Browsing by Subject "Immunohistochemistry"

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  • Koivisto, Maria (Helsingin yliopisto, 2020)
    Immunohistochemistry (IHC) is a widely used research tool for detecting antigens and can be used in medical and biochemical research. The co-localization of two separate proteins is sometimes crucial for analysis, requiring a double staining. This comes with a number of challenges since staining results depend on the pre-treatment of samples, host-species where the antibody was raised and spectral differentiation of the two proteins. In this study, the proteins GABAR-α2 and CAMKII were stained simultaneously to study the expression of the GABA receptor in hippocampal pyramidal cells. This was performed in PGC-1α transgenic mice, possibly expressing GABAR-α2 excessively compared to wildtype mice. Staining optimization was performed regarding primary and secondary antibody concentration, section thickness, antigen retrieval and detergent. Double staining was performed successfully and proteins of interest were visualized using a confocal microscope after which image analyses were performed using two different methods: 1) a traditional image analysis based on intensity and density of stained dots and 2) a novel convolutional neural network (CNN) machine learning approach. The traditional image analysis did not detect any differences in the stained brain slices, whereas the CNN model showed an accuracy of 72% in categorizing the images correctly as transgenic/wildtype brain slices. The results from the CNN model imply that GABAR-α2 is expressed differently in PGC-1α transgenic mice, which might impact other factors such as behaviour and learning. This protocol and the novel method of using CNN as an image analysis tool can be of future help when performing IHC analysis on brain neuronal studies.
  • Mazzoni, Annalisa; Maravic, Tatjana; Tezvergil-Mutluay, Arzu; Tjäderhane, Leo; Scaffa, Polliana Mendes Candia; Seseogullari-Dirihan, Roda; Bavelloni, Alberto; Gobbi, Pietro; Pashley, David H.; Tay, Franklin R.; Breschi, Lorenzo (2018)
    Objectives: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. Methods: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. Results: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (similar to 20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. Conclusion: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. Clinical significance: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.
  • Wirtz, Ralph M.; Sihto, Harri; Isola, Jorma; Heikkila, Paivi; Kellokumpu-Lehtinen, Pirkko-Liisa; Auvinen, Paivi; Turpeenniemi-Hujanen, Taina; Jyrkkio, Sirkku; Lakis, Sotiris; Schlombs, Kornelia; Laible, Mark; Weber, Stefan; Eidt, Sebastian; Sahin, Ugur; Joensuu, Heikki (2016)
    The biological subtype of breast cancer influences the selection of systemic therapy. Distinction between luminal A and B cancers depends on consistent assessment of Ki-67, but substantial intra-observer and inter-observer variability exists when immunohistochemistry (IHC) is used. We compared RT-qPCR with IHC in the assessment of Ki-67 and other standard factors used in breast cancer subtyping. RNA was extracted from archival breast tumour tissue of 769 women randomly assigned to the FinHer trial. Cancer ESR1, PGR, ERBB2 and MKI67 mRNA content was quantitated with an RT-qPCR assay. Local pathologists assessed ER, PgR and Ki-67 expression using IHC. HER2 amplification was identified with chromogenic in situ hybridization (CISH) centrally. The results were correlated with distant disease-free survival (DDFS) and overall survival (OS). qPCR-based and IHC-based assessments of ER and PgR showed good concordance. Both low tumour MKI67 mRNA (RT-qPCR) and Ki-67 protein (IHC) levels were prognostic for favourable DDFS [hazard ratio (HR) 0.42, 95 % CI 0.25-0.71, P = 0.001; and HR 0.56, 0.37-0.84, P = 0.005, respectively] and OS. In multivariable analyses, cancer MKI67 mRNA content had independent influence on DDFS (adjusted HR 0.51, 95 % CI 0.29-0.89, P = 0.019) while Ki-67 protein expression had not any influence (P = 0.266) whereas both assessments influenced independently OS. Luminal B patients treated with docetaxel-FEC had more favourable DDFS and OS than those treated with vinorelbine-FEC when the subtype was defined by RT-qPCR (for DDFS, HR 0.52, 95 % CI 0.29-0.94, P = 0.031), but not when defined using IHC. Breast cancer subtypes approximated with RT-qPCR and IHC show good concordance, but cancer MKI67 mRNA content correlated slightly better with DDFS than Ki-67 expression. The findings based on MKI67 mRNA content suggest that patients with luminal B cancer benefit more from docetaxel-FEC than from vinorelbine-FEC.
