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Background: Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry. However, FF samples are comprised of multiple cell types, making it difficult to ascertain the proteomic profiles of specific cells. Conversely, OCT-embedded (Optimal Cutting Temperature compound) specimens can undergo laser microdissection (LMD) to capture and study specific cell types separately from the cell mixture. In the current study, we compared proteomic data obtained from FF and OCT samples to determine if samples that are stored and processed differently produce comparable results. Methods: Proteins were extracted from FF and OCT-embedded invasive breast tumors from 5 female patients. FF samples were lysed via homogenization (FF/HOM) while OCT-embedded specimens underwent LMD to collect only tumor cells (OCT/LMD-T) or both tumor and stromal cells (OCT/LMD-TS) followed by incubation at 37°C. Proteins were extracted using the illustra triplePrep kit and then trypsin-digested, TMT-labeled, and processed by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Proteins were identified and quantified with Proteome Discoverer v1.4 and comparative analyses performed to identify proteins that were significantly differentially expressed amongst the different processing methods. Results: Among 4,950 proteins consistently quantified across all samples, 216 and 171 proteins were significantly differentially expressed (adjusted p-value < 0.05; |log2 FC| > 1) between FF/HOM vs. OCT/LMD-T and FF/HOM vs. OCT/LMD-TS, respectively, with most proteins being more highly abundant in the FF/HOM samples. PCA and unsupervised hierarchical clustering analysis with these 216 and 171 proteins were able to distinguish FF/HOM from OCT/LMD-T and OCT/LMD-TS samples, respectively. Likewise, PCA analysis and unsupervised clustering analysis using the 402 and 60 significantly differentially enriched GO terms (adjusted p-value (BH) < 0.2) in the FF/HOM vs. OCT/LMD-T and FF/HOM vs OCT/LMD-TS comparisons, respectively, not only distinguished OCT/LMD from FF/HOM samples but also separated LA and LB1 breast cancer subtypes within each storage/preparation method from one another. Although FF/HOM appears to be more similar to OCT/LMD-TS than OCT/LMD-T based on the number of differentially enriched proteins (216 vs. 171; p=0.022) and GO terms (402 vs. 60; p < 2.2 x 10-16), FF/HOM shows no greater similarity to OCT/LMD-TS than OCT/LMD-T based on PCA analysis with either proteins or GO terms ( based on weighted distance for pairwise samples, p = 0.97 from paired t-test). No significantly differentially enriched proteins or GO terms were detected between the OCT/LMD-T and OCT/LMD-TS samples but trended differences were detected. Conclusions: The proteomic profiles of the OCT/LMD-TS samples were more similar to those from OCT/LMD-T samples than FF/HOM samples, suggesting a strong influence from the sample processing methods. These results indicate that in LC-MS/MS proteomic studies, FF/HOM samples exhibit different protein profiles from OCT/LMD samples and thus, results from these two different methods cannot be directly compared. Our study also provides preliminary data for designing new studies to explore why OCT/LMD-TS samples are more similar to OCT/LMD-T than to FF/HOM samples, and to separate LA from LB1 samples. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DOD or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Lori A. Sturtz, Guisong Wang, Punit Shah, Richard Searfoss, Praveen-Kumar Raj-Kumar, Jeffrey A. Hooke, J. Leigh Fantacone-Campbell, Brenda Deyarmin, Mary Lou Cutler, Rangaprasad Sarangarajan, Niven R. Narain, Hai Hu, Michael A. Kiebish, Albert J. Kovatich, Craig D. Shriver. Comparative analysis of differentially abundant proteins quantified by LC-MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-38.

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist's toolbox.

Increasing studies have utilized mass spectrometry imaging (MSI) that is a label-free tool to investigate drug penetration and drug biotransformation in multicellular tumor spheroids (MCTS). Currently, the gelatin-assisted sectioning method is widely used to prepare frozen sections of MCTS for MSI. However, owing to the limited transparency of frozen gelatin, MCTS with diameters less than 500 μm that closely mimic solid tumors are difficult to be detected when cryosectioning. In order to identify the presence of MCTS, hematoxylin and eosin staining for frozen sections and dye pretreatment for MCTS were employed in previous works, which either increased the analytical time and cost in sample preparation or caused signal suppression in sample analysis. Herein, a new sectioning method was developed to prepare MCTS frozen sections. MCTS was coated with ice to ensure good visibility for small-size MCTS. The optimal cutting temperature compound was added around the ice block to assist the formation of frozen sections. A precast frozen mold was prepared to allow the acquisition of complete MCTS frozen sections. The developed method was applied to investigate lipid distribution in MCTS by using matrix-assisted laser desorption/ionization MSI. Compared to the gelatin-assisted sectioning method, our method did not cause signal suppression and analyte delocalization. Thus, this method provides an easy, universal, and innovative strategy to prepare MCTS frozen sections for further MSI analysis. Besides, we applied our method to investigate the penetration of bisphenol A in MCTS.

