Abstract Tumor initiating cells (TICs) drive tumor initiation, growth, therapy resistance, and metastasis, yet their molecular characteristics remain poorly defined in clear cell renal cell carcinoma (ccRCC). ccRCC is the most common form of kidney cancer, leading to 179,000 cancer-related deaths annually. We aim to identify ccRCC-TICs and their essential genes using a cell marker-agnostic strategy.We performed scRNA-seq on five ccRCC patient-derived xenograft (PDX) models and applied RNA velocity and CytoTRACE analyses to identify malignant cells that are origin for other cancer cells in each tumor. We identified a minor cell population (less than 15% of cells) as the origin of other tumor cells, representing putative CICs. Pathway and network analyses on the genes that were specifically up-regulated in the origin cells, suggested that a core network of proteins, regulated by PLK1-FOXM1 axis, conventionally known to regulate mitosis, are highly active in TICs and may be essential for their function.We validated the role if these proteins in tumor initiation in PDX and 3D patient-derived models of ccRCC, through functional experiments. These proteins were expressed in spheroids established following a TIC enrichment protocol. Spheroid cells exhibited higher tumorigenicity and colony formation ability compared to parental tumor cells, as confirmed by in-vivo injection in nude mice and in-vitro colony formation assays. Successive in vivo passaging confirmed the self-renewal capacity of spheroid-derived tumors. Pharmacological blockade of PLK1 with Volasertib elicited dose-dependent inhibitory effect on spheroid and colony formation, with in-vivo validation showing that blocking PLK1 significantly delayed tumor growth and more efficiently prevented tumor formation in nude mice. Cultivation of tumor cells isolated from treated PDX demonstrated loss of self-renewal and tumor-propagating capacity in these cells. Notably, RCC-TICs remained vulnerable to PLK1 inhibition despite bypassing mitotic arrest using TCMps1-12, revealing that PLK1 elicit its TIC-related functions independently from its canonical G2/M regulatory roles. RNA-seq analysis of treated models identified pathways in adhesion, cytokine signaling, immune evasion, and developmental self-renewal circuits, as potential processes mediating PLK1 roles in RCC stemness.Overall, we identified and validated essential proteins in RCC-TICs using single-cell transcriptome data, RCC spheroids, and PDX models. Targeting PLK1 disrupts the fundamental self-renewal and tumor-initiating capacities of ccRCC, offering a promising avenue for therapeutic intervention through PLK1 inhibition. Furthermore, our data uncover novel and non-canonical functions for PLK1, which upon further characterization may open new ways for combinational therapeutic strategies involving PLK1 and its effectors with standard of care in advanced ccRCC. Citation Format: Zohreh Mehrjoo, Hellen Kuasne, Ariel M. Aguirre, Ozge Saatci, Ali Shahini, Matthew G. Annis, Anne-Marie N. Fortier, Tianyuan Lu, Larisa M. Soto, Hong Zhao, Dongmei Zuo, Virginie Pilon, Matthew Dankner, Tamiko Nishimura, Kevin Petrecca, Jonathan D. Spicer, Peter Siegel, Simon Tanguay, Hamed S. Najafabadi, Sahin Ozgur, Morag Park, Yasser Riazalhosseini. Defining cancer initiating cells and their vulnerabilities in renal cell carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2184.
Mehrjoo et al. (Fri,) studied this question.