Determination of Combretastatin A4 Influence on Human Lung Cancer and Mouse Melanoma as in vitro Cell Culture Models by SPME-LC-HRMS Karol Jaroch (Presenter) Dep. of Pharmacodynamics and Molecular Pharmacolog >> POSTER (PDF) Presenter Bio: In 2015, under the supervisor of Prof. Barbara Bojko at the Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Nicolaus Copernicus University in Torun, I have started my research as a PhD Candidate. During my studies I had the great opportunity of having Internships at the Department of Pharmaceutical Chemistry, School of Pharmacy, Oslo University and also at the Department of Chemistry, University of Waterloo, Canada. From 2017 I am a Principal Investigator at a grant financed by the Polish National Science Centre concerning the use of SPME method. During HPLC 2017 symposium in Prague, Czech Republic I was awarded the Best Poster Award.

Authors: Karol Jaroch, Barbara Bojko
Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Toruń, Poland

Use of solid phase microextraction (SPME) in combination with high-resolution mass spectrometry (HRMS) for cell culture metabolomics analysis allows getting more sophisticated data from in vitro assays. SPME was employed for determination of metabolomic changes after induction of non-small cell lung cancer cell line (A549) or melanoma (B16F10) cell lines with combretastatin A4 phosphate and combretastatin A4. Both, up- and downregulation of aminoacids (e.g.: valine, proline,) low molecular mass acids (pyroglutamic acid) and amides (palmitamide) was found as distinguishing between treated and non-treated cells. The whole procedure can be considered as HTS and „non-invasive” for cells. The use of SPME-(LC)-HRMS with cell cultures in high-throughput manner were presented for the first time. Work has been financed by Nicolaus Copernicus University statutory grant No 451.

Introduction

The use of solid phase microextraction (SPME) in combination with high-resolution mass spectrometry (HRMS) for cell culture metabolomics analysis allows getting more sophisticated data from in vitro assays. Due to fact that SPME is an equilibrium based non-exhaustive microextraction technique it enables performing multiple extractions from a single sample with no influence on the tested cells. Moreover, the opportunity of time course analysis from a single sample reduces the total number of samples, and eliminates inter-batch variability, for which cell culturing can be significant.

Methods

Solid phase microextraction was employed for determination of metabolomic changes after induction of non-small cell lung cancer cell line (A549) or melanoma (B16F10) cell lines with combretastatin A4 phosphate and combretastatin A4. Extraction protocol was established directly in 96-well plate, for small molecules extraction, a SPME fiber format was utilized with multiple analysis from the same sample. For determination of effective concentration of CA4, the MTT-based cytotoxicity assay was performed. To ensure that extraction protocol will not decrease the CA4 amount to non-toxic level the protein binding and extraction recovery were determined. The SPME was performed directly from cell culture medium causing no effect on cell growth. The whole procedure can be considered as HTS and non-disturbing for cells. Next, metabolomics analysis by LC-HRMS (Q-Exactive Focus) was performed.

Results

While there was no difference in cell number, it was observed that the extracellular metabolic pattern of both cell lines was changed after administration of the tested drug. This suggests a pharmacological activity of administered compound towards studied cell line models. The principal component analysis showed a difference between cells exposed and unexposed to CA4. The most important metabolomics changes (both, up- and downregulation) were reflected in the levels of amino acids (e.g.: valine, proline,) low molecular mass acids (pyroglutamic acid) and amides (palmitamide).

Conclusions & Discussion

The results indicate, that using a direct SPME immersion from cell cultures after exposure to drug allows obtaining information changes during single experiment without affecting cells growth, so the whole procedure can be considered as HTS and „non-invasive” for cells. Currently, there is ongoing work on the biological interpretation of the results towards identifying cellular metabolic pathways altered by CA4 and CA4P. The use of SPME-(LC)-HRMS with cell cultures in a high-throughput manner were presented for the first time.

Work has been financed by Nicolaus Copernicus University (Toruń, Poland) statutory grant No 451.