Transcriptomic and proteomic study of cancer cell lines exposed to actinomycin D and nutlin-3a reveals numerous, novel candidates for p53-regulated genes.

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI-H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/ß-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.

Proteomics of urinary small extracellular vesicles in early diagnosis of kidney diseases in children-expectations and limitations.

The primary function of the kidneys is to maintain systemic homeostasis (disruption of renal structure and function results in multilevel impairment of body function). Kidney diseases are characterized by a chronic, progressive course and may result in the development of chronic kidney disease (CKD). Evaluation of the composition of the proteome of urinary small extracellular vesicles (sEVs) as a so-called liquid biopsy is a promising new research direction. Knowing the composition of sEV could allow localization of cellular changes in specific sections of the nephron or the interstitial tissue before fixed changes, detectable only at an advanced stage of the disease, occur. Research is currently underway on the role of sEVs in the diagnosis and monitoring of many disease entities. Reports in the literature on the subject include: diabetic nephropathy, focal glomerulosclerosis in the course of glomerulopathies, renal fibrosis of various etiologies. Studies on pediatric patients are still few, involving piloting if small groups of patients without validation studies. Here, we review the literature addressing the use of sEV for diagnosis of the most common urinary disorders in children. We evaluate the clinical utility and define limitations of markers present in sEV as potential liquid biopsy.

Immune capture and protein profiling of small extracellular vesicles from human plasma.

Extracellular vesicles (EVs), the key players in inter-cellular communication, are produced by all cell types and are present in all body fluids. Analysis of the proteome content is an important approach in structural and functional studies of these vesicles. EVs circulating in human plasma are heterogeneous in size, cellular origin, and functions. This heterogeneity and the potential presence of contamination with plasma components such as lipoprotein particles and soluble plasma proteins represent a challenge in profiling the proteome of EV subsets by mass spectrometry. An immunocapture strategy prior to mass spectrometry may be used to isolate a homogeneous subpopulation of small EVs (sEV) with a specific endocytic origin from plasma or other biofluids. Immunocapture selectively separates EV subpopulations in biofluids based on the presence of a unique protein carried on the vesicle surface. The advantages and disadvantages of EV immune capture as a preparative step for mass spectrometry are discussed.

Prognostic Value of Molecular Intratumor Heterogeneity in Primary Oral Cancer and Its Lymph Node Metastases Assessed by Mass Spectrometry Imaging.

Different aspects of intra-tumor heterogeneity (ITH), which are associated with the development of cancer and its response to treatment, have postulated prognostic value. Here we searched for potential association between phenotypic ITH analyzed by mass spectrometry imaging (MSI) and prognosis of head and neck cancer. The study involved tissue specimens resected from 77 patients with locally advanced oral squamous cell carcinoma, including 37 patients where matched samples of primary tumor and synchronous lymph node metastases were analyzed. A 3-year follow-up was available for all patients which enabled their separation into two groups: with no evidence of disease (NED, n = 41) and with progressive disease (PD, n = 36). After on-tissue trypsin digestion, peptide maps of all cancer regions were segmented using an unsupervised approach to reveal their intrinsic heterogeneity. We found that intra-tumor similarity of spectra was higher in the PD group and diversity of clusters identified during image segmentation was higher in the NED group, which indicated a higher level of ITH in patients with more favorable outcomes. Signature of molecular components that correlated with long-term outcomes could be associated with proteins involved in the immune functions. Furthermore, a positive correlation between ITH and histopathological lymphocytic host response was observed. Hence, we proposed that a higher level of ITH revealed by MSI in cancers with a better prognosis could reflect the presence of heterotypic components of tumor microenvironment such as infiltrating immune cells enhancing the response to the treatment.

Proteomic and Metabolomic Profiles of T Cell-Derived Exosomes Isolated from Human Plasma.