  • Emre, Dusunceli (Helsingin yliopisto, 2022)
    The degree of neurogenesis in the adult hippocampal dentate gyrus (DG) is the center of the discussion in the field of adult neurogenesis. Although there is an on-going controversy, accumulating evidence suggests that the neural stem cells (NSCs) in the adult human DG are very few. The question remains open as to why there are so few NSCs in the adult human DG when compared with the rodent DG. In order to address these questions, it seems necessary to understand the developmental process of the NSCs in the adult human DG. In this thesis, the neural stem and progenitor cells in the fetal human DG are characterized. In addition to these findings, a semi-automatic method for counting and categorizing cells in their expressions of immunochemistry markers is developed.
  • Passador-Santos, F.; Gronroos, M.; Irish, J.; Gilbert, R.; Gullane, P.; Perez-Ordonez, B.; Makitie, A.; Leivo, I. (2016)
    Myoepithelial carcinoma (MCA) is a rare malignancy of salivary glands that was included in the WHO Classification of Head and Neck Tumors in 1991. MCA has shown a broad spectrum of clinical outcomes, but attempts to identify prognostic markers for this malignancy have not resulted in significant progress. Conventional histopathological characteristics such as tumour grade, nuclear atypia, mitotic index and cell proliferation have failed to predict the outcome of MCA. In this study, we reviewed the histopathology of 19 cases of MCA focusing on nuclear atypia, mitotic count, tumour necrosis, nerve and vascular invasion and occurrence of a pre-existing pleomorphic adenoma in connection to the MCA. Histopathological characteristics and clinical information were correlated with the immunohistochemical expression of cell cycle proteins including c-Myc, p21, Cdk4 and Cyclin D3. The proportion of tumour cells immunoreactive for these markers and their intensity of staining were correlated with clinical information using logistic regression, Kaplan-Meier and Cox regression. Using logistic regression analysis, cytoplasmic c-Myc expression was associated with the occurrence of metastases (P = 0.019), but limitations of semi-quantitation of immunostaining and the limited number of cases preclude definitive conclusions. Our data show that the occurrence of tumour necrosis predicts poor disease-free survival in MCA (P = 0.035).
  • Candia Scaffa, Polliana Mendes; Breschi, Lorenzo; Mazzoni, Annalisa; Pimenta Vidal, Cristina de Mattos; Curci, Rosa; Apolonio, Fabianni; Gobbi, Pietro; Pashley, David; Tjaderhane, Leo; dos Santos Tersariol, Ivarne Luis; Nascimento, Fabio Dupart; Carrilhon, Marcela Rocha (2017)
    It has been hypothesized that cysteine cathepsin's (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin. Activities of CTs and MMPs in dentin extracts were evaluated spectrofluorometrically. In addition, in situ gelatinolytic activity of dentin was assayed by zymography. The results revealed the distribution of CT-B and CT-K along the dentin organic matrix and also indicated co-occurrence of MMPs and CTs in that tissue. The enzyme kinetics studies showed proteolytic activity in dentin extracts for both classes of proteases. Furthermore, it was observed that, at least for sound human dentin matrices, the activity of MMPs seems to be predominant over the CTs one. (C) 2016 Elsevier Ltd. All rights reserved.
  • Majala, Susanna; Vesterinen, Tiina; Seppänen, Hanna; Mustonen, Harri; Sundström, Jari; Schalin-Jäntti, Camilla; Gullichsen, Risto; Schildt, Jukka; Kemppainen, Jukka; Arola, Johanna; Kauhanen, Saila (2022)
    Purpose: The aim of this study was to correlate immunohistochemical (IHC) tissue levels of SSTR1-5 with the receptor density generated from [68Ga]Ga-DOTANOC uptake in a prospective series of NF-PNENs. Methods: Twenty-one patients with a total of thirty-five NF-PNEN-lesions and twenty-one histologically confirmed lymph node metastases (LN+) were included in this prospective study. Twenty patients were operated on, and one underwent endoscopic ultrasonography and core-needle biopsy. PET/CT with both [68Ga]Ga-DOTANOC and [18F]F-FDG was performed on all patients. All histological samples were re-classified and IHC-stained with monoclonal SSTR1-5 antibodies and Ki-67 and correlated with [68Ga]Ga-DOTANOC and [18F]F-FDG PET/CT. Results: Expression of SSTR1-5 was detected in 74%, 91%, 80%, 14%, and 77% of NF-PNENs. There was a concordance of SSTR2 IHC with positive/negative [68Ga]Ga-DOTANOC finding (Spearman’s rho 0.382, p = 0.043). All [68Ga]Ga-DOTANOC-avid tumors expressed SSTR2 or SSTR3 or SSTR5. Expression of SSTR5 was higher in tumors with a low Ki-67 proliferation index (PI) (−0.353, 95% CI −0.654–0.039, p = 0.038). The mean Ki-67 PI for SSTR5 positive tumors was 2.44 (SD 2.56, CI 1.0–3.0) and 6.38 (SD 7.25, CI 2.25–8.75) for negative tumors. Conclusion: SSTR2 was the only SSTR subtype to correlate with [68Ga]Ga-DOTANOC PET/CT. Our prospective study confirms SSTR2 to be of the highest impact for SST PET/CT signal.
