Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma

Katia Mariniello¹, James F H Pittaway¹, Barbara Altieri², Kleiton Silva Borges^{3

4}, Irene Hadjidemetriou¹, Claudio Ribeiro^{3

4}, Gerard Ruiz-Babot^{3

5}, David S Tourigny⁶, Jiang A Lim¹, Julie Foster⁷, Julie Cleaver⁷, Jane Sosabowski⁷, Nafis Rahman⁸, Milena Doroszko⁸, Constanze Hantel⁹, Sandra Sigala¹⁰, Andrea Abate¹⁰, Mariangela Tamburello¹⁰, Katja Kiseljak-Vassiliades^{11

12}, Margaret Wierman^{11

12}, Charlotte Hall¹, Laila Parvanta¹³, Tarek E Abdel-Aziz¹⁴, Teng-Teng Chung¹⁵, Aimee Di Marco¹⁶, Fausto Palazzo¹⁶, Celso E Gomez-Sanchez¹⁷, David R Taylor¹⁸, Oliver Rayner¹⁸, Cristina L Ronchi¹⁹, Carles Gaston-Massuet¹, Silviu Sbiera², William M Drake¹, Emanuel Rognoni²⁰, Matthias Kroiss^{2

21}, David T Breault^{3

4}, Martin Fassnacht², Leonardo Guasti¹

Affiliations

¹ Centre for Endocrinology, William Harvey Research Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, London, UK.
² Division of Endocrinology and Diabetes, Dept. of Medicine, University Hospital, University of Würzburg, Würzburg, Germany.
³ Division of Endocrinology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
⁴ Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.
⁵ Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technical, University Dresden, Dresden, Germany.
⁶ School of Mathematics, University of Birmingham, Birmingham, UK.
⁷ Centre for Cancer Biomarkers and Biotherapeutics, Barts Cancer Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, UK.
⁸ Institute of Biomedicine, University of Turku, Turku, Finland.
⁹ Department of Endocrinology, Diabetology and Clinical Nutrition, University Hospital Zurich (USZ) and University of Zurich (UZH), Zurich, Switzerland.
¹⁰ Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
¹¹ Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, USA.
¹² Division of Endocrinology, Metabolism and Diabetes at Rocky Mountain Regional Veterans Affair Medical Center, Washington, District of Columbia, USA.
¹³ Department of Surgery, St Bartholomew's Hospital, West Smithfield, London, UK.
¹⁴ Department of Surgery, University College London Hospitals NHS Foundation Trust, London, UK.
¹⁵ Department of Endocrinology, University College London Hospitals NHS Foundation Trust, London, UK.
¹⁶ Department of Endocrine and Thyroid Surgery, Hammersmith Hospital, Imperial College London, London, UK.
¹⁷ Endocrine Section, G.V. (Sonny) Montgomery VA Medical Center and the Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, USA.
¹⁸ Department of Clinical Biochemistry (Synnovis Analytics), King's College Hospital, London, UK.
¹⁹ Institute of Metabolism and System Research College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
²⁰ Centre for Cell Biology & Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
²¹ Department of Internal Medicine IV, LMU University Hospital, LMU Munich, München, Germany.

PMID: 40035383
PMCID: PMC12187577
DOI: 10.1002/cac2.70012

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma

Katia Mariniello et al. Cancer Commun (Lond). 2025 Jun.

. 2025 Jun;45(6):663-668.

doi: 10.1002/cac2.70012. Epub 2025 Mar 4.

