Development and validation of a tumor-derived CXCL1 qPCR assay to support patient selection for anti-CXCL1 therapeutics in bladder cancer

Date:	June 2026
PMCID:	https://pmc.ncbi.nlm.nih.gov/articles/PMC13310799/
Category:	N/A
Authors:	Toru Sakatani, Sunao Tanaka, Kaoru Murakami, Cynthia Jinno, Francisco Aguilar, Catherine Bresee, Owen T M Chan, Daniel J Luthringer, Alberto Contreras-Sanz, Peter Black, Charles J Rosser, Hideki Furuya
Abstract:

The CXCR2-CXCL1 axis is critical in tumorigenesis, specifically, CXCL1 is a secreted chemokine implicated in tumor progression, angiogenesis, and immune modulation. Although elevated CXCL1 expression has been associated with poor outcomes in cancers, its evaluation by immunohistochemistry (IHC) is limited by diffusion and stromal expression. We developed a qPCR-based assay to objectively quantify CXCL1 mRNA in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. We analytically validated the CXCL1 qPCR assay using FFPE tissues from muscle-invasive bladder cancer (MIBC) patients. Assay performance was evaluated across preanalytical (tissue preparation, storage conditions), analytical (RNA input, quality, necrosis), and reproducibility parameters. The assay was then applied to a cohort of 47 MIBC patients who underwent radical cystectomy after neoadjuvant chemotherapy, and qPCR expression values were validated against RNA-seq TPM. The assay showed high concordance between fresh frozen and FFPE tissues (P = 0.78), stable RNA profiles under appropriate storage, and robust performance across a 5-100 ng input range when RNA DV200 exceeded 15%. Expression remained reliable in samples with up to 80% necrosis. Inter-operator reproducibility was strong (Pearson r = 0.80). In the clinical cohort, CXCL1 expression by qPCR showed excellent agreement with RNA-seq expression in 47 MIBC patients (Pearson r = 0.88), confirming the assay's robustness and cross-platform validity. We present a robust, analytically validated qPCR assay for quantifying CXCL1 expression in FFPE tumor specimens. The assay demonstrated excellent concordance with RNA-seq expression values, supporting its reliability for measuring CXCL1 mRNA expression in bulk tumor specimens and its potential utility as a companion diagnostic in future CXCR-2-CXCL1-targeted clinical trials.

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Acknowledgements:

The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute, or the National Institute of Health.

The Translational and Basic Science Research in Early Lesions (TBEL) Research Consortia is supported and funded by grants from the National Cancer Institute and the National Institutes of Health under the following award numbers:

Project Number:	Awardee Organization
U54CA274374	Fred Hutchinson Cancer Center
U54CA274375	Houston Methodist Research Institute
U54CA274370	Johns Hopkins University
U54CA274371	UT MD Anderson Cancer Center
U54CA274367	Vanderbilt University Medical Center