Fanconi anemia (FA) patients have DNA repair mutations predisposing them to oral cancers so treatment with non-DNA damaging agents is highly desired. As cancer experimental therapeutics advance towards study paradigms that more carefully mimic physiology, e.g. organoids and spheroids. Presently, we are using a 3D cell culture method emphasizing layers of extracellular matrix (ECM) as FA premalignant lesions retain epithelial architecture. We conducted 3D screening studies using 2 layered geometries: 1) nanofibers coated with 1 ng/mL 2:1 Collagen III/IV (C3C4) then 100 pg/mL 2.75:1 Tenascin C/Fibronectin (TNFN) and 2) polystyrene layered with 50 ug/mL C3C4 then 3.5 ug/mL TNFN. We developed these coatings to maximize lipid raft recovery, a drug transport feature lost in 2D culture. The ECM impact on FA cell drug transport was characterized by comparing effective nuclear delivery of a 20nm nanoencapsulated FITC-labeled RNAi compound (s50-TBG-RNAi3UTR), known to require lipid rafts, to the same oligo formulated with Dotap in a FA HNSCC cell line (FA1). Using confocal microscopy and IMARS software to segment delivery at maximal nuclear intensity into nuclear or endolysosomal compartments, we found, for FA1 cells on ECM, nuclear oligo signal colocalization with nuclear compartment ± coefficient of variation was 62 ± 2.2% for the capsule vs 9.8 ± 8.5% for Dotap oligo. On glass, nuclear delivery was 10 ± 52% for s50 capsule vs 25 ± 32% for Dotap. Endolysosomal compartment sequestration was respectively 28 ± 12, 62 ± 10, 44 ± 19 and 51 ± 15%. This illustrates a relevant decrease in variation in FA cell biology from ECM addition. Next, we studied metformin (Met) and pioglitazone (Pio)(agents in current oral cancer prevention studies) combined with G2/M blockade inhibitors, MK1775 (Wee1 kinase inhibitor) and GSK461364 (PLK inhibitor) in FA1s. Inhibiting G2/M blockade, already induced by FA-derived DNA damage, could promote inappropriate cell division, mitotic catastrophe (MTOC) and death. In initial 2D FA1 growth studies, increased death was observed with Met plus MK1775. 3D studies, in contrast, showed increased cell death for FA1s treated with Pio plus either MK1775 or s50-TBG-RNAiCK2 (RNAiCK2). Confocal mechanistic studies in cells plated on ECM and treated for 18 hours, indicated Pio + MK1775 had inhibited Survivin and ß-catenin upregulation and FA1 cells did proceed into MOC and death. For Pio (PPARg agonist) + RNAiCK2, RXRa was dephosphorylated at S260, enabling its escape from cytosolic sequestration to bind nuclear PPARg, initiating differentiation. Differentiation was indicated by upregulation of panKeratin and Transglutaminase-3. We conclude combination therapies with high interest agents in FA-associated oral cancer can be performed in 3D culture systems and might confirm drug mechanisms of action, thus augmenting other standard methods of cancer drug evaluation and screening (e.g. cell proliferation and clonogenicity).
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