Joanna Bogusiewicz1, Magdalena Gaca1, Paulina Goryńska1, Karol Jaroch1, Krzysztof Goryński1, Dariusz Paczkowski2, Jacek Furtak2, Marek Harat2,3, Barbara Bojko1

1 Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland

2 Department of Neurosurgery, 10th Military Research Hospital and Polyclinic in Bydgoszcz, Poland

3 Department of Public Health, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland

j.bogusiewicz@cm.umk.pl, bbojko@cm.umk.pl

Solid Phase Microextraction - Low Invasive Sampling Method for Brain

Tumor Differentiation

Meningiomas and gliomas are the most common types of benign and malignant brain tumors, respectively. It is important to

diagnose them as soon as possible and differentiate their types correctly. This procedure allows physicians to choose appropriate

treatment and expand life expectancy. Imaging techniques (magnetic resonance, computer tomography) or histopathology with

genetic tests are used for these purposes. Unfortunately, even with the use of such advanced methods the life expectancy for

patients with Glioblastoma multiforme (IV grade glioma) is only few months. That is why development of new diagnostic

methods which would allow early, accurate and reliable diagnose and having high prognostic value in treamtent choice and

assessment of its effectiveness are highly desired.

Data analysis

- Cancer progression

- Higher tumor grade

- Positive response on treatment

…

Extraction

SPME fiber – diameter ca. 200 μm

Sorbent – C18, 7mm

Time – 30 minutes

Desorption

LC-MS analysis

Solvent – 200 μl of IPA: MeOH; 1:1

Time – 60 minutes

Storage delayed

Acknowledgments: The National Science Center Poland, within the research task No. 2015/18/M/ST4/00059 entitled “New analytical solutions in oncology:

from basic research to rapid intraoperative diagnostics“.

Fig. 2 PCA score plot

Fig. 5 PC and PE profile of summary area under peaks

Fig. 1 Profile of detected fatty acids of Phosphatidylcholines

SPME procedure

HILIC RPLC

Cer 74 68

PC 86 32

PE 82 26

PG 7 6

PS 2 9

SM 22 10

MG - 2

DG - 23

TG - 18

TOTAL

COUNT 273 188

Identified lipids in positive ion mode*,**

HILIC - HRMS Column – SeQuant ZIC-cHILIC, 3μm

100x2.1 mm

Mobile Phases – A: ACN; kmmmmmkkkkk

B: 5mM ammonium acetate in water

Flow rate – 0.4ml/min

Column oven temperature – 40°C

RPLC - HRMS Column – Waters, XSelect CSH C18, 3.5µm, 2.1x75mm

Mobile phases – A: ACN:MeOH:water; 30:30:40; B: IPA:MeOH;

90:10; to both of them: +10mM ammonium acetate and 1mM acetic

acid.

Flow rate – 0.2 ml/min

Column oven temperature – 55ºC

Fig. 3 OPLS score plot Fig. 4 S-plot

GLIOMA MENINGIOMA

Results

Conclusions

* putative ID based on LipidSearch 4.1.30 parent search **FA, PA were deteced in negative ionization mode

• Two outliers were obsreved. Retrospective analysis of sampling workflow

showed about 30 minutes delay in sampling of these tumors, which could have

caused changes in the lipidome (ref. 5)

• This sample was excluded from further statistical analysis.

• It can be observed that gliomas are more diverse while meningiomas cluster more thightly.

• Particular groups of lipids differentiate these two types of tumors; SM and CER are more characteristic for meningiomas and PC and

PE for gliomas.

1. Alves A. et al: Biophysics in cancer: The relevance of drug-membrane interaction studies. Biochim Biophys Acta. 2016, 1858(9), 2231-2244

2. Cheng M. et al: Targeting Phospholipid Metabolism in Cancer. Frontiers in Oncology, 2016, 6(266),1-17

3. Li Y. et al: Imaging Tumor Metabolism using in vivo MR Spectroscopy. Cancer J. 2015, 21(2): 123–128.

4. Reyes-Garcés N. et al: Advances in Solid Phase Microextraction and Perspective on Future Directions. Anal Chem. 2018,90(1):302-360

5. Roszkowska A. et al.: Tissue storage affects lipidome profiling in comparison to in vivo microsampling approach. Sci. Rep. 2018, 8, 6980

Conclusions

Solid Phase Microextraction (SPME) seems to be useful method in lipidomics studies. It was proven it allows:

• to extract lipids from all groups with wide range of fatty acids,

• to differentiate brain tumor types: benign meningiomas and malignant gliomas.

Moreover, SPME pocedure is easy, fast, there is no need for labor- and time-intensive sample preparation step, and the

amount of used solvents is small comparing to other extraction methods.

Future plans: to expand studied group of patients to perform more in-depth analysis, with particular focus on gliomas

(lipidomic phenotype vs. genotype and histological data).

References

The goal of this work is to test a Solid Phase Microextraction (SPME) fibers as a low invasive tool for lipidomic analysis of

brain tumors.

(ref. 2)

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