The extracellular matrix landscape in salivary gland carcinomas is defined by cellular differentiation via expression of three distinct protein modules.

The extracellular matrix (ECM) is an integral part of the tumor microenvironment of carcinomas. Even though salivary gland carcinomas (SGCs) display a range of tumor cell differentiation and distinct extracellular matrices, their ECM landscape has not been characterized in depth. The ECM composition of 89 SGC primaries, 14 metastases, and 25 normal salivary gland tissues was assessed using deep proteomic profiling. Machine learning algorithms and network analysis were used to detect tumor groups and protein modules that explain specific ECM landscapes. Multimodal in situ studies to validate exploratory findings and to infer a putative cellular origin of ECM components were applied. We revealed two fundamental SGC ECM classes which align with the presence or absence of myoepithelial tumor differentiation. We describe the SGC ECM through three biologically distinct protein modules that are differentially expressed across ECM classes and cell types. The modules have a distinct prognostic impact on different SGC types. Since targeted therapy is rarely available for SGC, we used the proteomic expression profile to identify putative therapeutic targets. In summary, we provide the first extensive inventory of ECM components in SGC, a difficult-to-treat disease that encompasses tumors with distinct cellular differentiation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

High-resolution MRI of the human palatine tonsil and its schematic anatomic 3D reconstruction.

The palatine tonsils form an important part of the human immune system. Together with the other lymphoid tonsils of Waldeyer's tonsillar ring, they act as the first line of defense against ingested or inhaled pathogens. Although histologically stained sections of the palatine tonsil are widely available, they represent the tissue only in two dimensions and do not provide reference to three-dimensional space. Such a representation of a tonsillar specimen based on imaging data as a 3D anatomical reconstruction is lacking both in scientific publications and especially in textbooks. As a first step in this direction, the objective of the present work was to image a resected tonsil specimen with high spatial resolution in a 9.4 T small-bore pre-clinical MRI and to combine these data with data from the completely sectioned and H&E stained same palatine tonsil. Based on the information from both image modalities, a 3D anatomical sketch was drawn by a scientific graphic artist. In perspective, such studies could help to overcome the difficulty of capturing the spatial extent and arrangement of anatomical structures from 2D images and to establish a link between three-dimensional anatomical preparations and two-dimensional sections or illustrations, as they have been found so far in common textbooks and anatomical atlases.

CD138 Is Expressed in Different Entities of Salivary Gland Cancer and Their Lymph Node Metastases and Therefore Represents a Potential Therapeutic Target.

Advanced salivary gland carcinomas (SGC) often lack therapeutic options. Agents targeting CD138 have recently shown promising results in clinical trials for multiple myeloma and a preclinical trial for triple-negative breast cancer. Immunohistochemistry for CD138 was performed for all patients who had undergone primary surgery for SGC with curative intent. Findings were validated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. Overall, 111 primary SGC and 13 lymph node metastases from salivary duct carcinomas (SaDu) were evaluated. CD138 expression was found in 60% of all SGC with differing expression across entities (p < 0.01). A mean of 25.2% of the tumor cells in mucoepidermoid carcinoma (MuEp) were positive, followed by epithelial-myoepithelial carcinoma (20.9%), acinic cell carcinoma (16.0%), and SaDu (15.2%). High-/intermediate-grade MuEp showed CD138 expression in a mean of 34.8% of tumor cells. For SaDu, lymph node metastases showed CD138 expression in a mean of 31.2% of tumor cells which correlated with CD138 expression in their primaries (p = 0.01; Spearman's ρ = 0.71). MALDI-MS imaging confirmed the presence of the CD138 protein in SGC. No significant association was found between clinicopathological data, including progression-free survival (p = 0.50) and CD138 expression. CD138 is expressed in the cell membrane of different entities of SGC and SaDu lymph node metastases and therefore represents a potential target for CD138 targeting drugs.

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome.

Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.

Multimodal Scanning Microscope Combining Optical Coherence Tomography, Raman Spectroscopy and Fluorescence Lifetime Microscopy for Mesoscale Label-Free Imaging of Tissue.

Multimodal optical imaging of tissue has significant potential to become an indispensable diagnostic tool in clinical pathology. Conventional bright-field microscopy provides contrast based on attenuation or reflectance of light, having no depth-related information and no molecular specificity. Recent developments in biomedical optics have introduced a variety of optical modalities, such as Raman spectroscopy (RS), fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorophores, optical coherence tomography (OCT), and others, which provide a distinct characteristic, i.e., molecular, chemical, and morphological information, of the sample. To harvest the full analytical potential of those modalities, we have developed a novel multimodal imaging system, which allows the co-registered acquisition of OCT/FLIM/RS on a single device. The present implementation allows the investigation of biological tissues in the mesoscale range, 0.1-5 mm in a correlated manner. Due to the co-registered acquisition of the modalities, it is possible to directly compare and evaluate the corresponding information between the three modalities. Moreover, by additionally preparing and characterizing entire pathological hematoxylin and eosin (H&E) slides of head and neck biopsies, it is also possible to correlate the multimodal spectroscopic information to any location of the ground truth H&E information. To the best of our knowledge, this is the first development and implementation of a compact and clinically applicable multimodal scanning microscope, which combines OCT, FLIM, and RS together with the possibility for co-registering H&E information for a morpho-chemical tissue characterization and a correlation with the pathological ground truth (H&E) of the underlying signal origin directly in a clinical environment.