  • Reinert, Jochim; Richard, Bernhard C.; Klafki, Hans W.; Friedrich, Beate; Bayer, Thomas A.; Wiltfang, Jens; Kovacs, Gabor G.; Ingelsson, Martin; Lannfelt, Lars; Paetau, Anders; Bergquist, Jonas; Wirths, Oliver (2016)
    In Alzheimer's disease (AD) a variety of amyloid beta-peptides (A beta) are deposited in the form of extracellular diffuse and neuritic plaques (NP), as well as within the vasculature. The generation of A beta from its precursor, the amyloid precursor protein (APP), is a highly complex procedure that involves subsequent proteolysis of APP by beta-and gamma-secretases. Brain accumulation of A beta due to impaired A beta degradation and/or altered ratios between the different A beta species produced is believed to play a pivotal role in AD pathogenesis. While the presence of A beta 40 and A beta 42 in vascular and parenchymal amyloid have been subject of extensive studies, the deposition of carboxyterminal truncated A beta peptides in AD has not received comparable attention. In the current study, we for the first time demonstrate the immunohistochemical localization of A beta 37 and A beta 39 in human sporadic AD (SAD). Our study further included the analysis of familial AD (FAD) cases carrying the APP mutations KM670/671NL, E693G and I716F, as well as a case of the PSEN1 Delta Exon9 mutation. A beta 37 and A beta 39 were found to be widely distributed within the vasculature in the brains of the majority of studied SAD and FAD cases, the latter also presenting considerable amounts of A beta 37 containing NPs. In addition, both peptides were found to be present in extracellular plaques but only scarce within the vasculature in brains of a variety of transgenic AD mouse models. Taken together, our study indicates the importance of C-terminally truncated A beta in sporadic and familial AD and raises questions about how these species are generated and regulated.
  • Sidorova, Y.; Domanskyi, A. (2020)
    Oxidative stress is prominent in many neurodegenerative diseases. Along with mitochondrial dysfunction and pathological protein aggregation, increased levels of reactive oxygen and nitrogen species, together with impaired antioxidant defense mechanisms, are frequently observed in Alzheimer’s, Parkinson’s, Huntington’s disease and amyotrophic lateral sclerosis. The presence of oxidative stress markers in patients’ plasma and cerebrospinal fluid may aid early disease diagnoses, as well as provide clues regarding the efficacy of experimental disease-modifying therapies in clinical trials. In preclinical animal models, the detection and localization of oxidatively damaged lipids, proteins and nucleic acids helps to identify most vulnerable neuronal populations and brain areas, and elucidate the molecular pathways and the timeline of pathology progression. Here, we describe the protocol for the detection of oxidative stress markers using immunohistochemistry on formaldehyde-fixed, paraffin-embedded tissue sections, applicable to the analysis of postmortem samples and tissues from animal models. In addition, we provide a simple method for the detection of malondialdehyde in tissue lysates and body fluids, which is useful for screening and the identification of tissues and structures in the nervous system which are most affected by oxidative stress. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
  • Ukkola, Iiris; Nummela, Pirjo; Pasanen, Annukka; Kero, Mia; Lepistö, Anna; Kytölä, Soili; Bützow, Ralf; Ristimäki, Ari (2021)
    Universal testing of microsatellite instability (MSI) is recommended for colorectal cancer (CRC) and endometrial cancer (EC) to screen for Lynch syndrome and to aid in assessing prognosis and optimal treatment. We compared the performance of Idylla MSI test to immunohistochemistry (IHC) of mismatch repair (MMR) proteins in consecutive series of 100 CRC and 108 EC samples, as well as in retrospective series of 28 CRC and 33 EC specimens with known deficient MMR protein expression. The concordance between the Idylla test and IHC was 100% in all CRC samples (n=128) but lower in EC samples (87.2%; n=141). In the EC samples, sensitivity of Idylla test was 72.7% and specificity 100%. EC MSI/dMMR agreement was 85.4% for MLH1, 87.5% for MSH2, and only 35.3% for MSH6. When we analyzed 14 EC samples that were discrepant, i.e., dMMR using IHC and microsatellite stable using Idylla, with microsatellite markers BAT25 and BAT26, we found four cases to be replication error (RER) positive. All RER positive cases were deficient for MSH6 protein expression. We also re-analyzed EC samples with variable tumor cellularity to determine the limit of detection of the Idylla test and found that a 30% or higher tumor cellularity is required. We conclude that Idylla MSI test offers a sensitive and specific method for CRC diagnostics but is less sensitive in EC samples especially in the case of MSH6 deficiency.