Exosomes that are released by T cells are key messengers involved in immune regulation. However, the molecular profiling of these vesicles, which is necessary for understanding their functions, requires their isolation from a very heterogeneous mixture of extracellular vesicles that are present in the human plasma. It has been shown that exosomes that are produced by T cells could be isolated from plasma by immune capture using antibodies that target the CD3 antigen, which is a key component of the TCR complex that is present in all T lymphocytes. Here, we demonstrate that CD3(+) exosomes that are isolated from plasma can be used for high-throughput molecular profiling using proteomics and metabolomics tools. This profiling allowed for the identification of proteins and metabolites that differentiated the CD3(+) from the CD3(-) exosome fractions that were present in the plasma of healthy donors. Importantly, the proteins and metabolites that accumulated in the CD3(+) vesicles reflected the known molecular features of T lymphocytes. Hence, CD3(+) exosomes that are isolated from human plasma by immune capture could serve as a "T cell biopsy".

Radiation-Induced Bystander Effect Mediated by Exosomes Involves the Replication Stress in Recipient Cells.

Exosomes released by irradiated cells mediate the radiation-induced bystander effect, which is manifested by DNA breaks detected in recipient cells; yet, the specific mechanism responsible for the generation of chromosome lesions remains unclear. In this study, naive FaDu head and neck cancer cells were stimulated with exosomes released by irradiated (a single 2 Gy dose) or mock-irradiated cells. Maximum accumulation of gamma H2A.X foci, a marker of DNA breaks, was detected after one hour of stimulation with exosomes from irradiated donors, the level of which was comparable to the one observed in directly irradiated cells (a weaker wave of the gamma H2A.X foci accumulation was also noted after 23 h of stimulation). Exosomes from irradiated cells, but not from control ones, activated two stress-induced protein kinases: ATM and ATR. Noteworthy is that while direct irradiation activated only ATM, both ATM and ATR were activated by two factors known to induce the replication stress: hydroxyurea and camptothecin (with subsequent phosphorylation of gamma H2A.X). One hour of stimulation with exosomes from irradiated cells suppressed DNA synthesis in recipient cells and resulted in the subsequent nuclear accumulation of RNA:DNA hybrids, which is an indicator of impaired replication. Interestingly, the abovementioned effects were observed before a substantial internalization of exosomes, which may suggest a receptor-mediated mechanism. It was observed that after one hour of stimulation with exosomes from irradiated donors, phosphorylation of several nuclear proteins, including replication factors and regulators of heterochromatin remodeling as well as components of multiple intracellular signaling pathways increased. Hence, we concluded that the bystander effect mediated by exosomes released from irradiated cells involves the replication stress in recipient cells.

Intra-Tumor Heterogeneity Revealed by Mass Spectrometry Imaging Is Associated with the Prognosis of Breast Cancer.

Intra-tumor heterogeneity (ITH) results from the coexistence of genetically distinct cancer cell (sub)populations, their phenotypic plasticity, and the presence of heterotypic components of the tumor microenvironment (TME). Here we addressed the potential association between phenotypic ITH revealed by mass spectrometry imaging (MSI) and the prognosis of breast cancer. Tissue specimens resected from 59 patients treated radically due to the locally advanced HER2-positive invasive ductal carcinoma were included in the study. After the on-tissue trypsin digestion of cellular proteins, peptide maps of all cancer regions (about 380,000 spectra in total) were segmented by an unsupervised approach to reveal their intrinsic heterogeneity. A high degree of similarity between spectra was observed, which indicated the relative homogeneity of cancer regions. However, when the number and diversity of the detected clusters of spectra were analyzed, differences between patient groups were observed. It is noteworthy that a higher degree of heterogeneity was found in tumors from patients who remained disease-free during a 5-year follow-up (n = 38) compared to tumors from patients with progressive disease (distant metastases detected during the follow-up, n = 21). Interestingly, such differences were not observed between patients with a different status of regional lymph nodes, cancer grade, or expression of estrogen receptor at the time of the primary treatment. Subsequently, spectral components with different abundance in cancer regions were detected in patients with different outcomes, and their hypothetical identity was established by assignment to measured masses of tryptic peptides identified in corresponding tissue lysates. Such differentiating components were associated with proteins involved in immune regulation and hemostasis. Further, a positive correlation between the level of tumor-infiltrating lymphocytes and heterogeneity revealed by MSI was observed. We postulate that a higher heterogeneity of tumors with a better prognosis could reflect the presence of heterotypic components including infiltrating immune cells, that facilitated the response to treatment.

