Analysis Of Human Tissue Samples Research Articles

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

Abstract SY36-01: Somatic evolution, cancer, and our inevitable decline with age: Inextricably linked

Abstract Why do we get cancer? Why is cancer highly associated with old age? Aging is associated with the accumulation of more mutations; some of which can contribute to cancer phenotypes. However, we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices, or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. To better understand the evolutionary forces that control somatic cell evolution, we used mouse models of cancer initiation, mathematical models of cellular evolution, and analyses of human tissue samples. We have shown that aging- and inflammation-dependent changes in stem cells and their tissue environments dramatically dictate whether cancer-causing mutations are advantageous to stem cells in our tissues, starting the cells down the path to cancer and deepening our insight into cancer risk. Recent studies from many labs have shown how as we age our tissues become dominated by clones bearing mutations in known cancer-associated genes. I will present studies from our labs exploring how cigarette smoking and aging can alter mutational landscapes in the non-malignant lung. To observe somatic mutations in lung biopsies from individuals with different smoking histories, we used a rare-mutation detection technique called Duplex Sequencing to analyze somatic variants from over 200 lung samples, including from subjects from the BDRE (Colorado), PEACE, and TRACERx cohorts. Using this method, we have identified large numbers of mutations in each brushing or biopsy, many of which are cataloged in the Catalogue Of Somatic Mutations In Cancer (COSMIC), and most of these are predicted to disrupt protein function. We also observe pervasive positive selection acting on mutations in many but not all of the cancer-associated genes, with different patterns of selection in the lungs of ever-smokers and never-smokers. Ongoing studies are using primary human and mouse lung epithelial cultures and mouse models to study how these mutations interact with smoking and age to alter associated clonal expansions, tissue integrity, and malignant progression. We propose a model whereby aging and microenvironmental changes induced through lifestyle choices like smoking can promote selection for cells with adaptive mutations, which can contribute to not only cancer risk but also tissue aging. Thus, while young tissues impede selection for such adaptive mutations, old age is associated with a feed-forward loop of aging tissue-mediated selection for mutant clones that then increase tissue aging. Thus, understanding the forces controlling clonal selection as we age and due to lifetime exposures could be critical for controlling multiple diseases of old age. [EJE, EL and FM contributed equally.These studies were led by CS and JD.] Citation Format: Edward J. Evans, Emilia Lim, Fabio Marongiu, Shi Biao Chia, William Hill, Clare Weeden, Oriol Pich, Andrew Rowan, Faiz Jabbar, Cristina Naceur-Lombardelli, Raju Veeriah, Moumita Ghosh, Daniel Merrick, York E. Miller, Robert Keith, Mariam Jamal-Hanjani, Charles Swanton, James V. DeGregori. Somatic evolution, cancer, and our inevitable decline with age: Inextricably linked [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY36-01.

Abstract 101: A novel humanized mouse model recapitulates the unique TME found in patients with HIV-associated NSCLC

