Aim:For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared.Materials & methods:‘Case study’ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed.Results:Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC–MS. Western blotting and nanoLC–MS also revealed negative exosome markers regarding both isolation approaches.Conclusion:The two isolation methods had an overall similar performance, but we hesitate to use the term ‘exosome isolation’ as impurities may be present with both isolation methods. NanoLC–MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.
Glioblastoma is the most common and
malignant brain tumor, and
current therapies confer only modest survival benefits. A major obstacle
is our ability to monitor treatment effect on tumors. Current imaging
modalities are ambiguous, and repeated biopsies are not encouraged.
To scout for markers of treatment response, we used NMR spectroscopy
to study the effects of a survivin inhibitor on the metabolome of
primary glioblastoma cancer stem cells. Applying high resolution NMR
spectroscopy (1H resonance frequency: 800.03 MHz) to just
3 million cells per sample, we achieved sensitive and high resolving
determinations of, e.g., amino acids, nucleosides, and constituents
of the citric acid cycle. For control samples that were cultured,
prepared, and measured at varying dates, peak area relative standard
deviations were 15–20%. Analyses of unfractionated lysates
were performed for straightforward compound identification with COLMAR
and HMDB databases. Principal component analysis revealed that citrate
levels were clearly upregulated in nonresponsive cells, while lactate
levels substantially decreased following treatment for both responsive
and nonresponsive cells. Hence, lactate and citrate may be potential
markers of successful drug uptake and poor response to survivin inhibitors,
respectively. Our metabolomics approach provided alternative biomarker
candidates compared to spectrometry-based proteomics, underlining
benefits of complementary methodologies. These initial findings make
a foundation for exploring in vivo MR spectroscopy
(MRS) of brain tumors, as citrate and lactate are MRS-visible. In
sum, NMR metabolomics is a tool for addressing glioblastoma.
Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the "organ-in-a-column" units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated "on column" with heroin, followed by on-line monitoring of the drug's phase 1 metabolism. Enzymatic metabolism of heroin produced in the "organ-in-a-column" units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.
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