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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

We also compared our mouse EMT signatures to human pan-cancer EMT programs (Gavish et al., Nature 2023). The mouse late EMT state closely matched the human "complete EMT" program, and PITX1/KLF5 vs. NFATC1/CREB3L1 activity patterns were conserved too. (16/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

Is this specific to skin cancer? We re-analyzed a public mouse pancreatic adenocarcinoma dataset, and found the same logic: Klf5 marks epithelial/early states, Nfatc1 and Creb3l1 mark late EMT. Same regulatory grammar, different tumor type. (15/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

On the other side, Nfatc1 and Creb3l1 were predicted (and confirmed by immunostaining) to be active specifically in hybrid/late EMT cells. Knocking them out blocked progression to late EMT, cells got stuck in earlier, more epithelial states. (12/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

Even more striking: Pitx1 and Klf5 knockout tumors formed far fewer lung metastases. So these "early EMT" TFs aren't just markers of an epithelial state, they're functionally required for efficient metastatic colonization. (11/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

Using CRISPR/Cas9, we knocked out Pitx1 and Klf5 in tumor cells. Both are normally expressed in epithelial + early hybrid EMT cells. Their loss caused a strong shift toward more advanced (hybrid) EMT states, both in vitro and in transplanted tumors in vivo. (10/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

In silico knockout simulations predicted that losing early regulators (Klf5, Pitx1) pushes cells toward more mesenchymal states, while losing late regulators (Nfatc1, Creb3l1) pulls cells back toward epithelial states. Time to test this for real. (9/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

This let us build a predicted TF-TF interaction network: epithelial TFs (Grhl1, Trp63, Klf5, Pitx1) form a tight circuit that gets repressed by Zeb1 as cells transition, while late-EMT TFs (Nfatc1, Ebf1, Creb3l1) get switched on downstream. (8/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

We then added single-cell multiome (RNA + ATAC together) to map the chromatin landscape, which enhancers open and close as cells move through EMT, and combined it with SCENIC+ to build enhancer-driven gene regulatory networks (eRegulons). (7/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

To find the TFs driving these transitions, we ran SCENIC (gene regulatory network inference) on the single-cell data. This recovered known regulators (Grhl2, Zeb1, Twist1...) but also flagged new candidates: Klf5, Pitx1, Nfatc1, Creb3l1. (6/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 13/07/2026

Unsupervised clustering revealed 4 discrete EMT states: Epithelial → Early hybrid → Hybrid → Late EMT. Each has its own gene signature, and we confirmed all 4 exist at the protein level by immunostaining in tumors. (4/20)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 27/03/2025

Finally, we used this dataset to run the first single-cell benchmark of inferCNV, one of the most commonly used tools for inferring CNAs from single-cell transcriptome data, and discovered that these inference methods have limited accuracy. (7/9)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 27/03/2025

For the first time, we used matched transcriptome data to study gene dosage effects at single-cell resolution. We found a link between the 22q11.21 amplicon & drug-tolerant cell state plasticity (6/9)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 27/03/2025

Using Gtag&T, we uncovered small focal alterations on chromosomes 13 & 22 that were detectable at single-cell resolution. These differences in size, copy-number & structure were specific to different cancer subclones (5/9)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 27/03/2025

We applied Gtag&T-seq to a melanoma xenograft model before, during, and after treatment. Our findings reveal important differences in cell state plasticity and treatment response across cancer subclones (4/9)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 27/03/2025

Gtag&T significantly lowers the cost of single-cell genome & transcriptome sequencing while improving coverage uniformity compared to the original G&T-seq. More bang for your research buck! (3/9)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 27/03/2025

In this paper, we present #Gtag&T-seq — a revamped version of G&T-seq that enables simultaneous profiling of genome & transcriptome from the same single cell using direct genomic tagmentation (2/9)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

In addition, NP137 therapy in mice transplanted with human lung cancer cells previously treated with TGFB1 (to induce EMT) decreases EMT in cancer cells (20/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

Finally, we examined the human and clinical relevance of our findings by correlating netrin-1 and UNC5B expression with several EMT signatures in human cancers. Although there was no correlation for netrin-1, there was a strong association for UNC5B expression and EMT (19/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

A large overlap of genes down- or upregulated in both conditions was revealed by bulk RNA-seq of Ntn1-KD and Unc5b-KD tumour cells, indicating that netrin-1 and UNC5B regulate similar transcriptional programmes and signalling pathways (18/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

EPCAM+ tumour cells isolated from our mouse model go through EMT in vitro. Ntn1 or Unc5b knockdown significantly reduced this ability. Furthermore, we saw a decrease in cell migration that was not exacerbated by NP137 administration (17/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

Krt15 expression was high in the epithelial-B1 state, and GO term analysis of epithelial-B2 marker genes revealed increased glycolysis and keratinization (15/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

In the control tumour, lineage trajectory analysis revealed two distinct lineage trajectories, one from epithelial cells to hybrid EMT and the other from epithelial cells to late full EMT (13/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

In terms of EMT states, NP137 administration increased the proportion of the epithelial state while inhibiting the occurrence of the late EMT state, thereby blocking EMT progression at the hybrid EMT state (12/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

Following netrin-1 inhibition, we found a significant reduction in the expression of genes associated with EMT, hypoxia, angiogenesis, and inflammatory response across all tumour cells (11/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

The effect of NP137 treatment on the tumour microenvironment was studied using 10x scRNA-seq. In treated mice, we discovered a significant decrease in tumour cells and an increase in CAFs (10/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

NP137 treatment in combination with cisplatin and 5-FU decreased the resistance to chemotherapy in tumours with EMT (9/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

Furthermore, this pharmacological inhibition of netrin-1 reduced the number of metastasis, demonstrating that NP137 treatment directly inhibits metastasis formation (8/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

Administration of NP137, a blocking antibody targeting Netrin-1 treatment reduced the number of tumours per mouse as well as the proportion of tumour cells that underwent EMT (7/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

We first tested the role of Netrin-1 in promoting EMT by overexpressing it in our mouse model. Netrin1 overexpression results in an increase in the number of tumours per mouse and the proportion of tumour cells undergoing EMT (6/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

Our primary focus was on secreted factors predominantly expressed by mesenchymal tumour cells, with specific therapies already in use for human cancer treatment. Our most promising discovery was Netrin-1 (Ntn1) and its receptor Unc5b. (4/23)

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Sebastiaan (Seb) Vanuytven @svanuytven.eurosky.social · 19/11/2024

In our quest for potential novel therapies, we performed RNA-seq on tumour cells from the skin squamous cell carcinoma model, previously characterised in the ground-breaking work of the fantastic Prof. Pastushenko and the 'bioinfomagician' Prof. Sifrim in 2018 (3/23)

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