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Mass spectrometry imaging with desorption electrospray ionization mass spectrometry (DESI-MS) is used to characterize cancer from ex vivo slices of tissues. The process is time-consuming. The use of tissue smears for DESI-MS analysis has been proposed as it eliminates the time required to snap-freeze and section the tissue. To assess the utility of tissue smears for rapid cancer characterization, principal component analysis (PCA) was performed to evaluate the concordance between DESI-MS profiles of breast cancer from tissue slices and smears prepared on various surfaces. PCA suggested no statistical discrimination between DESI-MS profiles of tissue sections and tissue smears prepared on glass, polytetrafluoroethylene (PTFE), and porous PTFE. However, the abundances of cancer biomarker ions varied between sections and smears, with DESI-MS analysis of tissue sections yielding higher ion abundances of cancer biomarkers compared with smears. Coefficient of variance (CV) analysis suggests DESI-MS profiles from tissue smears are as reproducible as the ones from tissue sections. The limit of detection with smear samples from single pixel analysis is comparable to tissue sections that average the signal from a tissue area of 0.01 mm2. The smears prepared on the PTFE surface possessed a higher degree of homogeneity compared with the smears prepared on the glass surface. This allowed single MS scans (~1 s) from random positions across the surface of the smear to be used in rapid cancer typing with good reproducibility, providing pathologic information for cancer typing at speeds suitable for clinical utility.

Targeted and localized mass spectrometry imaging allows faster characterization of cancer compared to conventional methods., While mass spectrometers can detect chemical signatures within milliseconds of data acquisition time, the non-targeted nature of mass spectrometry imaging (MSI) necessitates probing the entire surface of the sample to reveal molecular composition even if the information is only sought from a sample subsection. This leads to long analysis times. Here, we used polarimetry to identify, within a biological tissue, areas of polarimetric heterogeneity indicative of cancer. We were then able to target our MS analysis using polarimetry results to either the cancer region itself or to the cancer margin. A tandem of polarimetry and Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) enables fast (10 fold compared to non-targeted imaging), and accurate pathology assessment (cancer typification in less than 2 minutes compared to 30 minutes for histopathology) of ex vivo tissue slices, without additional sample preparation. This workflow reduces the overall analysis time of MSI as a research tool.

Squamous cell carcinomas constitute a major class of head & neck cancers, where the tumour stroma ratio (TSR) carries prognostic information. Patients affected by stroma-rich tumours exhibit a poor prognosis and a higher chance of relapse. As such, there is a need for a technology platform that allows rapid determination of the tumour stroma ratio. In this work, we provide a proof-of-principle demonstration that Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) can be used to determine tumour stroma ratios. Slices from three independent mouse xenograft tumours from the human FaDu cell line were subjected to DESI-MS imaging, staining and detailed analysis using digital pathology methods. Using multivariate statistical methods we compared the MS profiles with those of isolated stromal cells. We found that m/z 773.53 [PG(18:1)(18:1) - H]-, m/z 835.53 [PI(34:1) - H]- and m/z 863.56 [PI(18:1)(18:0) - H]- are biomarker ions that can distinguish FaDu cancer from cancer associated fibroblast (CAF) cells. A comparison with DESI-MS analysis of controlled mixtures of the CAF and FaDu cells showed that the abundance of the biomarker ions above can be used to determine, with an error margin of close to 5% compared with quantitative pathology estimates, TSR values. This proof-of-principle demonstration is encouraging and must be further validated using human samples and a larger sample base. At maturity, DESI-MS thus may become a stand-alone molecular pathology tool providing an alternative rapid cancer assessment without the need for time-consuming staining and microscopy methods, potentially further conserving human resources.

Spatially Targeted Mass Spectrometry (MS) analysis using survey scans with an imaging modality often requires consecutive tissue slices, because of the tissue damage during survey scan or due to incompatible sample preparation requirements between the survey modality and MS. We report two spatially targeted MS analysis workflows based on polarized light imaging guidance that use the same tissue sample for survey and targeted analysis. The first workflow is applicable for thin-slice analysis, and uses transmission-polarimetry-guided Desorption ElectroSpray Ionization Mass Spectrometry (DESI-MS), and confirmatory H&E histopathology analysis on the same slice; this is validated using quantitative digital pathology methods. The second workflow explores a polarimetry-guided MS platform for thick tissue assessment by developing reflection-mode polarimetric imaging coupled with a hand-held Picosecond InfraRed Laser (PIRL) MS ablation probe that requires minimal tissue removal to produce detectable signal. Tissue differentiation within 5–10 s of sampling with the hand-held probe is shown using multivariate statistical methods of the MS profiles. Both workflows were tasked with differentiating necrotic cancer sites from viable cancers using a breast tumour model, and their performance was evaluated. The use of the same tissue surface addresses mismatches in guidance due to intrinsic changes in tissue morphology over consecutive sections.

Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is associated with aggressive forms of cancer and unfavourable outcomes. To facilitate rapid detection of necrosis with Mass Spectrometry (MS), we report the lipid MS profile of necrotic breast cancer with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging validated with statistical analysis and correlating pathology. This MS profile is characterized by (1) the presence of the ion of m/z 572.48 [Cer(d34:1) + Cl]− which is a ceramide absent from the viable cancer subregions; (2) the absence of the ion of m/z 391.25 which is present in small abundance only in viable cancer subregions; and (3) a slight increase in the relative intensity of known breast cancer biomarker ions of m/z 281.25 [FA(18:1)-H]− and 303.23 [FA(20:4)-H]−. Necrosis is accompanied by alterations in the tissue optical depolarization rate, allowing tissue polarimetry to guide DESI-MS analysis for rapid MS profiling or targeted MS imaging. This workflow, in combination with the MS profile of necrosis, may permit rapid characterization of necrotic tumors from tissue slices. Further, necrosis-specific biomarker ions are detected in seconds with single MS scans of necrotic tumor tissue smears, which further accelerates the identification workflow by avoiding tissue sectioning and slide preparation.