Authors

Affiliations

¹ Centre for Endocrinology, William Harvey Research Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, London, UK.
² Division of Endocrinology and Diabetes, Dept. of Medicine, University Hospital, University of Würzburg, Würzburg, Germany.
³ Division of Endocrinology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
⁴ Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.
⁵ Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technical, University Dresden, Dresden, Germany.
⁶ School of Mathematics, University of Birmingham, Birmingham, UK.
⁷ Centre for Cancer Biomarkers and Biotherapeutics, Barts Cancer Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, UK.
⁸ Institute of Biomedicine, University of Turku, Turku, Finland.
⁹ Department of Endocrinology, Diabetology and Clinical Nutrition, University Hospital Zurich (USZ) and University of Zurich (UZH), Zurich, Switzerland.
¹⁰ Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
¹¹ Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, USA.
¹² Division of Endocrinology, Metabolism and Diabetes at Rocky Mountain Regional Veterans Affair Medical Center, Washington, District of Columbia, USA.
¹³ Department of Surgery, St Bartholomew's Hospital, West Smithfield, London, UK.
¹⁴ Department of Surgery, University College London Hospitals NHS Foundation Trust, London, UK.
¹⁵ Department of Endocrinology, University College London Hospitals NHS Foundation Trust, London, UK.
¹⁶ Department of Endocrine and Thyroid Surgery, Hammersmith Hospital, Imperial College London, London, UK.
¹⁷ Endocrine Section, G.V. (Sonny) Montgomery VA Medical Center and the Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, USA.
¹⁸ Department of Clinical Biochemistry (Synnovis Analytics), King's College Hospital, London, UK.
¹⁹ Institute of Metabolism and System Research College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
²⁰ Centre for Cell Biology & Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
²¹ Department of Internal Medicine IV, LMU University Hospital, LMU Munich, München, Germany.

PMID: 40035383
PMCID: PMC12187577
DOI: 10.1002/cac2.70012

No abstract available

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Conflict of interest statement

The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.

