Notwithstanding the breakthroughs in understanding cancer, diagnosis, and disease management over the last couple of decades, the disease progression especially tumor development, remains to a great extent a mystery [24,25,26,27,28,29]. The number of identified cancer subtypes is ever-increasing due to the discovery of new singularities, such as genetic mutation or disease-specific biomarkers. As a consequence, a more precise diagnostic and subsequent treatment course can be devised. Typically, to reach a precise diagnosis, a series of immunohistological tests are required, followed by a detailed evaluation by a pathologist, which is often very time-consuming, delaying the start of the time-sensitive treatment. On top of that, the process can require a significant amount of tissue for the analysis, which is often not available in tumor biopsy samples.

For proteomic profiling using mass spectrometry imaging, the amount of required tissue is very small, namely, a single 2–3 μm thick tissue section that can afterward still be used for histological or immunohistochemical evaluation, DNA analysis, or further proteomic investigation [30,31,32,33]. Additionally, the number of analytes measured in a single run is considerably higher when compared to current methods where one or a small set of protein signatures is evaluated in one measurement. For these reasons, a significant part of the efforts has been directed toward training machine learning (ML) methods using the proteomic or metabolomic profiles obtained directly from the tissue by Mass Spectrometry Imaging (MSI). ML algorithms look for traits in the spectra data that can categorize the information in subgroups or classes either by employing unsupervised machine learning where the data are grouped by their affinity without requiring any preliminary analysis of the sample or through the use of supervised ML algorithms where previously acquired information (e.g., tumor diagnosis, staging, or treatment outcome) is used to train the algorithms.

One of the clinical applications of MSI is to assist in intraoperative consultation for the assessment of tumor margins to guarantee complete tumor resection [34,35,36]. One of the hallmarks in tissue diagnosis is the development of small devices that can quickly differentiate tumor from normal tissue, such as the iKnife and MasSpec Pen. These devices guide the surgeons by quickly classifying the tissue while performing resection surgery of various organs (such as the colon, breast, stomach, liver, lung, and brain) [37,38,39,40]. The apparatus relies on mass spectrometry measurements associated with ML algorithms to discriminate the tissue material from frozen tissue sections or directly in vivo [41,42,43]. As an example, King et al. utilized ambient ionization mass spectrometry to ionize samples ex vivo to assist in the differentiation of tumor resection margins of pancreatic ductal adenocarcinoma (PDAC) (n = 53) from the bile duct (n = 23) and nontumor samples of pancreatic tissue (n = 58). Samples collected prospectively were measured using the MasSpec Pen technology, and used for training and testing the least absolute shrinkage and selection operator (lasso) classifiers. The method yielded over 98% agreement with histological evaluation when classifying normal pancreas and PDAC in the training set (78 tissue analyses; PDAC samples with >70% tumor cells) and 78.8% in the validation set (33 tissue analyses; samples with mixed cellularity and low epithelial tumor cell concentration) [44]. A second lasso classifier was employed to differentiate bile duct (n = 16) and PDAC (n = 27) with accuracy of 98% in the training set, and accuracy of 91% in the validation set (bile duct, n = 7; PDCA, n = 17; PDAC invading bile duct, n = 8). Classifiers built on ex vivo samples were then utilized to classify tissue in the operating room in vivo and ex vivo (64 analyses), achieving an overall agreement of 93.8%. These studies consolidate that molecular signatures detected by MSI can be applied in real-time to discern tumor margins and tissue with different provenience within approximately only 3 seconds [45].

The differentiation between malignant and benign skin lesions also presents a challenge that can be addressed by MSI characterization. Margulis et al. applied DESI–MSI to measure the lipid and metabolite profile of basal cell carcinoma (BCC), a common skin cancer, and normal skin, with the objective of identifying micrometer-sized tumor aggregates of malignant skin lesions [46]. Arachidonic acid and glycerophosphoglycerol were markedly abundant in BCC compared to normal skin regions [46]. In this study, a lasso regression based on solely 24 mass features was able to discriminate BCC aggregates from adjacent normal skin. The authors reported that this approach could be employed as a fast intraoperative process during Mohs surgeries, complementing the histopathological evaluation.