Abstract Among people with HIV (PWH), non-small cell lung cancer (NSCLC) is increasing in incidence, presents with more advanced disease, and portends a worse prognosis compared to the general NSCLC population. Despite effective control of viral replication with antiretroviral therapy (ART), PWH have evidence of immune dysfunction, and it is unknown whether these immune perturbations impact the tumor microenvironment (TME) to influence disease disparities.Here we preclinically model HIV-associated NSCLC using MISTRG6-A2, a humanized mouse system that is highly optimized for development of functional innate and adaptive immune cells. We compare tumor growth and immune features of HIV-infected, ART-suppressed MISTRG6-A2 hosts to uninfected hosts, with parameters informed by parallel analyses of human tissue samples obtained from PWH with NSCLC, and non-HIV controls.MISTRG6-A2 mice were engrafted with human CD34+ HSPCs from HLA-A*02-expressing donors on post-natal day 2 and intravenously infected with HIV-1 at 6 weeks of age. When HIV viral titers were >106 copies of viral RNA/ml plasma and hCD4 T cells were depleted, ART was initiated (RAL/FTC/TDF). When plasma viral RNA was undetectable and CD4 T cells recovered, NSCLC PDX tissue from HLA-A*02-expressing tumor was implanted into these HIV-infected, ART-suppressed MISTRG6-A2 mice as well as non-infected littermate controls which had received HSPCs from the same donor. HIV-infected mice displayed enhanced growth of PDX tissue (mean tumor size 206.7 mm3 vs 110.2 mm3; p < 0.05).Quantitative immunofluorescence of the TME from these hosts revealed significantly increased infiltration of CD4 and CD8 T cells in tumors of HIV-NSCLC mice (p<0.01 and 0.05, respectively); of note, enhanced T cell infiltration was restricted to tumors, with similar frequency of CD4 and CD8 T cells detected in spleen, lung and liver tissues of HIV-infected vs uninfected hosts. Staining of tumor epitopes revealed increased expression of B2M and EGFR in tumors from HIV-NSCLC mice (mean qIF scores in NSCLC vs HIV-NSCLC 3.8 × 106 vs 1.2 × 107 for B2M, p<0.001; 440 vs 1,971 for EGFR, p<0.01), consistent with our findings in samples from HIV-NSCLC tumor tissues vs NSCLC tumor tissues. Of note, elevated EGFR staining was only present in tumor tissue, not found in stromal cells or other tissues. Single cell transcriptomic analyses of the TME revealed prominent interferon and antigen presentation signatures in HIV-NSCLC tumors, as well as differential expression of immunoregulatory molecules.These results demonstrate the fidelity of the HIV-NSCLC MISTRG6-A2 system as a model for HIV-associated NSCLC and suggest EGFR-directed therapies as potentially relevant in this neglected disease. Citation Format: Melani Juric, Gabriel Kaufmann, Li Zhu, Kishu Ranjan, Kriti Agrawal, Barani Kumar Rajendran, Jyothi K. Rajashekar, Hongyu Zhao, Yuval Kluger, Brinda Emu, Kurt A. Schalper, Priti Kumar, Richard A. Flavell, Michael Chiorazzi. A novel humanized mouse model recapitulates the unique TME found in patients with HIV-associated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 101.

Quantitative proteomics I.: Concept, design, and planning of quantitative proteomics experiments.

Quantitative proteomics I.: Concept, design, and planning of quantitative proteomics experiments.

Evaluation of different analytical approaches using total reflection X-ray fluorescence systems for multielemental analysis of human tissues with different adipose content

Elemental content plays an important role in biological processes, and so, the multielemental analysis of human tissue samples is required in biomedical research. Still, the small amount of available biological samples and the adipose content of the samples can be major setbacks for the accurate determination of elemental content.In this study, we explored the potential of several analytical approaches combined with total reflection X-ray fluorescence spectrometry (TXRF) for multielemental analysis of human tissues with different adipose content (colon, heart, liver, lung, muscle, intestine, skin, stomach, uterus, bladder and aorta). The capabilities and limitations of different sample treatment procedures (suspension and acidic digestion) and two TXRF systems with different anode configurations (Mo and W X-ray tubes) have been evaluated for such purpose. Results showed that for tissues with a higher fat content (e.g., skin, and intestine) the best strategy was the acidic digestion of the sample before TXRF analysis. However, for other tissues, acceptable results were obtained by suspending 20 mg of powdered material in 1 mL of 2 M nitric acid. A further enhancement of the limits of detection and accuracy of the results was achieved if using Mo-TXRF systems, especially for the determination of low Z elements (e.g., K, and Ca) and of elements present at low concentrations (e.g., Cu) in the human tissues.Finally, results by TXRF analysis were compared with those obtained with μ-EDXRF and ICP-OES, and a good agreement was obtained.