Microdissection-An Essential Prerequisite for Spatial Cancer Omics.

The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function, different mutations, different mRNA expression and the highest intricacy in the protein pattern. Prior to genomic and proteomic analyses, it is therefore indispensable to identify the exact part of the tissue or even the exact cell. Laser-based microdissection is a tried and tested technique able to produce pure and well-defined cell material for further analysis with proteomic and genomic techniques. It sheds light on the heterogeneity of cancer or other complex diseases and enables the identification of biomarkers. This review aims to raise awareness for the reconsideration of laser-based microdissection and seeks to present current state-of-the-art combinations with omic techniques.

Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer.

Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.

Integration of 3D multimodal imaging data of a head and neck cancer and advanced feature recognition.

In the last years, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) became an imaging technique which has the potential to characterize complex tumor tissue. The combination with other modalities and with standard histology techniques was achieved by the use of image registration methods and enhances analysis possibilities. We analyzed an oral squamous cell carcinoma with up to 162 consecutive sections with MALDI MSI, hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) against CD31. Spatial segmentation maps of the MALDI MSI data were generated by similarity-based clustering of spectra. Next, the maps were overlaid with the H&E microscopy images and the results were interpreted by an experienced pathologist. Image registration was used to fuse both modalities and to build a three-dimensional (3D) model. To visualize structures below resolution of MALDI MSI, IHC was carried out for CD31 and results were embedded additionally. The integration of 3D MALDI MSI data with H&E and IHC images allows a correlation between histological and molecular information leading to a better understanding of the functional heterogeneity of tumors. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

The immunoregulator soluble TACI is released by ADAM10 and reflects B cell activation in autoimmunity.

BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF-APRIL receptor TACI, was applied in clinical trials. However, disease activity in multiple sclerosis unexpectedly increased, whereas in systemic lupus erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-κB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF-APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted autoimmune disease multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation.

Polymorphisms in melanoma differentiation-associated gene 5 link protein function to clearance of hepatitis C virus.

UNLABELLED: Among patients newly infected with hepatitis C virus (HCV), only 20-30% clear the infection spontaneously. In the remaining 70% the infection persists, causing chronic liver inflammation and disease. It is well established that polymorphisms in host genes, especially in components of the innate immune response, contribute to the phenomenon of spontaneous HCV clearance. Retinoic acid inducible gene-I (RIG-I)-like helicases such as melanoma differentiation-associated gene 5 (MDA-5) are cytoplasmic sensors of viral RNA that are critical for triggering innate immune responses after infection with RNA viruses. We analyzed 14 nonsynonymous single-nucleotide polymorphisms in RIG-I-like helicase-pathway-genes comparing European patients who spontaneously cleared HCV (n = 285) or had persistent infection (n = 509). We found that polymorphic haplotypes in the MDA-5 gene IFIH1 encoding histidine at position 843 and threonine at position 946 strongly correlate with the resolution of HCV infection (odds ratio [OR]: 16.23; 95% confidence interval [CI]: 3.67-71.87; P = 1.1 × 10(-6) ). Overexpression of MDA-5 genetic variants in HEK 293 cells and in a tissue culture model of HCV infection revealed that the histidine 843/threonine 946 variant leads to increased baseline and ligand-induced expression of interferon-induced genes and confers an increased ability to suppress HCV replication. CONCLUSION: These data suggest that MDA-5 plays a significant role in the defense against HCV and that polymorphisms in MDA-5 can influence the outcome of HCV infection.

Fingolimod induces neuroprotective factors in human astrocytes.