Due to the histological similarities between some tumor entities and the lack of specific markers, it can be challenging and very time-consuming for pathologists to reach a diagnosis. Medulloblastoma and pineoblastoma share clinical features and show identical histological characteristics [47]. The analysis of the lipid profile of pediatric medulloblastoma and pineoblastoma indicated that MALDI–MSI could be a suitable tool to support the diagnosis. To further understand both tumor types, the authors of this study performed a receiver operating characteristic (ROC) analysis of the mass spectrometry features, concluding that glycerophosphoglycerols and glycerophosphocholines exhibited higher intensity, and could, therefore, become potential markers for medulloblastoma, while sphingolipids showed higher expression in pineoblastoma [47].

Another diagnostic conundrum in clinical pathology is to correctly characterize chromophobe renal cell carcinoma (chRCC) from renal oncocytoma (RO). RO is a benign kidney lesion that, from a histomorphological perspective, closely resembles the malignant neoplasia chRCC. This can result in the overtreatment of RO patients. On the basis of metabolite and lipid profiles obtained by the DESI–MSI of 71 patients with renal cell neoplasia, Zhang et al. were able to discriminate benign from malignant tumors with 100% accuracy [48].

PDAC also exhibits close morphology and histological resemblance to cholangiocarcinoma (CC). Both entities arising from the epithelium of the pancreaticobiliary tree have aggressive behavior, and an incorrect diagnosis can have strong implications on the patient’s prognosis and therapeutic course. Bollwein et al. utilized the proteomic profile of 82 patients measured by MALDI–TOF to train and test classification algorithms to differentiate between the two tumor types with an accuracy of approximately 90% [49]. The authors also advanced a feature importance list calculated by the mean decrease in the impurity of gradient-boosting classification, which revealed that histone H2A and the collagen α-1 (I) chain are more intensely expressed in PDAC when compared with CC, which could be disease-related biomarkers [49].

MSI has matured and is taking confident strides in assisting in tumor diagnosis by either providing further understanding of the molecular composition of complex tumor structures or merely helping in simpler tasks such as the identification of tumor regions; studies applying MSI have presented encouraging results that it could be the clinicians’ right hand for tumor analysis and classification [31,50].

An essential part of the clinical diagnosis of tumors is to predict how the tumor will progress and to predict the patient’s response to a certain treatment. For that, assessing the development stage of the disease is imperative, but it is also necessary to characterize the predictive molecular variation within the patient’s tumor to foresee the reactivity of a certain treatment.

MSI-based models were explored as a tool to accurately evaluate predictive molecular variations [51,52,53]. In a recent study by Erlmeier et al., MALDI–MSI was used in correlation with Kaplan–Meier curves to estimate predictive metabolic profiles for the prognosis of renal cell carcinoma (RCC) [54]. An increase in nucleotides (namely, cyclic guanosine monophosphate) was associated with a poor prognosis. The authors were also able to detect some metabolic pathways specific to some tumor types, particularly the glutathione metabolism, which is increased in late-stage clear cell RCC and associated with poor outcomes [54].

As the range of molecules characterized by MSI is not restricted to metabolites, prognostic studies based on glycan, protein, and lipid activity were carried out as well. One of these studies was performed by Phillips and coworkers, where prognostic features of triple-negative breast cancer (TNBC) were evaluated through the analysis of tryptic digested proteins utilizing a MALDI–TOF–TOF instrument [55]. The authors were able to identify 14 proteins that distinguish TNBC from benign lesions, and the correlation between these proteins and the Kaplan–Meier curves showed that COL1A1, COL1A2, COL6A3, ATIC, CCDC24, PLEKHG2, SOX11, and UBR4 are correlated with poor patient outcomes [55]. The results are supported by the literature, as COL1A1 and COL1A2, two components of Type I collagen, are upregulated in invasive breast cancer, with a potential role in spinal metastasis [56]. Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is also necessary for cell proliferation [57], and SOX11 plays a role in breast cancer growth and invasion, and in regulating the basal-like phenotype [58].