Abstract SY24-01: Changes in adaptive landscapes and somatic evolution as we age

Abstract Why do we get cancer? Why is cancer highly associated with old age, and why are insults like smoking, obesity, and sunlight associated with increased risk of cancers? Of course, these contexts all cause mutations, and some of these mutations can contribute to malignant phenotypes. But we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are microenvironmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, somatic evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. We will discuss the evidence from many labs for microenvironmental change driven selection in the evolution of cancer, additional support from epidemiological and clinical studies, as well as the limitations, inconsistencies and unknowns that require the attention of cancer researchers. My own lab has been using mouse models of cancer initiation, mathematical models of clonal evolution, and analyses of human tissue samples to better understand the evolutionary forces that control somatic cell evolution and thus cancer risk. Using Monte Carlo modeling, we have explored the impact of key somatic evolutionary parameters on multi-stage carcinogenesis, revealing that two additional major mechanisms, aging-dependent somatic selection and life history-dependent evolution of species-specific tumor suppressor mechanisms, need to be incorporated into the multi-stage model of carcinogenesis to make it capable of generalizing cancer incidence across tissues and species. Using mouse models, we have shown that aging and inflammation dependent changes in tissue microenvironments dramatically impact the fitness impact of oncogenic mutations, leading to aging and inflammation driven clonal expansions in the hematopoietic compartment. These studies have also uncovered molecular mechanisms underlying oncogene-driven adaptation to aged and inflammatory tissue environments. In all, these studies indicate that strategies to prevent or treat cancers will need to incorporate interventions that alter tissue microenvironments. While we largely cannot prevent mutation accumulation through our lives, we do have the ability to manipulate tissue microenvironments so as to change the evolutionary trajectories of oncogenically-mutated cells. Citation Format: James V. DeGregori. Changes in adaptive landscapes and somatic evolution as we age [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-01.

Abstract IA012: Aging, tissue ecology, and the evolution of cancer within us

Abstract Why do we get cancer? Why is cancer highly associated with old age? Of course, aging is associated with the accumulation of more mutations, and some of these mutations can contribute to cancer phenotypes. But we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. We have been using mouse models of cancer initiation, mathematical models of cellular evolution, and analyses of human tissue samples to better understand the evolutionary forces that control somatic cell evolution and thus cancer risk. We have shown that aging and inflammation dependent changes in tissue environments dramatically dictate whether cancer-causing mutations are advantageous to stem cells in our tissues, starting the cells down the path to cancer. Our studies have focused on cancer initiation within the hematopoietic system and the lung. These studies have also uncovered molecular explanations for mutation-driven adaptation to aged and inflammatory tissue environments. In all, these studies indicate that strategies to prevent or treat cancers will need to incorporate interventions that alter tissue microenvironments. Citation Format: James DeGregori, Catherine Pham-Danis, Andrii I Rozhok, Edward J. Evans, Fabio Marongiu, Hannah Scarborough, Curtis J. Henry. Aging, tissue ecology, and the evolution of cancer within us [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA012.

Abstract IA13: In the light of evolution: Why do we get more cancers in old age?

Abstract Why do we get cancer? Why is cancer highly associated with old age? Of course, aging is associated with the accumulation of more mutations, and some of these mutations can contribute to cancer phenotypes. But we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. We have been using mouse models of cancer initiation, mathematical models of cellular evolution, and analyses of human tissue samples to better understand the evolutionary forces that control somatic cell evolution and thus cancer risk. We have shown that aging and inflammation dependent changes in tissue environments dramatically dictate whether cancer-causing mutations are advantageous to stem cells in our tissues, starting the cells down the path to cancer. Our studies have focused on cancer initiation within the hematopoietic system and the lung. These studies have also uncovered molecular explanations for mutation-driven adaptation to aged and inflammatory tissue environments. In all, these studies indicate that strategies to prevent or treat cancers will need to incorporate interventions that alter tissue microenvironments. While we largely cannot prevent mutation accumulation through our lives, we do have the ability to manipulate tissue environments so as to change the evolutionary trajectories of cells with cancer-causing mutations. Citation Format: Catherine Pham-Danis, Andrii Rozhok, Hannah Scarborough, Nathaniel Little, Curtis Henry, Travis Nemkov, Kirk Hansen, James DeGregori. In the light of evolution: Why do we get more cancers in old age? [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA13.

Stem cell derived oligodendrocytes to study myelin diseases.

Oligodendroglial pathology is central to de- and dysmyelinating, but also contributes to neurodegenerative and psychiatric diseases as well as brain injury. The understanding of oligodendroglial biology in health and disease has been significantly increased during recent years by experimental in vitro and in vivo preclinical studies as well as histological analyses of human tissue samples. However, for many of these diseases the underlying pathology is still not fully understood and treatment options are frequently lacking. This is at least partly caused by the limited access to human oligodendrocytes from patients to perform functional studies and drug screens. The induced pluripotent stem cell technology (iPSC) represents a possibility to circumvent this obstacle and paves new ways to study human disease and to develop new treatment options for so far incurable central nervous system (CNS) diseases. In this review, we summarize the differences between human and rodent oligodendrocytes, provide an overview of the different techniques to generate oligodendrocytes from human progenitor or stem cells and describe the results from studies using iPSC derived oligodendroglial lineage cells. Furthermore, we discuss future perspectives and challenges of the iPSC technology with respect to disease modeling, drug screen, and cell transplantation approaches.