Proteomic profile of melanoma cell-derived small extracellular vesicles in patients' plasma: a potential correlate of melanoma progression.

Molecular profiling of small extracellular vesicles (sEV) isolated from plasma of cancer patients emerges as promising strategy for biomarkers discovery. We investigated the proteomic profiles of sEV immunoselected using anti-CSPG4 antibodies from 15 melanoma patients' plasma. The proteomes of sEV separated into melanoma cell-derived (MTEX) and non-malignant cell-derived (NMTEX) were compared using high-resolution mass spectrometry. Paired analysis identified the MTEX-associated profile of 16 proteins that discriminated MTEX from NMETEX. We also identified the MTEX profile that discriminated between seven patients with no evidence of melanoma (NED) after therapy and eight with progressive disease (PD). Among 75 MTEX proteins overexpressed in PD patients, PDCD6IP (ALIX) had the highest discriminating value, while CNTN1 (contactin-1) was upregulated only in MTEX of NED patients. This is the first report documenting that proteomes of tumour-derived sEV in patients' plasma discriminate cancer from non-cancer and identify proteins with potential to serve as prognostic biomarkers in melanoma.

Classification of Thyroid Tumors Based on Mass Spectrometry Imaging of Tissue Microarrays; a Single-Pixel Approach.

The primary diagnosis of thyroid tumors based on histopathological patterns can be ambiguous in some cases, so proper classification of thyroid diseases might be improved if molecular biomarkers support cytological and histological assessment. In this work, tissue microarrays representative for major types of thyroid malignancies-papillary thyroid cancer (classical and follicular variant), follicular thyroid cancer, anaplastic thyroid cancer, and medullary thyroid cancer-and benign thyroid follicular adenoma and normal thyroid were analyzed by mass spectrometry imaging (MSI), and then different computation approaches were implemented to test the suitability of the registered profiles of tryptic peptides for tumor classification. Molecular similarity among all seven types of thyroid specimens was estimated, and multicomponent classifiers were built for sample classification using individual MSI spectra that corresponded to small clusters of cells. Moreover, MSI components showing the most significant differences in abundance between the compared types of tissues detected and their putative identity were established by annotation with fragments of proteins identified by liquid chromatography-tandem mass spectrometry in corresponding tissue lysates. In general, high accuracy of sample classification was associated with low inter-tissue similarity index and a high number of components with significant differences in abundance between the tissues. Particularly, high molecular similarity was noted between three types of tumors with follicular morphology (adenoma, follicular cancer, and follicular variant of papillary cancer), whose differentiation represented the major classification problem in our dataset. However, low level of the intra-tissue heterogeneity increased the accuracy of classification despite high inter-tissue similarity (which was exemplified by normal thyroid and benign adenoma). We compared classifiers based on all detected MSI components (n = 1536) and the subset of the most abundant components (n = 147). Despite relatively higher contribution of components with significantly different abundance and lower overall inter-tissue similarity in the latter case, the precision of classification was generally higher using all MSI components. Moreover, the classification model based on individual spectra (a single-pixel approach) outperformed the model based on mean spectra of tissue cores. Our result confirmed the high feasibility of MSI-based approaches to multi-class detection of cancer types and proved the good performance of sample classification based on individual spectra (molecular image pixels) that overcame problems related to small amounts of heterogeneous material, which limit the applicability of classical proteomics.

Molecular Heterogeneity of Papillary Thyroid Cancer: Comparison of Primary Tumors and Synchronous Metastases in Regional Lymph Nodes by Mass Spectrometry Imaging.

