Patient-derived organoid-immune cell co-cultures and spatial biology support a CD44v9 cancer stem cell-directed therapeutic strategy for adamantinomatous craniopharyngioma

Background: Craniopharyngiomas are rare epithelial tumors that are categorized as adamantinomatous craniopharyngioma (ACP) or papillary craniopharyngioma (PCP) subtype with distinct histological, pathological and genetic differences. The treatment of craniopharyngioma remains a medical challenge and is associated with the complex clinical outcomes that are related to the unique and undefined tumor biology. Significant advancement has been made for the treatment of PCPs. While patients with PCPs often respond well to BRAF-MEK inhibitors, the absence of the BRAF V600E mutation in ACPs presents a challenge. Alternative mRNA splicing of Cluster of Differentiation (CD) 44 transmembrane glycoprotein (CD44) generates the variant isoform CD44v9. Expressed in several tumors including craniopharyngiomas, CD44v9 is a known cancer stem cell (CSC) marker that regulates tumor cell proliferation, metastasis and chemotherapy resistance. Early studies also propose the potential therapeutic benefit of targeting PD-L1 and/or PD-1 in ACPs. Objective: The potential therapeutic benefit of targeting PD-L1 and/or PD-1, and CD44v9 for the treatment of ACPs was identified. Method: Rarecyte Orion ™ Multiplex Immunofluorescence (MxIF) and CosMx ™ Whole Transcriptome Spatial Molecular Imager (CosMx ™ WTX SMI) was performed using tumor tissues collected from patients with ACPs and PCPs. Craniopharyngioma patient-derived organoids (CrPDOs) were generated and co-cultured with autologous immune cells (IMM) generated from peripheral blood mononuclear cells. CrPDO/IMM co cultures were used to predict the potential therapeutic benefit in response to tyrosine kinase inhibitor cabozantinib (Cabo) plus pembrolizumab (Pembro) or atezolizumab (Atezo) combinatorial therapy. CD44v9-directed antibody-drug conjugates (CD44v9-ADCs; Abzena) designed to target senescence were tested using CrPDOs. Results: CosMx ™ WTX SMI analysis showed expression of CD44 and TROP2 positive cells, cancer associated fibroblasts and myeloid derived suppressor cells (MDSCs) consistent with the staining pattern of PD-L1. MxIF of matched CrPDOs and tissues showed expression of CD44v9 within senescent CD44 positive spatially mapped cell populations. Combinatorial treatment of CrPDO/IMM co-cultures with Cabo plus Pembro or Atezo led to depletion of MDSCs correlating with increased cytotoxic T lymphocyte proliferation and tumor cell death with a persistent CD44v9+ CSC population. Treatment of PDOs with CD44v9-ADCs resulted in tumor cell death, as measured by high-content confocal microscopy by nuclear irregularity index and propidium iodide uptake. Conclusion: CD44v9-directed ADCs, carrying senolytic and antioxidant defense-inhibiting payloads are a potential therapeutic approach for targeting residual CSCs and preventing recurrence of ACPs.