Abstract LB-328: Loss of TFF1 leads to activation of RAGE in mouse and human gastric tumorigenesis

Abstract Background: The receptor for advanced glycation end products (RAGE) is a central signaling molecule in the innate immune system and is highly expressed in various cancers including gastric cancer. RAGE is a pattern recognition receptor that binds to multiple ligands leading to its activation and onset of pro-inflammatory response. Trefoil factor 1 (TFF1) is a small secreted protein expressed in the gastrointestinal tract which plays a key role in protection of gastric mucosal membrane and provides protection from injury and damage, therefore acts as tumor suppressor gene. In this study we investigated the role of TFF1 in regulation and degradation of RAGE receptor in vivo and in vitro. Material and Results: Using quantitative real-time PCR, we observed significant increase of RAGE expression in gastric tissue from Tff1 knockout from non-dysplastic lesion; this increase of expression of RAGE was more notable in dysplastic samples, as compared to normal tissues (p<0.001). The Tff1-KO mice tissues with gastritis and low grade dysplasia (LGD) and high grade dysplasia (HGD) showed significantly progressive higher expression of RAGE, as compared to normal tissues (p<0.001). Analysis of human tissue samples demonstrated significant overexpression of RAGE, as compared to normal gastric tissues (p=0.01). Furthermore, we analyzed paired tumor and normal samples, and we found a significant up-regulation of RAGE mRNA expression in tumors compared to their corresponding normal samples (p=0.01). We also detected an inverse relationship between TFF1 and RAGE levels in mouse and human tissue samples. We next determined the levels of RAGE ligands, S100A2 and A4. The results showed significant increase in expression levels of S100A2 and A4 in human and mouse neoplastic gastric lesions, as compared to normal tissue samples (P<.01). To determine a causal relationship between TFF1 and RAGE, we used in vitro cell models. Using Western blot and RT-PCR analysis, the reconstitution of TFF1 in AGS cells showed a decrease in expression levels of RAGE, S100A2, and A4, as compared to control cells. Treatment of cancer cell lines (AGS, MKN28, and MKN45) with RAGE inhibitor Azeliragon led to significant reduction in cell viability with IC50 range AGS= 1.65µM, MKN28=1.22µM, MKN45= 1.37µM . Conclusion: Our data demonstrate, for the first time, that high levels of RAGE and its ligands S100A2 and A4 are mechanistically related to TFF1 levels. RAGE inhibitors demonstrated a therapeutic efficacy in pre-clinical in vitro models. Additional in vitro and in vivo studies are planned to determine the functional, mechanistic and therapeutic outcomes of RAGE in gastric tumorigenesis. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Zheng Chen, Shoumin Zhu, Huma Naz, Ahmed Gomaa, Sen Wang, Barry I. Hudson, El-Rifai Wael. Loss of TFF1 leads to activation of RAGE in mouse and human gastric tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-328.

Effects of the IL-23-IL-17 pathway onbone in spondyloarthritis.

Over the past several years, a pathophysiological role for the IL-23-IL-17 pathway in human disease has been defined. A subset of rheumatic diseases, including psoriatic arthritis (PsA) and ankylosing spondylitis (AS), are now acknowledged to be triggered by dysregulated IL-23-IL-17 pathway activation. Genetic evidence links the IL-23-IL-17 pathway to inflammation in these rheumatic diseases, and mechanistic data from mice support a functional role for IL-23-IL-17 pathway activation in the development of enthesitis and in entheseal bone formation. Furthermore, analysis of human tissue samples, as well as data from clinical trials, also supports a role for activation of the IL-23-IL-17 pathway in these diseases. The unique bone phenotype that occurs in PsA and AS is a surprising coexistence of both systemic bone loss and periosteal and entheseal bone formation and is likely to be the result of the actions of IL-23 and/or IL-17 on bone. However, the effects of these cytokines on bone cells are complex, and controversy remains regarding their exact roles in the specific bone microenvironments relevant to PsA and AS.