Intra-tumor heterogeneity results from both genetic heterogeneity of cancer (sub)clones and phenotypic plasticity of cancer cells that could be induced by different local microenvironments. Here, we used mass spectrometry imaging (MSI) to compare molecular profiles of primary tumors located in the thyroid gland and their synchronous metastases in regional lymph nodes to analyze phenotypic heterogeneity in papillary thyroid cancer. Two types of cancerous (primary tumor and metastasis) and two types of not cancerous (thyroid gland and lymph node) regions of interest (ROIs) were delineated in postoperative material from 11 patients, then the distribution of tryptic peptides (spectral components) was analyzed by MSI in all tissue regions. Moreover, tryptic peptides identified by shotgun proteomics in corresponding tissue lysates were matched to components detected by MSI to enable their hypothetical protein annotation. Unsupervised segmentation of all cancer ROIs revealed that different clusters dominated in tumor ROIs and metastasis ROIs. The intra-patient similarity between thyroid and tumor ROIs was higher than the intra-patient similarity between tumor and metastasis ROIs. Moreover, the similarity between tumor and its metastasis from the same patients was lower than similarities among tumors and among metastases from different patients (inter-patient similarity was higher for metastasis ROIs than for tumor ROIs). Components differentiating between tumor and its metastases were annotated as proteins involved in the organization of the cytoskeleton and chromatin, as well as proteins involved in immunity-related functions. We concluded that phenotypical heterogeneity between primary tumor and lymph node metastases from the same patient was higher than inter-tumor heterogeneity between primary tumors from different patients.

MS-Based Proteomic Analysis of Serum and Plasma: Problem of High Abundant Components and Lights and Shadows of Albumin Removal.

Blood serum or plasma proteome is a gold mine of disease biomarkers. However, complexity and a huge dynamic range of their components, combined with multiple mechanisms of degradation and posttranslational modifications, further complicated by the presence of lipids, salts, and other metabolites, represent a real challenge for analytical sensitivity, resolution, and reproducibility. This problem exists particularly in the case of potential disease-specific markers, most typically represented by low-abundant proteins (LAPs), whose detection is usually impaired by the dominance of albumins, immunoglobulins, and other high-abundant serum/plasma proteins (HAPs). Hence, analysis of biomarker candidates in serum/plasma samples frequently requires separation of their components, usually including depletion of albumin in a fraction of interest. Such "preprocessing" of serum/plasma specimens is critical in proteomic analysis based on mass spectrometry. This approach is very potent; nevertheless a wide range of protein concentrations in serum/plasma represents a particular challenge, since high-abundant proteins (mostly albumin) dominate in a sample subjected to mass spectrometry and suppress peptide ions originating from low-abundant proteins, thus limiting probability and reliability of their detection. An emerging approach in serum-/plasma-based biomarker-oriented studies is the proteome component of exosomes - nanovesicles secreted by cells and involved in multiple aspects of intercellular communication. However, the presence of albumin, frequent contaminant of exosomes isolated from human serum/plasma, represents a real challenge also in this type of study. A similar problem is encountered in proteomic studies based on exosomes obtained in in vitro experiments where culture media are normally supplemented with fetal bovine serum containing growth factors and hormones. In this case exosomes are frequently contaminated with bovine serum albumin and other bovine serum proteins which should be removed before proteomic analysis of exosome cargo.

Proteomes of exosomes from HPV(+) or HPV(-) head and neck cancer cells: differential enrichment in immunoregulatory proteins.