  • Abdel-Rahman, Wael M.; Ruosaari, Saila; Knuutila, Sakari; Peltomäki, Päivi T (2012)
  • Kaprio, Tuomas; Lindström, Alexander M.; Rasila, Tiina; Saavalainen, Olga; Beilmann-Lehtonen, Ines; Mustonen, Harri; Haglund, Caj; Andersson, Leif C. (2021)
    Background Colon cancer (CC) is one of the most commonly diagnosed malignancies worldwide. Several biomarkers have been suggested for improved prognostic evaluation, but few have been implemented in clinical practice. There is a need for biomarkers that predict the tumor behavior in CC and allow stratification of patients that would benefit from adjuvant therapy. We recently identified and functionally characterized a previously unknown protein that we called ASTROPRINCIN (APCN) due to its abundance in astrocytes. APCN, also annotated as FAM171A1, is found in trophoblasts of early placenta. We demonstrated that high expression levels of APCN in cancer cells induced motility and ability of invasive growth in semisolid medium. Methods We screened by immunohistochemistry a tissue microarray material from the tumors of 429 CC patients with clinical follow-up in a test series and 255 CC patients in a validation series. Results We showed that low or absent APCN expression correlates with a favorable prognosis while high APCN expression was a sign of an adverse outcome. Cox uni- and multivariable analysis revealed that elevated tumor expression of APCN constitutes a robust marker of poor prognosis independent of stage, grade, patient's age, or gender. Conclusion Our findings demonstrate that APCN is a novel independent prognostic marker in CC and could potentially select patients for more intense postoperative adjuvant treatment and follow-up.
  • Mäkinen, Lotta (Helsingin yliopisto, 2017)
    Granulosa cell tumor (GCT) is a rare ovarian malignancy, which is considered to be of low malignant potential but has a tendency to recurrence. In this study, we studied the variation of estrogen receptors in adult type GCTs in correlation with clinical data. A tumor tissue microarray containing 194 AGCT tumor samples was immunostained with antibodies against ER-β and GPER1. 93% of the tumors expressed ER-β in either their nucleus (47%) or cytoplasm (78%), and 94% of tumors expressed GPER1. The cytoplasmic expressions of ER-β and GPER1 correlated positively. Nuclear expression of ER-β was more abundant than cytoplasmic expression in tumors from premenopausal women, when compared to tumors from postmenopausal women. Overall survival tended to be worse for patients with high GPER1 expression. On the basis of these findings, we prove that AGCTs have different estrogen receptor profiles that may have an impact on clinical findings.