A database of tissue-specific rhythmically expressed human genes has potential applications in circadian medicine.

The discovery that half of the mammalian protein-coding genome is regulated by the circadian clock has clear implications for medicine. Recent studies demonstrated that the circadian clock influences therapeutic outcomes in human heart disease and cancer. However, biological time is rarely given clinical consideration. A key barrier is the absence of information on tissue-specific molecular rhythms in the human body. We have applied the cyclic ordering by periodic structure (CYCLOPS) algorithm, designed to reconstruct sample temporal order in the absence of time-of-day information, to the gene expression collection of 13 tissues from 632 human donors. We identified rhythms in gene expression across the body; nearly half of protein-coding genes were shown to be cycling in at least 1 of the 13 tissues analyzed. One thousand of these cycling genes encode proteins that either transport or metabolize drugs or are themselves drug targets. These results provide a useful resource for studying the role of circadian rhythms in medicine and support the idea that biological time might play a role in determining drug response.

PO-281 Automated multiparametric tissue imaging platform using existing microscope hardware for the detection of spatially resolved single-cell resolution data

IntroductionCharacterising complexities of the tumour microenvironment is elemental to understanding disease mechanisms. The spatial relationships between infiltrating immune cells and the remodelling of the cellular matrix is widely recognised as a key component to defining tumour heterogeneity. Current methodologies for analysing the spatial dimension in tissues, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limited to a few parameters at a time, restricting the scope of identifiable cells. Conversely, single-cell technologies like mass cytometry and NGS-based tools provide multiplexing capabilities, but at the expense of the associated spatial information. Here we present a novel multiplexed imaging technology, termed CODEX, (CO-Detection by indEXing) that combines the high-parameter space of single-cell methodologies with the spatial analysis.Material and methodsThe CODEX technology involves labelling antibodies with oligonucleotide-based tags followed by a single staining step. Over 50 parameters are simultaneously measured in a single specimen in Akoya’s novel, fully automated process. Unlike other cyclic IF approaches involving multiple antibody staining and stripping steps, the CODEX platform involves a single initial staining step and subsequent gentle manipulation of the tissue thereafter. This provides a faster workflow and prevents tissue degradation due to the harsh conditions used to remove adhered antibody. Other multiplexed imaging technologies, including imaging cytometry and MIBI, require expensive equipment precluding their routine use across various labs. The CODEX technology developed by Akoya is comprised of a fluidics instrument that interfaces with existing microscope hardware as well as a suite of associated specialised reagent offerings.Results and discussionsHere we present the results of an analysis of human tissue samples, including both fresh-frozen (FF) and formalin-fixed paraffin embedded (FFPE) specimens using the CODEX platform. Panels of antibodies were validated against a variety of markers and used to stain human tonsil, lymph node and tumour tissues. These data were processed using a custom analysis pipeline from image registration through cell segmentation. Single-cell data was analysed using clustering algorithms to define cell types.ConclusionThis CODEX study showcases the impact of high-parameter spatially resolved analysis of clinically relevant tissues enables the detection of unique cell types and cell niches within the tumour microenvironment.