RATIONALE Biobanks of patient tissues have emerged as essential resources in biomedical research. Optimal cutting temperature compound (OCT) blends have shown to provide stability to the embedded tissue and are compatible with spectroscopic methods, such as infrared (IR) and Raman spectroscopy. Data derived from omics-methods are only useful if tissue damage caused by storage in OCT blends is minimal and well understood. In this context, we investigated the suitability of OCT storage for heart tissue destined for liquid chromatography/tandem mass spectrometry (LC/MS/MS) lipidomic studies. METHODS To determine the compatibility of OCT storage with LC/MS/MS lipidomics studies. The lipid profiles of macaque heart tissue snap-frozen in liquid nitrogen or stored in an OCT blend were evaluated. RESULTS We have evaluated a lipid extraction protocol suitable for OCT-embedded tissue that is compatible with LC/MS/MS. We annotated and evaluated the profiles of 306 lipid species from tissues stored in OCT or liquid nitrogen. For most of the lipid species (95.4%), the profiles were independent of the storage conditions. However, 4.6% of the lipid species; mainly plasmalogens, were affected by the storage method. CONCLUSIONS This study shows that OCT storage is compatible with LC-MS/MS lipidomics of heart tissue, facilitating the use of biobanked tissue samples for future studies.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1—non-muscle invasive bladder cancer (NMIBC), and T2/T3—muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers., Open access funding provided by Uppsala University., This work was supported by Magnus Bergvall Foundation (S.B.L, 2017-02330, 2018-02726, 2019-03296), Clas Groschinsky memory foundation (S.B.L., M1603, M1742), and Swedish Foundation for Strategic research (S.B.L., SB16-0039). A.V. would like to acknowledge the Ministry of Science and Innovation for a “Juan de la Cierva” postdoctoral Grant.

Background: Primary aldosteronism (PA) is the most common cause of secondary hypertension, accounting for 5-8% of all hypertension. PA is most commonly attributed to an aldosterone-producing adenoma (APA) or to bilateral hyperaldosteronism (BHA). Mutations in the inward-rectifying K+ channel (mKCNJ5), which increase autonomous aldosterone production, are most frequently detected in APAs. APAs with mKCNJ5 display aberrant expression of aldosterone synthase (CYP11B2) and 17α-hydroxylase (CYP17A1), which are involved in aldosterone and cortisol synthesis, respectively. Co-expression of these enzymes results in the production of a set of “hybrid” steroids, which have been proposed as serum biomarkers. The relative production of hybrid steroids in adrenal tumors vs. adjacent normal adrenal (NA) tissue has not been investigated. Objectives: To determine the utility of OCT-embedded adrenal tumor tissue for steroid profiling. To use immunohistochemistry (IHC)-guided OCT tumor capture for intratumoral hybrid steroid profiling in mKCNJ5 APA and NA tissue. Methods: OCT-embedded adrenal tissue from 9 patients (8 women, Age 45.9 ± 3.3 years) with APAs harboring known KCNJ5 mutations were used for the study. Where available OCT-embedded normal adrenal (NA) tissue adjacent to APAs were used as controls (n=4). IHC was performed for CYP11B2 and CYP17A1 on OCT tissue allowing guided APA capture from serial sections. Steroids were extracted from APA and adjacent NA tissue, and quantified by liquid chromatography/tandem mass spectrometry. Steroids measured were normalized to the protein content of the extracted tissue. Results: Compared to NA, APA tissue demonstrated 23-, 5.6- and 6.4-fold higher levels of aldosterone, 11-deoxycorticosterone, and 18-hydroxycorticosterone, respectively (P

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 µm thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 µm apart were prepared to assess a total of >104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts ÷ by the number of wild type (blue) crypts in a treatment group.

Although numerous studies have examined global mRNA expression in the whole endometrium, very few studies aimed at characterising the global mRNA profile at specific cellular types within this tissue. In this study, we hypothesised that metabolic status during the postpartum period can affect specifically gene expression of endometrial luminal, glandular epithelial and stromal cells. To achieve this, endometrial biopsies were performed in 22 postpartum cows receiving either a control diet (n = 12, aiming for 35 kg/d of energy-corrected milk, ECM) or a mild restricted diet (n = 10, aiming for 25 kg/d of ECM). The diet affected the metabolic status of the cows as documented in Ntallaris et al. (2017 Theriogenology 90, 276-283). Three endometrial biopsies were collected for each cow between 12 and 15 days post-oestrus (oestrus was synchronized with CIDR and all cows had progesterone concentrations >5 ng mL−1 at time of biopsy). Biopsies were frozen in Optimal Cutting Temperature compound and then 10-µm sections were obtained by using cryostat. Slides were fixed in ethanol and stained using cresol violet. Luminal and glandular epithelial cells as well as stromal cells were harvested by laser capture microdissection using the PixCell II LCM System. Following capture, mRNA was extracted from each cellular types using the Arcturus PicoPure RNA Isolation (Thermo Fisher Scientific, Waltham, MA, USA). A genome-wide transcriptomic profile of endometrial cells from biopsies was generated with RNAseq. Samples were sequenced in 50-length paired. The average number of reads across all samples was near 100 million. Reads were mapped on the UMD3.1 assembly of the Bos taurus genome using Tophat. Quantification of gene expression has been performed using HTSeq with annotations from Ensembl. The comparisons of interest have been performed using DESEqn 2 (R package) with the statistical method proposed by Love et al. (2014 Genome Biol. 15, 550). With an adjusted P-value of 0.05, 8, 571, and 2658 genes were found significantly differentially expressed between animals fed with low or control diets for glandular, stromal, and luminal cells respectively. These results provide novel insights into mechanisms regulating endometrium physiology in postpartum dairy cows. The number of differentially expressed genes according to diet was much higher in luminal epithelial and stromal cells than in glandular cells providing evidence that these cells express differential responses to the metabolic environment to which they are exposed. Therefore, transcriptomic analyses based only on full tissue, as possibly biased by differences in cell numbers of different types could be not fully relevant. We acknowledge the financial support of ‘Prolific’ (EU grant KBBE; 31176) and RMUTSV (Thailand)

