Background Wilms tumour (WT) relapse occurs more frequently in patients with blastemal-type WTs. The presence of cancer stem cells (CSCs) is linked to tumour survival and relapse, and CSCs may be found in greater numbers in blastemal cell foci. CSC-associated phenotypes have been described in untreated WT, but their persistence, organisation and relevance after neoadjuvant chemotherapy is unknown.

Methods We analysed 23 formalin-fixed paraffin-embedded blocks from 18 chemotherapy-treated patients where WTs were enriched for viable blastema, using human fetal kidney as developmental control. Immunohistochemistry and -fluorescence analysis determined progenitor (PAX2, SIX2, CITED1) and CSC-associated (NCAM, ALDH1, CD133) marker expression. We qualitatively and semi-quantitatively evaluated spatial expression patterns and co-localisation across tumour compartments.

Results PAX2 and SIX2 were co-expressed in blastema in most cases (15/18), with PAX2 expression higher at the periphery of blastemal foci and SIX2 expression found uniformly in central aspects. CITED1 expression was also associated with SIX2 in blastema tissues (14/18). NCAM was blastema-enriched (15/18) with higher central intensity, frequently adjacent to PAX2-expressing peripheral zones. ALDH1 expression was present across blastema and epithelium while NCAM-, ALDH1-double-positive cells were rarely observed (4/18). CD133 expression was less commonly seen (2/18), localising near epithelial/nephrogenic structures.

Conclusions After neoadjuvant chemotherapy, WT blastema retained overlapping but non-identical progenitor/CSC-associated marker landscapes with reproducible peripheral–centre gradients. These spatial arrangements suggest a blastemal niche for CSCs that may sustain a therapy-resistant state. Our analysis provides the foundation for future functional validation and molecular profiling to define key lineage relationships and therapeutic vulnerabilities in post-chemotherapy WT. [250/250 words]

The authors have declared no competing interest.

This work was supported by project grant NWCRKRNW2021.02 funded by Northwest Cancer Research and Kidney Research Northwest, and by project grant CCLGA 2023 17 Wilm, funded by the Little Princess Trust UK CCLG.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Parent/guardian informed consent was in place for the specimens collected from 18 WT index patients and the human foetal kidney specimen. Ethical approval of the study was provided by the UK VIVO Biobank Sample and Data Access Committee (date: 29.3.2023, project number 23-VIVO-10, project title: 'Investigating whether chemotherapy-induced senescence affects the behaviour of cancer stem cells in Wilms Tumour') and the London Bridge Research Ethics Committee for the UK IMPORT study for renal tumours of childhood (dates: 2.5.2012; 3.6.2019 and 18.11.2022, REC reference 12/LO/0101, title: 'Improving population outcomes for renal tumours of childhood - applying P-medicine tools to improve treatment decisions for Wilms tumour and other renal tumours of childhood and young adults: a SIOP renal tumours study group prospective clinical study'), while for the foetal human kidney specimen we obtained approval from the London South East Research Ethics Committee (date: 28 April 2025, REC reference 25/PR/0493, London South East Research Ethics Committee, title: Control Samples for Analysis of Wilms Tumour tissues).

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