The diagnosis and prognosis of prostate cancer are performed on the basis of histological evaluation following the Gleason scoring system. The Gleason score (GS) is based on the evaluation of changes in the morphology of tumor glands, but it does not provide any information about the metabolic pathways that caused the alteration. To explain the metabolic alterations, studies correlating the GS and molecular features have been carried out. The tryptic digestion of tissue microarrays of 729 human prostate cancer specimens measured by MALDI–TOF–MSI identified four molecular signatures associated with a low Gleason score, early disease stage, and the low proliferation marker Ki-67. One molecular feature was associated with high Ki-67, and another signal could be correlated with a prolonged time for prostate-specific antigen (PSA) recurrence [59]. In an independent pilot study, the lipid profile of prostate cancer samples was correlated with the Gleason score. The authors showed that phosphatidylcholines, phosphatidic acids, phosphatidylserines, phosphatidylinositols, and cardiolipins were overexpressed in GS (4 + 3), suggesting their involvement in the disease progression, and the possibility for them to be utilized as markers of prostate cancer aggressiveness [60]. The results from this study agree with the outcome of another pilot study by Wang and coworkers [61].

The glycan analysis of pancreatic ductal adenocarcinoma (PDAC) measured by MALDI–Fourier-transform ion cyclotron resonance (FT–ICR) MSI revealed 8 glycan fragments uniquely expressed in the stroma, and 18 glycan fragments exclusively present in PDAC tumor cells [62]. Sun et al. reported that hyaluronan and chondroitin sulfate overexpression was correlated with worse survival rates, higher concentrations of HexS in the stroma were associated with better prognosis, while HexNAcS and HexAHexNAcS abundance predicted worse survival [62].

González de Vega et al. demonstrated that laser ablation inductively coupled plasma (LA–ICP) MSI could be used for the detection of matrix metalloproteinase-11 (MMP-11) to differentiate between the metastatic and nonmetastatic lesions of human breast cancer as a complement to the current approaches. The authors employed prelabelled antibodies with nanoparticles to increase the sensitivity and facilitate the direct correlation with immunohistochemistry markers [63].

The complexity of the disease renders diverse molecular imbalances, so different studies proceeded to target different molecular classes using a wide range of mass spectrometers. These efforts, such as the ones described here and many others, provide concrete and complementary information to perceive the metabolism of cancer progression.

From identification of disease-specific markers, prognostic markers, and implementation with machine learning approaches to assist with clinical assignments, mass spectrometry imaging studies provide meaningful contributions to the understanding of the different tumors at the molecular level. However, the methodology is not yet approved for clinical use. 

As mentioned before, the amount of tissue used for diagnosis is limited and often not enough to run the immunohistochemical panels and additional molecular analyses required for a complete diagnostic workup. Especially in biopsy samples, the amount is very limited, and the tumor content present is also sometimes scarce. For this reason, multiplexed approaches where different analytes can be detected from the same sample section are highly attractive. MSI measurements can detect metabolites, peptides/proteins, glycans, and lipids; however, most of the studies consider only one class of analytes. Efforts on maximizing the amount of information obtained from one slide achieved robust protocols that facilitate the measurement of several analytical groups using the same slide [64,65,66,67]. Clift et al. developed a multienzyme workflow for the measurement of extracellular matrix constitution of a single section of FFPE tissue [68]. In this study, sequential digestions with chondroitinase ABC, PNGaseF, elastase, and collagenase Type III were performed. Following each digestion, a matrix was applied, and the sample was measured by MALDI–FT–ICR–MSI [68]. Furthermore, as it is a non-destructive methodology, it is still possible to use the very same section for pathology analysis via histology. The importance of devising such protocols also exceeds the mere fact of saving tissue in routine diagnosis; it opens an unprecedented opportunity to easily colocalize different types of analytes in the very same tissue section, and better understand the biology supporting the molecular changes.