  • Nummela, Pirjo; Leinonen, Hannele; Järvinen, Petrus; Thiel, Alexandra; Järvinen, Heikki; Lepisto, Anna; Ristimaki, Ari (2016)
    Pseudomyxoma peritonei is a fatal clinical syndrome with mucinous tumor cells disseminated into peritoneal cavity and secreting abundant mucinous ascites. The serum tumor markers CEA, CA19-9, and CA125 are used to monitor pseudomyxoma peritonei remission, but their expression at tissue level has not been well characterized. Herein, we analyzed expression of these proteins and the adenocarcinoma marker EpCAM in 92 appendix-derived pseudomyxoma peritonei tumors by immunohistochemistry. All tumors were found to ubiquitously express CEA and EpCAM. In the majority of the tumors (94.6%), CEA showed polarized immunostaining, but in 5 aggressive high-grade tumors containing numerous signet ring cells, a nonpolarized staining was detected. We found preoperative CEA serum values to correlate with peritoneal cancer index. However, the serum values of the advanced cases with nonpolarized staining pattern were normal, and the patients died within 5 years after diagnosis. Thus, serum CEA measurements did not reflect aggressiveness of these tumors. CA19-9 showed strong immunopositivity in most of the tumors (91.3%), and mutated enzyme FUT3 was demonstrated from the cases showing negative or weak staining. CA125 Was infrequently expressed by tumor cells (focal staining in 6.5% of the cases), but in most of the cages (79.3%), adjacent nonneoplastic mesothelial cells showed immunopositivity. As a conclusion, CEA and EpCAM are invariably expressed by pseudomyxoma peritonei tumor cells and could be exploited to targeted therapies against this malignancy. (C) 2016 Elsevier Inc. All rights reserved.
  • Helin, Henrik O.; Tuominen, Vilppu J.; Ylinen, Onni; Helin, Heikki; Isola, Jorma (2016)
    Evaluation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) is subject to interobserver variation and lack of reproducibility. Digital image analysis (DIA) has been shown to improve the consistency and accuracy of the evaluation and its use is encouraged in current testing guidelines. We studied whether digital image analysis using a free software application (ImmunoMembrane) can assist in interpreting HER2 IHC in equivocal 2+ cases. We also compared digital photomicrographs with whole-slide images (WSI) as material for ImmunoMembrane DIA. We stained 750 surgical resection specimens of invasive breast cancers immunohistochemically for HER2 and analysed staining with ImmunoMembrane. The ImmunoMembrane DIA scores were compared with the originally responsible pathologists' visual scores, a researcher's visual scores and in situ hybridisation (ISH) results. The originally responsible pathologists reported 9.1 % positive 3+ IHC scores, for the researcher this was 8.4 % and for ImmunoMembrane 9.5 %. Equivocal 2+ scores were 34 % for the pathologists, 43.7 % for the researcher and 10.1 % for ImmunoMembrane. Negative 0/1+ scores were 57.6 % for the pathologists, 46.8 % for the researcher and 80.8 % for ImmunoMembrane. There were six false positive cases, which were classified as 3+ by ImmunoMembrane and negative by ISH. Six cases were false negative defined as 0/1+ by IHC and positive by ISH. ImmunoMembrane DIA using digital photomicrographs and WSI showed almost perfect agreement. In conclusion, digital image analysis by ImmunoMembrane can help to resolve a majority of equivocal 2+ cases in HER2 IHC, which reduces the need for ISH testing.
  • Birkman, Eva-Maria; Mansuri, Naziha; Kurki, Samu; Ålgars, Annika; Lintunen, Minnamaija; Ristamäki, Raija; Sundström, Jari; Carpen, Olli (2018)
    Gastric cancer is traditionally divided into intestinal and diffuse histological subtypes, but recent molecular analyses have led to novel classification proposals based on genomic alterations. While the intestinal- and diffuse-type tumours are distinguishable from each other at the molecular level, intestinal-type tumours have more diverse molecular profile. The technology required for comprehensive molecular analysis is expensive and not applicable for routine clinical diagnostics. In this study, we have used immunohistochemistry and in situ hybridisation in molecular classification of gastric adenocarcinomas with an emphasis on the intestinal subtype. A tissue microarray consisting of 244 gastric adenocarcinomas was constructed, and the tumours were divided into four subgroups based on the presence of Epstein-Barr virus, TP53 aberrations and microsatellite instability. The intestinal- and diffuse-type tumours were separately examined. The distribution of EGFR and HER2 gene amplifications was studied in the intestinal-type tumours. Epstein-Barr virus positive intestinal-type tumours were more common in male patients (p = 0.035) and most often found in the gastric corpus (p = 0.011). The majority of the intestinal-type tumours with TP53 aberrations were proximally located (p = 0.010). All tumours with microsatellite instability showed intestinal-type histology (p = 0.017) and were associated with increased overall survival both in the univariate (p = 0.040) and multivariate analysis (p = 0.015). In conclusion, this study shows that gastric adenocarcinomas can be classified into biologically and clinically different subgroups by using a simple method also applicable for clinical diagnostics.