Frozen human tissues are necessary for research purposes, but tissue banking methods have not changed for more than a decade. Many institutions use cryovial tubes or plastic molds with an optimal cutting temperature compound. However, these methods are associated with several problems, such as samples sticking to one another and the need for a larger storing space. We established an efficient tissue freezing and storing procedure ("tissue tablet method") applicable to both molecular analysis and frozen tissue microarray. Tissue samples were chopped into tiny fragments and embedded into tablet-shaped frozen optimal cutting temperature compound using our original tissue-freezing plate. These tablets can be sectioned and stored in cryovial tubes. We compared the tissue quality of tablet-shaped samples with that of conventional optimal cutting temperature blocks and found no significant difference between them. Tissue microarray is a key method to utilize tissue-banking specimens. However, most tissue microarrays require the coring out of cylindrically shaped tissues from formalin-fixed, paraffin-embedded tissue blocks. Antigenic changes and mRNA degradation are frequently observed with formalin-fixed, paraffin-embedded samples. Therefore, we have applied tablet-shaped samples to construct frozen tissue microarrays with our original mounting base. Constructed tissue microarray sections showed good morphology without obvious artifact and good immunohistochemistry and in situ hybridization results. These results suggest that the quality of arrayed samples was sufficiently appropriate for research purposes. In conclusion, the tissue tablet method and frozen tissue microarray procedure can save time, provides easy tissue handling and processing, and satisfies the demands of research methodologies and tissue banking.

Formalin is a routine fixative facilitating tissue preservation and histopathology. Proteomic techniques require freshly frozen specimens, which are often difficult to procure, and methods facilitating proteomic analysis of archival formalin-fixed brain tissue are lacking. We employed antigen-epitope-retrieval principles to facilitate proteomic analysis of brain tissue that had been fixed and stored in formalin for 3-7 years. Twenty-micrometer-thick cryopreserved OCT-embedded sections from inferior temporal cortex of human (7 years in formalin) or mouse brain specimens (3 years in formalin) were hematoxylin-/eosin-stained. Approximately 16-64-mm2 areas of the tissue sections were manually scraped off slides, or approximately 2 mm2 of human brain cortex was captured off membrane-coated slides using laser microdissection. Tissue was treated using various pH and temperature conditions prior to trypsin digestion and nano-LC-MS/MS. The largest number of proteins were retrieved by solubilization at pH 9 at 95 degrees C for 1 h; treatments at pH 4 or 6 at 25 or 65 degrees C were generally ineffective. Three-year formalin-fixed murine tissue did not yield more proteins compared to human tissue. Use of formalin-fixed tissue for proteomics is an invaluable tool for medical research. The combination of proteomics and microdissection enables selective enrichment and identification of novel, unique, or abundant proteins that may be important in pathogenesis.

Optimal cutting temperature (OCT) is a widely used embedding medium for tissues for histopathologic analysis. This investigation examined the effects that OCT storage can have upon the ability to perform subsequent molecular biological analyses. Tumor material was dissected into small pieces and stored at approximately 20 degrees C both with and without OCT. DNA and RNA were then extracted from the tissue fragments and analyzed by the polymerase chain reaction (PCR), using primer sets designed to amplify a range of product sizes, and also by reverse transcriptase-PCR (RT-PCR). The storage of pathological specimens in OCT compound was found to affect significantly and irreversibly the ability to amplify DNA in the PCR, particularly as the size of the amplified fragment increased. This effect appeared to occur as a result of greater degradation of DNA extracted from tissue embedded in OCT compared to DNA extracted from tissue stored without OCT. RNA quality appeared unaffected, which may be because of the extraction protocol employed. Our results suggest that OCT-embedded frozen-tissue samples may be used for RNA isolation for subsequent RT-PCR and for the in vitro amplification of DNA targets of approximately300 base pairs only. We strongly advise against the routine storage of any tissue biopsy material in OCT if molecular analyses may be required.