BACKGROUND: Fingolimod (FTY720) is the first sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of multiple sclerosis. The phosphorylated active metabolite FTY720-phosphate (FTY-P) interferes with lymphocyte trafficking. In addition, it accumulates in the CNS and reduces brain atrophy in multiple sclerosis (MS), and neuroprotective effects are hypothesized. METHODS: Human primary astrocytes as well as human astrocytoma cells were stimulated with FTY-P or S1P. We analyzed gene expression by a genome-wide microarray and validated induced candidate genes by quantitative PCR (qPCR) and ELISA. To identify the S1P-receptor subtypes involved, we applied a membrane-impermeable S1P analog (dihydro-S1P), receptor subtype specific agonists and antagonists, as well as RNAi silencing. RESULTS: FTY-P induced leukemia inhibitory factor (LIF), interleukin 11 (IL11), and heparin-binding EGF-like growth factor (HBEGF) mRNA, as well as secretion of LIF and IL11 protein. In order to mimic an inflammatory milieu as observed in active MS lesions, we combined FTY-P application with tumor necrosis factor (TNF). In the presence of this key inflammatory cytokine, FTY-P synergistically induced LIF, HBEGF, and IL11 mRNA, as well as secretion of LIF and IL11 protein. TNF itself induced inflammatory, B-cell promoting, and antiviral factors (CXCL10, BAFF, MX1, and OAS2). Their induction was blocked by FTY-P. After continuous exposure of cells to FTY-P or S1P for up to 7 days, the extent of induction of neurotrophic factors and the suppression of TNF-induced inflammatory genes declined but was still detectable. The induction of neurotrophic factors was mediated via surface S1P receptors 1 (S1PR1) and 3 (S1PR3). CONCLUSIONS: We identified effects of FTY-P on astrocytes, namely induction of neurotrophic mediators (LIF, HBEGF, and IL11) and inhibition of TNF-induced inflammatory genes (CXCL10, BAFF, MX1, and OAS2). This supports the view that a part of the effects of fingolimod may be mediated via astrocytes.

Novel workflow for combining Raman spectroscopy and MALDI-MSI for tissue based studies.

Molecular heterogeneity of cancer is a major obstacle in tumor diagnosis and treatment. To deal with this heterogeneity, a multidisciplinary combination of different analysis techniques is of urgent need because a combination enables the creation of a multimodal image of a tumor. Here, we develop a computational workflow in order to combine matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) imaging and Raman microspectroscopic imaging for tissue based studies. The computational workflow can be used to confirm a spectral histopathology (SHP) based on one technique with another technique. In this contribution, we confirmed a Raman spectroscopic based SHP with MALDI-imaging. Owing to this combination, we could demonstrate, for a larynx carcinoma sample, that tissue types and different metabolic states could be extracted from the Raman spectra. Further investigations with the help of MALDI spectra yield a better characterization of variable epithelial differentiation and a better understanding of ongoing dysplastic alterations.

Indocyanine green uptake by human tumor and non­tumor cell lines and tissue.

Indocyanine green (ICG) is a potential promising dye for a better intraoperative tumor border definition and an improved patient outcome by potentially improving tumor border visualization compared with traditional white light guided surgery. Here, the cellular uptake of ICG in human squamous cell carcinoma (SCC026) and immortalized non-cancer skin (HaCaT) cell lines was evaluated to study the tumor-specific cellular uptake of ICG. The spatial distribution of ICG inside tumor tissue was investigated in tissue sections of head and neck squamous cell carcinoma at a microscopic level. ICG uptake and internalization was observed in living cells after 2.5 h and in the nucleus after 24 h. In dead cells, higher and faster uptake was observed. In the tissue sections, higher ICG signal intensity could be detected in connective tissue and surrounding clusters and blood vessels. In conclusion, no distinct ICG uptake by tumor cells was detected in cancer cell lines and tumor tissue. ICG localization in certain regions of tumor tissue appears to be a result of enhanced tissue permeability and retention, but not specific to tumor cells.

Synergistic cross-kingdom host cell damage between Candida albicans and Enterococcus faecalis.

The fungus Candida albicans and the Gram-positive bacterium Enterococcus faecalis share mucosal niches in the human body. As opportunistic pathogens, both are found to expand population size during dysbiosis, and can cause severe systemic infections in susceptible individuals. Here, we show that the presence of C. albicans results in increased host cell damage by E. faecalis . Furthermore, E. faecalis aggravates oropharyngeal candidiasis in mice. Increased damage is mediated by enterococcal cytolysin, and involves both physical interaction and altered glucose availability. Physical interaction promotes accumulation of bacteria on host cells, facilitating contact of cytolysin with host cells. Glucose depletion by the metabolic activity of the fungus sensitized host cells to cytolysin. This work illustrates how a complex interplay between fungi and bacteria can result in detrimental consequences for the host.

Comparison of denoising tools for the reconstruction of nonlinear multimodal images.