  • Seppälä, Miia; Pohjola, Konsta; Laranne, Jussi; Rautiainen, Markus; Huhtala, Heini; Renkonen, Risto; Lemstrom, Karl; Paavonen, Timo; Toppila-Salmi, Sanna (2016)
    Tongue cancer has a poor prognosis due to its early metastasis via lymphatic vessels. The present study aimed at evaluating lymphatic vessel density, relative density of lymphatic vessel, and diameter of lymphatic vessels and its predictive role in tongue cancer. Paraffin-embedded tongue and lymph node specimens (n = 113) were stained immunohistochemically with a polyclonal antibody von Willebrand factor, recognizing blood and lymphatic endothelium and with a monoclonal antibody podoplanin, recognizing lymphatic endothelium. The relative density of lymphatic vessels was counted by dividing the mean number of lymphatic vessels per microscopic field (podoplanin) by the mean number of all vessels (vWf) per microscopic field. The high relative density of lymphatic vessels (aeyen80 %) was associated with poor prognosis in tongue cancer. The relative density of lymphatic vessels predicted poor prognosis in the group of primary tumor size T1-T2 and in the group of non-metastatic cancer. The lymphatic vessel density and diameter of lymphatic vessels were not associated with tongue cancer survival. The relative density of lymphatic vessels might have clinically relevant prognostic impact. Further studies with increased number of patients are needed.
  • Beilmann-Lehtonen, Ines; Hagström, Jaana; Mustonen, Harri K; Koskensalo, Selja; Böckelman, Camilla; Haglund, Caj (2021)
    Background: Colorectal cancer (CRC), the third most common cancer globally, caused 881,000 cancer deaths in 2018. Toll-like receptors (TLRs), the primary sensors of pathogen-associated molecular patterns and damage-associated molecular patterns, activate innate and adaptive immune systems and participate in the development of an inflammatory tumor microenvironment. We aimed to explore the prognostic value of TLR3, TLR5, TLR7, and TLR9 tissue expressions in CRC patients. Methods: Using immunohistochemistry, we analyzed tissue microarray samples from 825 CRC patients who underwent surgery between 1982 and 2002 at the Department of Surgery, Helsinki University Hospital, Finland. After analyzing a pilot series of 205 tissue samples, we included only TLR5 and TLR7 in the remainder of the patient series. We evaluated the associations between TLR5 and TLR7 tissue expressions, clinicopathologic variables, and survival. Using the Kaplan-Meier method, we generated survival curves, determining significance using the log-rank test. Univariate and multivariate survival analyses relied on the Cox proportional hazards model. Results: The 5-year disease-specific survival was 55.9% among TLR5-negative (95% confidence interval [CI] 50.6-61.2%) and 61.9% (95% CI 56.6-67.2%; p = 0.011, log-rank test) among TLR5-positive patients. In the Cox multivariate survival analysis adjusted for age, sex, stage, location, and grade, positive TLR5 immunoexpression (hazard ratio [HR] 0.74; 95% CI 0.59-0.92; p = 0.007) served as an independent positive prognostic factor. TLR7 immunoexpression exhibited no prognostic value in the survival analysis across the entire cohort (HR 0.97; 95% CI 0.78-1.20; p = 0.754) nor in subgroup analyses. Conclusions: We show for the first time that a high TLR5 tumor tissue expression associates with a better prognosis in CRC patients.
  • Aaltonen, Niina; Singha, Prosanta K.; Jakupovic, Hermina; Wirth, Thomas; Samaranayake, Haritha; Pasonen-Seppanen, Sanna; Rilla, Kirsi; Varjosalo, Markku; Edgington-Mitchell, Laura E.; Kasperkiewicz, Paulina; Drag, Marcin; Kälvälä, Sara; Moisio, Eemeli; Savinainen, Juha R.; Laitinen, Jarmo T. (2020)
    Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.
  • Aaltonen, Niina; Singha, Prosanta K; Jakupović, Hermina; Wirth, Thomas; Samaranayake, Haritha; Pasonen-Seppänen, Sanna; Rilla, Kirsi; Varjosalo, Markku; Edgington-Mitchell, Laura E; Kasperkiewicz, Paulina; Drag, Marcin; Kälvälä, Sara; Moisio, Eemeli; Savinainen, Juha R; Laitinen, Jarmo T (BioMed Central, 2020)
    Abstract Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.