Human papillomavirus (HPV) is an etiologic factor in head and neck squamous cell carcinoma (HNSCC). HPV(+) cancers respond favorably to therapy potentially due to more robust anti-tumor immune responses. We hypothesized that tumor-derived exosomes (TEX) produced by HPV(+) or HPV(-) HNSCCs differentially modulate anti-tumor immune responses. Proteomes of exosomes from HPV(+) and HPV(-) HNSCC cell lines were compared in search for proteins putatively involved in the communication with immune system. TEX were isolated from supernatants of HPV(+) (SCC-2, SCC-47, and SCC-90) or HPV(-) (PCI-13 and PCI-30) cells by size exclusion chromatography. A comparison of proteome profiles was performed by high-resolution mass spectrometry. The presence and biological activity of selected immunoregulatory proteins were validated by flow cytometry and co-incubation assays. Exosomes produced by SCC-90 and PCI-30 cells contained 711 proteins, including 80 proteins specific for HPV(+) exosomes and 77 specific for HPV(-) exosomes, associated with similar GO terms such as regulation of cell growth, metabolism, communication, and cellular signaling. Search for proteins localized in the membrane and involved in immune regulation identified a few proteins detected specifically in HPV(+) or HPV(-) exosomes. Only HPV(+) exosomes were enriched in immune effector cell-related CD47 and CD276 antigens; only HPV(-) exosomes contained tumor-protective/growth-promoting antigens, MUC-1 and HLA-DA. Flow cytometry and Western blots confirmed the reciprocal presence/paucity of these proteins in a whole panel of tumor cells and corresponding exosomes. The differential content of protein cargos in HPV(+) and HPV(-) exosomes might contribute to the disparity in immune responses that characterize HPV(+) and HPV(-) HNSCC.

Discrimination of normal oral mucosa from oral cancer by mass spectrometry imaging of proteins and lipids.

Identification of biomarkers for molecular classification of cancer and for differentiation between cancerous and normal epithelium remains a vital issue in the field of head and neck cancer. Here we aimed to compare the ability of proteome and lipidome components to discriminate oral cancer from normal mucosa. Tissue specimens including squamous cell cancer and normal epithelium were analyzed by MALDI mass spectrometry imaging. Two molecular domains of tissue components were imaged in serial sections-peptides (resulting from trypsin-processed proteins) and lipids (primarily zwitterionic phospholipids), then regions of interest corresponding to cancer and normal epithelium were compared. Heterogeneity of cancer regions was higher than the heterogeneity of normal epithelium, and the distribution of peptide components was more heterogeneous than the distribution of lipid components. Moreover, there were more peptide components than lipid components that showed significantly different abundance between cancer and normal epithelium (median of the Cohen's effect was 0.49 and 0.31 in case of peptide and lipid components, respectively). Multicomponent cancer classifier was tested (vs. normal epithelium) using tissue specimens from three patients and then validated with a tissue specimen from the fourth patient. Peptide-based signature and lipid-based signature allowed cancer classification with a weighted accuracy of 0.85 and 0.69, respectively. Nevertheless, both classifiers had very high precision (0.98 and 0.94, respectively). We concluded that though molecular differences between cancerous and normal mucosa were higher in the proteome domain than in the analyzed lipidome subdomain, imaging of lipidome components also enabled discrimination of oral cancer and normal epithelium. Therefore, both cancer proteome and lipidome are promising sources of biomarkers of oral malignancies.

Proteome profiles of different types of thyroid cancers.

Proteomics profiling of tissue specimens representative for major types of thyroid cancers: papillary (classical and follicular variant), follicular, anaplastic and medullary, as well as benign follicular adenoma, was performed using shotgun LC-MS/MS approaches. A combination of Orbitrap and MALDI-TOF approach allowed to identify protein products of 3700 unique genes and revealed large differences between medullary, anaplastic and epithelium-derived differentiated cancers (papillary and follicular). Proteins characteristic for medullary and anaplastic cancers included factors associated with neuroendocrine functions and factors typically associated with advanced malignancies, respectively. Proteomes of different types of epithelium-derived differentiated cancers and follicular adenoma were compared using multi-enzyme LC-MS/MS approach, which revealed products of 4800 unique genes. A comparable overall similarity of follicular cancers to both variants of papillary cancers was found. Moreover, follicular adenoma showed higher overall similarity to follicular cancer than to either variant of papillary cancer. Proteins discriminating differentiated thyroid neoplasms included factors associated with lipid and hormone metabolism, regulation of gene expression and maintenance of DNA structure. Importantly, proteome data matched several features of transcriptome and metabolome profiles of thyroid cancers contributing to systems biology of this malignancy.

Isolation of Exosomes for the Purpose of Protein Cargo Analysis with the Use of Mass Spectrometry.