Metal conjugated antibodies have been employed to study the spatial distribution of proteins in the tissue with high spatial accuracy and sensitivity, which is ideal for the quantification of proteins, especially when only residual expression is detected, and therefore to predict the response of a patient to chemical treatment. Along these lines, Bishop et al. resorted to LA–ICP–MSI to simultaneously quantify and localize dystrophin in muscle sections [69]. Duchenne muscular dystrophy is characterized by the absence or decreased expression of dystrophin; to evaluate therapy efficacy, it is necessary to quantify and locate dystrophin in skeletal muscle, but the current methods lack reproducibility and sensitivity. MSI outperformed current techniques with increased sensitivity and using less amount of a sample, which reduces the need for invasive surgical biopsies [69].

The high sensitivity of MSI approaches was also explored for single-cell analysis [70,71,72,73]. While most of the ongoing single-cell studies could provide more information about disease progression, intracellular mechanisms, and novel treatment targets, we can foresee that this level of detail could also be useful in the clinic, especially for early on-set diagnosis, treatment choice, and disease prognosis, especially to further assess the response of immune cells to a specific treatment, or in the diagnosis of small sections or sections with a low number of tumor cells present, e.g., in small precursor lesions. Using single-cell proteomic characterization, Brunner and coworkers showed that quantifying cellular heterogeneity following targeted perturbation enables the direct analyses of drug responses in single-cell hierarchies on the proteomic level [71]. The study also highlighted the stability of the proteome when compared to single-cell RNA [71]. Further single-cell characterization utilizing MSI was recently reviewed elsewhere [72,74,75].

Another very exciting prospect of this technology was covered in a report by Neuman et al., where they describe the integration of MSI with orthogonal approaches to maximize the information of each experiment [76]. The combination of MSI with microscopy, spectroscopy, transcriptomics, and electrochemistry adds a new layer of information and an exponentially better understanding of the sample. Likewise, achievements in the integration of MSI with spatially targeted tandem MS, the combination of different ionization methods, microextraction, and ion mobility separation are achieving a high level of resolution and opening new possibilities for the technology [76]. While some of the aforementioned multimodal MSI techniques are more useful from a research perspective, others, such as the integration with transcriptomics and microscopy, can revolutionize the way in which clinical diagnosis is conducted.

Personalized medicine for tumor assessment has understanding the tumor biology of one person as the main objective. As individual and unique as our DNA, disease expression can also be different from patient to patient, and that is also translated to the response to the treatment. Personalized medicine is devised to maximize the efficiency of disease management considering individual variabilities such as genetics, protein expression, and lifestyle. By utilizing the most state-of-the-art approaches within a relatively short time, accurate diagnosis, prognosis, and therapeutic options are provided to the patient [77,78].

Mass spectrometry imaging was utilized to elucidate questions relating to tumor diagnosis and stratification, diagnostic prediction, intratumor heterogeneity characterization, biomarker discovery, and intraoperative consultation. Since the technique allows for such a diverse yet complete overview of tissue composition, MSI has been capturing attention as a convenient approach for personalized medicine.

As the technology is not fully ready to be accepted in the clinic just yet, approaches that allow for the integration of current histopathology evaluation and MSI in a single slide can help in moving it toward that direction. The so-called immunohistochemical MSI resorts to photocleavable linkers connected to antibodies that facilitate fluorescent immunohistochemistry (IHC) analysis before performing targeted MSI [79]. An untargeted measurement of the sample is also possible, and it should be performed before adding the antibodies to the sample [79]. Highly multiplexed IHC reduces the number of tissue sections required for a diagnosis. When coupling it to MSI, it is possible to retrieve molecular information directly from the tissue and associate it with high-precision IHC.

New efforts should focus on establishing nationwide guidelines for standardized data collection and facilitate the data transfer between medical institutions and researchers without compromising the patient’s privacy. Furthermore, integration of proteomic analysis, namely MSI, with other databases that are being developed to advance digital pathology, will enable a broader understanding of the disease progression, allow better markers to be developed, and consequently better therapy, while expanding the machine learning tools available to keep improving the personalized care for every individual patient.