Mapping the spatiotemporal dynamics of CD44v9 reveals a senescent cell therapy-resistant niche in pancreatic cancer

Background: Despite decades of research and advances in cancer therapeutics, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with limited therapeutic response and a profoundly fibrotic and immunosuppressive tumor microenvironment (TME). Alternative mRNA splicing of cell adhesion molecule Cluster of Differentiation (CD) 44 transmembrane glycoprotein (CD44) generates variant isoform (CD44v) CD44v9. CD44v9 stabilizes the cystine/glutamate antiporter SLC7A11, enhancing glutathione synthesis to buffer reactive oxygen species and chemotherapeutic resistance. Although CD44v9 is a known cancer stem cell (CSC) marker and has been linked to chemotherapy resistance, a comprehensive spatiotemporal analysis of CD44v9 expression in the context of disease progression has not been completed. Objective: To identify CD44v9 as a potential therapeutic target and driver of chemotherapy resistance, progression of disease, and metastasis. Methods: The spatiotemporal dynamics of CD44v9 expression were measured using PDAC patient longitudinal tumor tissues pre- and post-SOC chemotherapy using the Rarecyte Orion ™ Multiplex Immunofluorescence (MxIF) and CosMx ™ Whole Transcriptome Spatial Molecular Imager (CosMx ™ WTX SMI). MxIF images were curated into Minerva Stories (https://uga-som-imtm.github.io/170-8T-211-09T/) for interactive data. Patient-derived organoids (PDO) harboring cancer associated fibroblasts (CAFs) generated from matched longitudinal tissue samples were used to identify the therapeutic benefit of targeting CD44v9. Four novel CD44v9-directed antibody-drug conjugates (CD44v9-ADCs; Abzena), each designed to target senescence or antioxidant defense were tested alone and in combination with standard-of-care (SOC) chemotherapeutic drugs. Results: CosMx ™ WTX SMI of longitudinal collected tumor tissues showed an increased expression of CD44 positive cells within cell niches. MxIF of matched tissue sections revealed expression of CD44v9 within the same CD44 positive spatially identified cell population. CD44v9+ CSCs exhibited a senescent-associated secretory phenotype with high expression of MIF and CEACAM genes, and resided within niches dense with cancer associated fibroblasts, Schwann cells, and an immune suppressive cells signature. Combination treatment of PDOs with CD44v9-ADCs resulted in sensitization to SOC-induced tumor cell death, as measured by high-content confocal microscopy by nuclear irregularity index, propidium iodide uptake and decreased circumference. Conclusion: The expression of CD44v9 marks a CSC population that persists within the patient PDAC TME post-SOC. CD44v9-directed ADCs, carrying senolytic and antioxidant defense-inhibiting payloads, are a potential therapeutic approach to prevent disease progression, recurrence, and metastasis.