Exosomes are intercellular messengers with a high potential for diagnostic and therapeutic utility. It is believed that exosomes present in body fluids are responsible for providing signals which inhibit immune cells, interfere with antitumor immunity, and thus influence the response to treatment and its effect. One of the most interesting issues in exosome studies is proper addressing of their cargo composed of nucleic acids and proteins. Effective and selective isolation of extracellular vesicles and identification of proteins present in exosomes has turned out to be a challenging aspect of their exploration. Here we propose a novel approach that is based on isolation of exosomes by mini-size-exclusion chromatography which allows efficient, rapid, and reliable isolation of morphologically intact and functionally active exosomes without the need of ultracentrifugation. The purpose of this chapter is to describe a simple and high-throughput method to isolate, purify, and identify exosomal proteins using a mass spectrometry approach. The proposed protocol compiles the expertise of two research groups specialized in exosome research and in mass spectrometry-based proteomics. The protocol combines differential centrifugation followed by ultrafiltration, centrifugation-based filtration, and gel filtration on Sepharose 2B in order to obtain exosomal fractions characterized by only low contamination with albumin.

Molecular profiles of thyroid cancer subtypes: Classification based on features of tissue revealed by mass spectrometry imaging.

Determination of the specific type of thyroid cancer is crucial for the prognosis and selection of treatment of this malignancy. However, in some cases appropriate classification is not possible based on histopathological features only, and it might be supported by molecular biomarkers. Here we aimed to characterize molecular profiles of different thyroid malignancies using mass spectrometry imaging (MSI) which enables the direct annotation of molecular features with morphological pictures of an analyzed tissue. Fifteen formalin-fixed paraffin-embedded tissue specimens corresponding to five major types of thyroid cancer were analyzed by MALDI-MSI after in-situ trypsin digestion, and the possibility of classification based on the results of unsupervised segmentation of MALDI images was tested. Novel method of semi-supervised detection of the cancer region of interest (ROI) was implemented. We found strong separation of medullary cancer from malignancies derived from thyroid epithelium, and separation of anaplastic cancer from differentiated cancers. Reliable classification of medullary and anaplastic cancers using an approach based on automated detection of cancer ROI was validated with independent samples. Moreover, extraction of spectra from tumor areas allowed the detection of molecular components that differentiated follicular cancer and two variants of papillary cancer (classical and follicular). We concluded that MALDI-MSI approach is a promising strategy in the search for biomarkers supporting classification of thyroid malignant tumors. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Detection of molecular signatures of oral squamous cell carcinoma and normal epithelium - application of a novel methodology for unsupervised segmentation of imaging mass spectrometry data.

Intra-tumor heterogeneity is a vivid problem of molecular oncology that could be addressed by imaging mass spectrometry. Here we aimed to assess molecular heterogeneity of oral squamous cell carcinoma and to detect signatures discriminating normal and cancerous epithelium. Tryptic peptides were analyzed by MALDI-IMS in tissue specimens from five patients with oral cancer. Novel algorithm of IMS data analysis was developed and implemented, which included Gaussian mixture modeling for detection of spectral components and iterative k-means algorithm for unsupervised spectra clustering performed in domain reduced to a subset of the most dispersed components. About 4% of the detected peptides showed significantly different abundances between normal epithelium and tumor, and could be considered as a molecular signature of oral cancer. Moreover, unsupervised clustering revealed two major sub-regions within expert-defined tumor areas. One of them showed molecular similarity with histologically normal epithelium. The other one showed similarity with connective tissue, yet was markedly different from normal epithelium. Pathologist's re-inspection of tissue specimens confirmed distinct features in both tumor sub-regions: foci of actual cancer cells or cancer microenvironment-related cells prevailed in corresponding areas. Hence, molecular differences detected during automated segmentation of IMS data had an apparent reflection in real structures present in tumor.

Tissue fixed with formalin and processed without paraffin embedding is suitable for imaging of both peptides and lipids by MALDI-IMS.

