Cisplatin-Induced\nMetabolic Responses Measured with\nRaman Spectroscopy in Cancer Cells, Spheroids, and Canine-Derived\nOrganoids

<i>Ex vivo</i> assessment of drug response\nwith conventional\ncell viability assays remains the standard practice for guiding initial\ntherapeutic choices. However, such ensemble approaches fail to capture\nheterogeneities in treatment response and cannot identify early markers\nof response. Here, we leverage Raman spectroscopy (RS) as an accurate,\nlow-cost, extraction-free, and label-free approach to track metabolic\nchanges in cancer cells, spheroids, and organoids in response to cisplatin\ntreatment. We identified 12 statistically significant metabolites\nin cells and 19 metabolites in spheroids and organoids as a function\nof depth. We show that the cisplatin treatment of 4T1 cells and spheroids\nresults in a shift in metabolite levels; metabolites including nucleic\nacids such as DNA, 783 cm<sup>–1</sup> with <i>p</i> = 0.00021 for cells; <i>p</i> = 0.02173 for spheroids,\nmajor amino acids such as threonine, 1338 cm<sup>–1</sup> with <i>p</i> = 0.00045 for cells; <i>p</i> = 0.01022 for\nspheroids, proteins such as amide III, 1248 cm<sup>–1</sup> with <i>p</i> = 0.00606 for cells; <i>p</i> =\n0.00511 for spheroids serve as early predictors of response. Our RS\nfindings were also applicable to canine-derived organoids, showing\nspatial variations in metabolic changes as a function of organoid\ndepth in response to cisplatin. Further, the metabolic pathways such\nas tricarboxylic acid (TCA)/citric acid cycle and glyoxylate and dicarboxylate\nmetabolism that drive drug response showed significant differences\nbased on organoid depth, replicating the heterogeneous treatment response\nseen in solid tumors where there is a difference from the periphery\nto the tumor core. Our study showcases the versatility of RS as a\npredictive tool for treatment response applicable from cells to organotypic\ncultures, that has the potential to decrease animal burden and readout\ntime for preclinical drug efficacy.