Figures

**FIGURE 1**
**Dlk1 is an adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma in mice and humans**. (A) Schematic of *Dlk1^CreERT2/+* ; *Rosa^tdTomato/+* mice (*Dlk1Cre*) injected with tamoxifen for fate mapping experiments. Dlk1⁺ cells and their progeny were labelled with tdTomato (visualized with an anti‐Red Fluorescence Protein [RFP] antibody) upon tamoxifen injection. (B‐E) When dams were injected with tamoxifen at E12.5, and adrenals were analyzed at both P10 and P38, clusters and columns of RFP⁺/Sf1⁺ cells (representing Dlk1 progeny) spanned the entire width of the cortex in both males (D) and females (E). As expected, RFP was also detected in the medulla. (F) Dlk1 progeny were significantly decreased at P38 compared to P10, with females showing a small, non‐significant trend toward more Dlk1 progeny than males. (G) In *BPCre* mice, Dlk1 expression increased stepwise from non‐metastatic primary ACC to metastatic primary ACC and then to metastatic lesions. (H) Intense DLK1 expression was observed in lung metastases. (I) In humans, DLK1 expression was consistent in primary (upper left) and recurrent (lower right) tumors in the same patient. 19 secondary disease specimens were available from patients whose primary tumors were included in the study. (J) DLK1 expression level in secondary tumors positively correlated with those in primary tumors. (K) Categorizing DLK1 expression levels into quartiles (based on median and interquartile range values), higher DLK1 levels were associated with stepwise increase in the risk of disease recurrence (median RFS: low DLK1 = 32.5 months, low‐intermediate DLK1 = 18.5 months, high‐intermediate DLK1 = 15 months, and high DLK1 = 9 months). This was significant by the log‐rank test for trend across the four groups (χ² = 9.263) and when comparing high versus low DLK1 expression groups directly. (L) Higher DLK1 expression was associated with an increased risk of disease progression in ENSAT stage I & II disease (n = 57, median PFS: high DLK1 = 10 months versus low DLK1 = 27.5 months, HR 1.863, 95% CI = 1.038‐3.340). (M) In ENSAT stage III & IV groups, median PFS was comparable (n = 121, high DLK1 = 6 months versus low DLK1 = 7 months, HR = 1.159, 95% CI = 0.793‐1.694). There was no significant difference in mean Ki‐67% between ENSAT stage I & II group (20.24 ± 16.68) and stage III & IV group (19.57 ± 16.28) in this cohort. (N) Illustration of DLK1 structure, highlighting the ectodomain cleaved by TACE. (O‐Q) Serum Dlk1 levels were significantly higher in *BPCre* mice than in age‐matched controls. This was also observed in subcutaneous tumor mouse models injected with BPCre tumor‐derived BCH‐ACC3A cells (P) and H295R human ACC cells (Q). (R) In humans, pre‐operative serum DLK1 levels in the London prospective discovery cohort were significantly higher in ACC (16.81 ± 4.876ng/mL) than in benign adrenocortical adenomas (10.54 ± 4.417ng/mL). (S) Receiver operating characteristic (ROC) curve using all pre‐operative values demonstrated that serum DLK1 predicted ACC diagnosis with high accuracy (AUC 0.824 ± 0.072, P < 0.001). (T) In the Würzburg validation cohort, serum DLK1 levels were higher in patients with ENSAT stage IV disease than in those with recurrent disease following primary surgery (11.46 ± 1.459ng/mL versus 6.749 ± 3.016ng/mL), disease‐free patients (6.666 ± 2.855ng/mL), and patients with isolated primary tumors (11.46 ± 1.459ng/mL versus 7.357 ± 2.913ng/mL). (U) Following primary ACC resection, serum DLK1 levels significantly decreased compared to pre‐operative levels (mean decrease = 6.568 ± 2.565ng/mL). (V) Pre‐operative serum DLK1 levels significantly correlated with primary ACC DLK1 H‐score in the same patients. (W) Volcano plot of differentially expressed genes in DLK1⁺ versus DLK1⁻ tumor areas using spatial transcriptomics. Applying a fold‐change cutoff of > 2 or < 2, 10 genes were significantly upregulated, and 17 genes were significantly downregulated. Among the 9 upregulated genes (excluding *DLK1*), 5 were involved in cholesterol synthesis (*EBP*, *DHCR7*, *DHCR24*, *MSMO1*) and fatty acids metabolism (*FADS2*). Other upregulated genes included those involved in steroidogenesis (*CYP17A1*), vesicular and cholesterol binding (*SYP*), and cathepsin (*CTSA*) and clusterin (*CLU*). Downregulated genes included pro‐apoptotic genes (*BNIP3*, *BNIP3L*, *NR4A1*) and transcriptional regulators of differentiation (*EGR1*, *FOS*, *JUN*). (X) Western blot analysis showing increased DLK1 expression across the indicated ACC cell lines when cultured in 3D spheroid compared to 2D culture. (Y) H295R cells were fluorescence‐activated cell sorted (FACS) into DLK1⁺ and DLK1⁻ populations. DLK1⁺ cells generated significantly more colony‐forming units than DLK1⁻ cells after 21 days in culture. Data are displayed as individual points, with horizontal bars representing the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: E, embryonic day; EGF, epidermal‐like growth factor; Med, Medulla; P, postnatal day; PFS, progression‐free survival; RFS, recurrence‐free survival; sc, subcutaneous; TACE, TNFα converting enzyme.

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Update of

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma.
Mariniello K, Pittaway JFH, Altieri B, Borges KS, Hadjidemetriou I, Ribeiro C, Ruiz-Babot G, Lim JA, Foster J, Cleaver J, Sosabowski J, Rahman N, Doroszko M, Hantel C, Sigala S, Abate A, Tamburello M, Kiseljak-Vassiliades K, Wierman M, Parvanta L, Abdel-Aziz TE, Chung TT, Di Marco A, Palazzo F, Gomez-Sanchez CE, Taylor DR, Rayner O, Ronchi CL, Gaston-Massuet C, Sbiera S, Drake WM, Rognoni E, Kroiss M, Breault DT, Fassnacht M, Guasti L. Mariniello K, et al. bioRxiv [Preprint]. 2024 Aug 22:2024.08.22.609117. doi: 10.1101/2024.08.22.609117. bioRxiv. 2024. Update in: Cancer Commun (Lond). 2025 Jun;45(6):663-668. doi: 10.1002/cac2.70012. PMID: 39229217 Free PMC article. Updated. Preprint.

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Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma

Affiliations

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma

Authors

Affiliations

Conflict of interest statement

Figures

Update of

References

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