Type and quality of sample preparation have significant impact on imaging mass spectrometry results. Though imaging of fresh-frozen tissues is considered to give the best results, they are incompatible with clinical practice, since routine diagnostics is most frequently performed using formalin-fixed tissues, and formalin-fixed paraffin-embedded material is a gold standard in histopathology. We aimed to assess utility of formalin-fixed tissue specimen processed without paraffin embedding (i.e., deep-frozen and cryo-sectioned) for MALDI imaging of both peptides and lipids. Peptide and lipid imaging was performed in fresh-frozen, FFPE and formalin-fixed/frozen samples of a mouse kidney, then composition of the resulting spectra was compared. We demonstrated similarity of spectra registered during peptide imaging in FFPE and formalin-fixed/frozen tissues, and similarity of spectra registered during lipid imaging in fresh-frozen and formalin-fixed/frozen material. Furthermore, molecular images of formalin-fixed/frozen tissue resembled the features of both fresh-frozen and FFPE tissue in the case of peptide imaging, and the features of fresh-frozen tissue in the case of lipid imaging. We conclude that tissue preserved by formalin fixation and processed without paraffin embedding can be considered as an alternative to both fresh-frozen and FFPE material.

Preparation of a new Cd(II)-imprinted polymer and its application to determination of cadmium(II) via flow-injection-flame atomic absorption spectrometry.

A new cadmium(II)-imprinted polymer based on cadmium(II) 2,2'-{ethane-1,2-diylbis[nitrilo(E)methylylidene]} diphenolate-4-vinylpyridine complex was obtained via suspension polymerization. The beads were used as a minicolumn packing for flow-injection-flame atomic absorption spectrometry (FI-FAAS) determination of cadmium(II) in water samples. Sorption effectiveness was optimal within pH range of 6.6-7.7. Nitric acid, 0.5% (v/v) was used as eluent. Fast cadmium(II) sorption by the proposed material enabled to apply sample flow rates up to 10mLmin(-1) without loss in sorption effectiveness. Enrichment factor (EF), concentration efficiency (CE) and limit of detection (LOD, 3sigma) found for 120-s sorption time were 117, 39.1min(-1) and 0.11microgL(-1), respectively. Sorbent stability was proved for at least 100 preconcentration cycles (RSD=2.9%). When compared to non-imprinted polymer the new Cd(II)-imprinted polymer exhibited improved selectivity towards cadmium(II) against other heavy metal ions, especially Cu(II) and Pb(II), as well as light metal ions. Accuracy of the method was tested for ground water and waste water certified reference materials and fortified water. The method was applied to Cd(II) determination in natural water samples.

Application of a metal ion-imprinted polymer based on salen-Cu complex to flow injection preconcentration and FAAS determination of copper.

A new Cu(II)-imprinted polymer (Cu-IIP) for preconcentration of copper by liquid-solid extraction via flow injection technique has been proposed. Cu-IIP was obtained by copolymerization of salen-Cu(II) complex with styrene and divinylbenzene using suspension polymerization technique. Granules fraction of 60-80 microm in diameter was used as a microcolumn packing. Cu(II) sorption was proved to be the most effective from solutions of pH 7, whereas similar elution effectiveness was observed when applying as eluents hydrochloric or nitric acid in the concentration range of 0.5-10% (v/v). The system exhibited good long-term stability and acid resistance. Batch sorbent capacity was found to be 0.11 mmol g(-1) of a dry polymer. Enrichment factor (EF) for 30 s loading time was 16. Preconcentration of Cu(II) and potentially interfering metal ions is strongly pH dependent. Examination of Cu(II) sorption in the presence of Pb(II), Cd(II), Zn(II) and Ag(I) showed significant influence of cadmium and zinc ions only and that was for the interferent concentrations above 0.5 mg L(-1) (Cu-IIP mass of ca. 35 mg). The interference effect was reduced with the sorbent mass increase. Fe(III) and Mn(II) ions, present in treated tap water in relatively high concentrations, did not interfere. Effective pH adjusting of the loaded solution in on-line mode, when applying diluted Clark-Lubs buffering solution, allowed accurate copper determination in tap water (compared to graphite furnace atomic absorption spectrometry, GFAAS) using standard addition or combination calibration method.