Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

As metabolic rewiring is crucial for cancer cell proliferation, metabolic phenotyping of patient-derived organoids is desirable to identify drug-induced changes and trace metabolic vulnerabilities of tumor subtypes. We established a novel protocol for metabolomic and lipidomic profiling of colorectal cancer organoids by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) facing the challenge of capturing metabolic information from a minimal sample amount (120% compared to matrix-derived signals present in blank samples. As a proof-of-concept, the method was applied to examine the early metabolic response of colorectal cancer organoids to 5-fluorouracil treatment. Statistical analysis revealed dose-dependent changes in the metabolic profiles of treated organoids including elevated levels of 2′-deoxyuridine, 2′-O-methylcytidine, inosine and 1-methyladenosine and depletion of 2′-deoxyadenosine and specific phospholipids. In accordance with the mechanism of action of 5-fluorouracil, changed metabolites are mainly involved in purine and pyrimidine metabolism. The novel protocol provides a first basis for the assessment of metabolic drug response phenotypes in 3D organoid models.

The tumor microenvironment is essential for mediating drug resistance and tumor progression. Here, we present a coculture system, which enables drug testing of colorectal cancer organoids and fibroblasts without additional matrix components such as Matrigel or basement membrane extracts. First, we describe steps to use a readout for high-throughput drug testing using a luminescence-based viability assay. Second, we detail a readout that uses flow cytometry to distinguish toxic effects on either colorectal cancer organoids or fibroblasts.