Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

ASBTRACTThe biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.

Carotenoids are natural pigments with provitamin A and antioxidant activities. Biosynthesized in plants as their all-trans isomers, carotenoids isomerize in solution and in humans to multiple cis isomers which can have different bioavailabilities and functions. Since separation and characterization of isomeric carotenoids using HPLC or LC-MS-MS is time consuming, the potential for ion mobility mass spectrometry (IM-MS) to resolve and characterize carotenoid isomers rapidly without chromatography was investigated using travelling-wave ion mobility spectrometry on a quadrupole time-of-flight mass spectrometer. The all-trans isomers of lycopene and β-carotene were separated by several milliseconds from the cis-isomers which were detected as partially overlapping peaks. The collision cross-section values of these carotenoid isomers were determined using IM-MS to be 180 Å2 and 236 Å2 for cis-lycopenes and all-trans-lycopene, and 181 Å2 and 225 Å2 for cis-β-carotene and all-trans-β-carotene, respectively. Collision-induced dissociation MS-MS of ion mobility-resolved isomers indicated that cis and all-trans carotenoid isomers can be distinguished by their fragmentation patterns. Previous MS-MS studies of cis- and all trans-carotenoids had suggested that they produced identical tandem mass spectra, but this appears to have been the result of isomerization during ionization. Introduction of specific cis or trans isomers by infusion or HPLC resulted in cis/trans isomerization in the ion source during electrospray, and the relative levels of cis carotenoids forming in the ion source compared to the all-trans isomers were temperature dependent.