Biophotonic multimodal imaging techniques provide deep insights into biological samples such as cells or tissues. However, the measurement time increases dramatically when high-resolution multimodal images (MM) are required. To address this challenge, mathematical methods can be used to shorten the acquisition time for such high-quality images. In this research, we compared standard methods, e.g., the median filter method and the phase retrieval method via the Gerchberg-Saxton algorithm with artificial intelligence (AI) based methods using MM images of head and neck tissues. The AI methods include two approaches: the first one is a transfer learning-based technique that uses the pre-trained network DnCNN. The second approach is the training of networks using augmented head and neck MM images. In this manner, we compared the Noise2Noise network, the MIRNet network, and our deep learning network namely incSRCNN, which is derived from the super-resolution convolutional neural network and inspired by the inception network. These methods reconstruct improved images using measured low-quality (LQ) images, which were measured in approximately 2 seconds. The evaluation was performed on artificial LQ images generated by degrading high-quality (HQ) images measured in 8 seconds using Poisson noise. The results showed the potential of using deep learning on these multimodal images to improve the data quality and reduce the acquisition time. Our proposed network has the advantage of having a simple architecture compared with similar-performing but highly parametrized networks DnCNN, MIRNet, and Noise2Noise.

Design and test of a rigid endomicroscopic system for multimodal imaging and femtosecond laser ablation.

Significance: Conventional diagnosis of laryngeal cancer is normally made by a combination of endoscopic examination, a subsequent biopsy, and histopathology, but this requires several days and unnecessary biopsies can increase pathologist workload. Nonlinear imaging implemented through endoscopy can shorten this diagnosis time, and localize the margin of the cancerous area with high resolution. Aim: Develop a rigid endomicroscope for the head and neck region, aiming for in-vivo multimodal imaging with a large field of view (FOV) and tissue ablation. Approach: Three nonlinear imaging modalities, which are coherent anti-Stokes Raman scattering, two-photon excitation fluorescence, and second harmonic generation, as well as the single photon fluorescence of indocyanine green, are applied for multimodal endomicroscopic imaging. High-energy femtosecond laser pulses are transmitted for tissue ablation. Results: This endomicroscopic system consists of two major parts, one is the rigid endomicroscopic tube 250 mm in length and 6 mm in diameter, and the other is the scan-head (10×12×6 cm3 in size) for quasi-static scanning imaging. The final multimodal image accomplishes a maximum FOV up to 650 µm, and a resolution of 1 µm is achieved over 560 µm FOV. The optics can easily guide sub-picosecond pulses for ablation. Conclusions: The system exhibits large potential for helping real-time tissue diagnosis in surgery, by providing histological tissue information with a large FOV and high resolution, label-free. By guiding high-energy fs laser pulses, the system is even able to remove suspicious tissue areas, as has been shown for thin tissue sections in this study.

5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I.

The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5'-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5'-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5'-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly "single-stranded" 5'-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts.

Multi-Class Cancer Subtyping in Salivary Gland Carcinomas with MALDI Imaging and Deep Learning.

Salivary gland carcinomas (SGC) are a heterogeneous group of tumors. The prognosis varies strongly according to its type, and even the distinction between benign and malign tumor is challenging. Adenoid cystic carcinoma (AdCy) is one subgroup of SGCs that is prone to late metastasis. This makes accurate tumor subtyping an important task. Matrix-assisted laser desorption/ionization (MALDI) imaging is a label-free technique capable of providing spatially resolved information about the abundance of biomolecules according to their mass-to-charge ratio. We analyzed tissue micro arrays (TMAs) of 25 patients (including six different SGC subtypes and a healthy control group of six patients) with high mass resolution MALDI imaging using a 12-Tesla magnetic resonance mass spectrometer. The high mass resolution allowed us to accurately detect single masses, with strong contributions to each class prediction. To address the added complexity created by the high mass resolution and multiple classes, we propose a deep-learning model. We showed that our deep-learning model provides a per-class classification accuracy of greater than 80% with little preprocessing. Based on this classification, we employed methods of explainable artificial intelligence (AI) to gain further insights into the spectrometric features of AdCys.

Intraoperative Assessment of Tumor Margins in Tissue Sections with Hyperspectral Imaging and Machine Learning.

The intraoperative assessment of tumor margins of head and neck cancer is crucial for complete tumor resection and patient outcome. The current standard is to take tumor biopsies during surgery for frozen section analysis by a pathologist after H&E staining. This evaluation is time-consuming, subjective, methodologically limited and underlies a selection bias. Optical methods such as hyperspectral imaging (HSI) are therefore of high interest to overcome these limitations. We aimed to analyze the feasibility and accuracy of an intraoperative HSI assessment on unstained tissue sections taken from seven patients with oral squamous cell carcinoma. Afterwards, the tissue sections were subjected to standard histopathological processing and evaluation. We trained different machine learning models on the HSI data, including a supervised 3D convolutional neural network to perform tumor detection. The results were congruent with the histopathological annotations. Therefore, this approach enables the delineation of tumor margins with artificial HSI-based histopathological information during surgery with high speed and accuracy on par with traditional intraoperative tumor margin assessment (Accuracy: 0.76, Specificity: 0.89, Sensitivity: 0.48). With this, we introduce HSI in combination with ML hyperspectral imaging as a potential new tool for intraoperative tumor margin assessment.