MSACL 2023 Abstract

Introduction
For molecular diagnosis-based stratified medicine, accurate diagnosis of the disease is essential to successfully treat patients. However, the presence of metastases significantly complicates treatment and as the primary tumour is often not identifiable, their investigation is critically important. This study aims to investigate the relations between the tumour, its metastasis and their primary cell lines established from these tumours using chemical imaging and ambient Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry (LA-REIMS). We aim to investigate whether conclusions can be drawn about the primary tumour solely from the metastasis and the cultured cell line.

Methods
Primary cell lines were established from snap frozen, spontaneous tumours taken from veterinary surgeries. Cells were frozen at each passage to allow monitoring changes along the establishment of cultures. Tumour tissues were sectioned; cell pellets were dried on slides to be characterized by OPO Laser-Assisted REIMS source coupled to an imaging platform and fitted with a XevoTM G2-XS TOF Mass Spectrometer (Waters Corporation). Samples were also measured using Automated Desorption Electrospray Ionization (AutoDESI) fitted with a XevoTM G2-XS TOF MS (Waters Corporation). The data acquired in negative ion mode, mass to charge range 50-1200, was processed with our in-house built software AMX (Waters) using multivariate statistics including Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). Ions were identified using MS/MS and exact mass measurements.
For research use only. Not for use in diagnostic procedures.

Preliminary Data
Spontaneous tumour - tumour metastasis pairs, a simplex tubulopapillar carcinoma and its skin metastasis, a complex adenocarcinoma and its lung metastasis, were examined. The tumours and the respective cell lines were measured using LA-REIMS and DESI imaging with 70 micrometre resolution which provided unique metabolic fingerprints and rich molecular profiles. Section measurements revealed great similarity between the primary tumour and its metastasis (PCA space distance 0.9 units), and between the cells of the immortalized primary tumour and metastasis cell lines as well, meanwhile tumour-cell relations unveiled significant differences (PCA space distance over 2.2 units).
The cross-validation results clearly demonstrate a connection between the metastasis, the primary tumour and their established cell lines. In general, the homogenous cell cultures are highly similar to each other regardless of their origin. The original tumour is clearly identifiable based on the patterns detected in the respective metastasis, however, cell lines show significantly different characteristics compared to the tumour tissues. Due to the heterogeneity of the tissue surrounding the tumour, the fingerprint of the homogenous cell culture (identical consistency) is highly different thus the metabolic fingerprints cannot be compared directly.
To understand these observations, the findings were examined at the molecular level. There were significant differences between the lipid profiles of the cells and tissues, including several phosphatidic acids (PA(34:1), PA(36:2), PA(36:1), PA(38:3) and PA(38:2)), which differences were characteristic to both tumour-metastasis pairs. Changes in other PAs, phosphatidyl-ethanolamines (PE) and ceramides (Cer) were also identified. These characteristic lipids are found in higher amounts in the tumour as compared to the healthy tissue, and even higher amounts in cell lines in the vast majority of cases, showing an alteration in the tumour lipid metabolism. The biological relevance of these molecules is still under investigation.

Conclusion and Perspectives
Results demonstrate that mass spectrometric fingerprints of metastases allow clear identification of the primary tumours. Although cultured cell lines derived from the primary tumour and the metastasis show highly similar patterns, these are significantly different from the tumour tissue signatures. At the same time, similarities allow to examine whether the sensitivity profiles of the cell lines and the tumour overlap. We aim to subject the cells to different dietary experiments and examine a successfully developed diet in vivo in an animal model for tumour suppression. This may subsequently indicate that cell lines could be generated from recurrent tumours and used to develop therapies that helps to reduce tumour recurrence, or to develop therapies even without the knowledge of the primary tumour.

Novel Aspect
Comparative molecular analysis of tumour - tumour metastasis pairs and cell lines derived from therein using Rapid Evaporative Ionization Mass Spectrometry.