlr4fl/flLyz2cre/wt vs. Tlr4-/-, P=0.676: birc5 Tlr4fl/flLyz2wt/wt vs. Tlr4fl/flLyz2cre/wt, P=0.531; Tlr4fl/flLyz2wt/wt vs. Tlr4-/-, P=0.0057; Tlr4fl/flLyz2cre/wt vs. Tlr4-/-, P=0.0726: tnf Tlr4fl/flLyz2wt/wt vs. Tlr4fl/flLyz2cre/wt, P=0.988; Tlr4fl/flLyz2wt/wt vs. Tlr4-/-, P=0.951; Tlr4fl/flLyz2cre/wt vs. Tlr4-/-, P=0.987.

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Extended Data Fig. 8 Effect of E. faecalis is not based on initiation of carcinogenesis by DNA damage.

(a, b) After transplantation of feces of HD, CLD, or HCC, or inoculation of E. faecalis strain V583, DEN was injected. One day later liver was harvested and stained with anti-phospho-H2AX antibody. (a) Representative pictures of phospho-H2AX staining from 2 independent experiments are shown. Bars, 50µm. (b) Percentages of phospho-H2AX-positive nuclei were compared between HD, CLD and HCC, or V583 WT and gelE-deleted V583. n=24 or 12, respectively. (c) After inoculation of V583, CCl4 was injected with or without DEN, and 8 months later number of liver tumors was counted. Means and SE are shown. n=12. (b) Kruskal-Wallis test for 3 groups and two-sided Mann-Whitney U test for 2 groups. (c) two-sided Mann-Whitney U test. ***, P < 0.001. Exact P-values were as follows: (b) HD vs. CLD, P>0.999; HD vs. HCC, P>0.999; CLD vs. HCC, P>0.999: V583WT vs. V583ΔgelE, P=0.932: (c) P<0.0001.

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Extended Data Fig. 9 Aberrant localization of tight junction protein ZO-1 in the colon of the mice colonized with gelE-positive E. faecalis.

Inoculation of E. faecalis V583 wild-type (wt) or ∆gelE strains (a–e, and g) or fecal transplantation from HD, CLD and HCC (f) was performed orally and rectally in microbiota-depleted WT or F2rl1-/- mice. After DEN injection once followed by CCl4 injection twice, colon was harvested. (a–d) Colon tissues were stained with anti-ZO-1 antibody (green) and DAPI (blue). Bar, 100µm. Arrows indicate internalization of ZO-1 in the epithelial cells. Representative pictures from 2 independent experiments are shown. (e) Immunoblotting of colonic protein was performed with anti-ZO-1 antibody or anti-GAPDH antibody. Representative pictures from 2 independent experiments are shown. (f, g) mRNA expression of colon tissues were determined by PCR. n=16, 15, 16 and 6 for HD, CLD, HCC and ABX in (f), biologically independent samples. n=9 mice per group in (g) and two independent experiments were combined. (h) CFU of E. faecalis strain V583 in feces of WT and F2rl1-/- mice after inoculation was determined. n=4–6. Four independent experiments were combined. (f to h) Means and SE are shown. (f and h), one-way ANOVA followed by Tukey’s test; (g), two-sided Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Exact P-values were as follows: (f) tnf HD vs. CLD, P=0.221; HD vs. HCC, P=0.0025; HD vs. ABX, P=0.997; CLD vs. HCC, P=0.309; CLD vs. ABX, P=0.365; HCC vs. ABX, P=0.023: il1b HD vs. CLD, P=0.213; HD vs. HCC, P=0.0099; HD vs. ABX, P=0.966; CLD vs. HCC, P=0.592; CLD vs. ABX, P=0.230; HCC vs. ABX, P=0.0283: il6 HD vs. CLD, P=0.472; HD vs. HCC, P=0.0201; HD vs. ABX, P=0.994; CLD vs. HCC, P=0.434; CLD vs. ABX, P=0.546; HCC vs. ABX, P=0.0749: (g) tnf V583WT vs. V583ΔgelE, P=0.0002: il1b V583WT vs. V583ΔgelE, P=0.0028: il6 V583WT vs. V583ΔgelE, P=0.297. ABX, antibiotics; GF, germ-free.

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Extended Data Fig. 10 Fibrosis, gene expression and tumor number in the liver of E. faecalis-inoculated NASH mouse model.

Streptozotocin was subcutaneously injected on 2 days after birth and high-fat diet was started in 4 week-old of age. Fecal transplantation from the HD, CLD, or HCC subjects (a, b) or inoculation of E. faecalis strain V583 (b–d) was performed after depletion of microbiota in 6 week-old of age. The mice were euthanized in 17 week-old of age. (a) Representative images of the murine liver after Sirius Red staining. Bar, 100µm. (b) Area of fibrosis was measured as Sirius-red-positive area and shown. n=11–12 or 6. (c) The number of liver tumors per mouse is plotted. n=6. (d) mRNA expression in the liver was analyzed by real-time PCR. n=6. Representative of 2 independent repeats with similar results. (e) Plasma LPS level was shown. n=6. Means and SE are shown. (b), left, Kruskal-Wallis test. Right, two-sided Mann-Whitney U test; (c–e), two-sided Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Exact P-values were as follows: (b) HD vs. CLD, P=0.0304; HD vs. HCC, P<0.0001; CLD vs. HCC, P=0.176: V583WT vs. V583ΔgelE, P=0.0260: (c) V583WT vs. V583ΔgelE, P=0.0286: (d) mki67, P=0.0022: ccnb1, P=0.0152: col1a1, P=0.132: birc5, P=0.0411: tnf, P=0.0260: ccl2, P=0.0411: (e) P=0.0022.

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Supplementary information

Supplementary Information

Supplementary Tables 1–9 and Methods.

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Iida, N., Mizukoshi, E., Yamashita, T. et al. Chronic liver disease enables gut Enterococcus faecalis colonization to promote liver carcinogenesis. Nat Cancer 2, 1039–1054 (2021). https://doi.org/10.1038/s43018-021-00251-3

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