Invited commentary

Although we have witnessed the evolution of the endovascular repair of abdominal aortic aneurysm (AAA) in the past two decades, there still is a great need for vigorous scientific research to establish the effective pharmacologic therapy for AAA. One common research strategy is to conduct high-quality observational studies using large databases. Such database analysis has been used with increasing frequency in many aspects of vascular surgery clinical research. An alternative method is to perform basic science research by conducting molecular and histologic analysis of the human tissue samples, animal models, and cell-based assays. In this article by Doderer and coauthors, the authors performed detailed analysis of human tissue samples and showed the role of adventitial fatty degeneration in AAA. The authors collected tissue samples from the open repair of AAA and popliteal artery aneurysm. By comparing the two similar but distinct diseases, they found the extensive adipocyte aggregation and fatty degeneration only in AAA. The molecular analyses of these tissues subsequently showed the enrichment of adipogenic mediators in AAA tissue. Adipogenesis is one of the key cell differentiation processes during which preadipocytes differentiate into mature adipocytes. This process has two steps: adipocyte determination and differentiation. These steps are controlled by a well-organized, multistep process that requires numerous transcription factors like CCAAT/enhancer-binding protein gene family and peroxisome proliferator-activated receptor γ. The authors identified an abundance of these transcriptional factors in the AAA wall with adipocyte clusters. Theoretically, any pharmacologic therapy for AAA without postponing surgery or preventing rupture is clinically worthless, and thus AAA research should also focus on rupture prevention. Currently, we use the maximum diameter of AAA as the sole indicator for surgical repair as it is known to correlate with the risk of rupture. In this article, the authors compared ruptured and nonruptured AAA tissues and showed the association between adipogenesis and rupture. Interestingly, another recent report also supports this finding by showing the adipocyte accumulation with associated macrophage accumulation in ruptured AAA in animal models.1Kugo H. Zaima N. Tanaka H. Mouri Y. Yanagimoto K. Hayamizu K. et al.Adipocyte in vascular wall can induce the rupture of abdominal aortic aneurysm.Sci Rep. 2016; 6: 31268Crossref PubMed Scopus (34) Google Scholar To summarize, the authors showed that fatty degeneration is a distinctive feature of AAA. Furthermore, they showed the upregulation of adipogenesis and peroxisome proliferator-activated receptor γ signaling pathways in ruptured AAA compared with nonruptured AAA. I hope the further exploration of these interesting findings will open up the next therapeutic target for AAA and will advance the diagnostic and therapeutic strategy of treating AAA. The opinions or views expressed in this commentary are those of the authors and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery or the Society for Vascular Surgery. Adventitial adipogenic degeneration is an unidentified contributor to aortic wall weakening in the abdominal aortic aneurysmJournal of Vascular SurgeryVol. 67Issue 6PreviewThe processes driving human abdominal aortic aneurysm (AAA) progression are not fully understood. Although antiinflammatory and proteolytic strategies effectively quench aneurysm progression in preclinical models, so far all clinical interventions failed. These observations hint at an incomplete understanding of the processes involved in AAA progression and rupture. Interestingly, strong clinical and molecular associations exist between popliteal artery aneurysms (PAAs) and AAAs; however, PAAs have an extremely low propensity to rupture. Full-Text PDF Open Archive

Human IgG Increases Virulence of Streptococcus pyogenes through Complement Evasion

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.

Matrixmetalloproteinases and tissue inhibitors of metalloproteinases: Immunhistochemical markers in the diagnosis of lethal myocardial infarctions?

Matrixmetalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of metalloproteinases (TIMP) 1 were tested as markers of myocardial early ischemia/infarctions. Experiments with an animal model, the isolated Langendorff heart, and analysis of human tissue samples drawn during autopsies were performed. Results of the experiments with the Langendorff model implied that the detectable amount of the markers might increase early after the onset of ischemia, in less than one hour, under ideal conditions. The results of the examined human cases showed that MMP-2 is constantly detectable in human myocardial tissue with an increased amount in case of an infarction with longer survival times. MMP-9 and TIMP-1 were negative in control cases, distinct positive staining results were obtained mainly in cases of infarctions with longer survival times and only rarely in those with a short survival time. According to these results MMPs and TIMPs do not qualify as first choice markers of myocardial infarctions. As an interesting side finding in the Langendorff experiments, positive staining results for all three markers were seen in myocardial areas that were mechanically traumatized by ECG-electrodes or ligation of blood vessels. These findings make the markers interesting for forensic wound age estimation.

Clinical significance of immunohistochemically detected extracellular matrix proteins and their spatial distribution in primary cancer.

Our understanding of cancer has evolved mainly from results of studies utilizing experimental models. Simplification inherent to in vitro cell culture models enabled potential ways of cell behaviour in response to various external stimuli to be described, but it has led also to disappointments in clinical trials, presumably due to the lack of crucial tissue components, including extracellular matrix (ECM). ECM and its role in healthy and diseased tissues are being explored extensively and significance of ECM for cell behaviour has been evidenced experimentally. Part of the information gathered in such research that is relevant for natural conditions of a human body can be identified by carefully designed analyses of human tissue samples. This review summarizes published information on clinical significance of ECM in cancer and examines whether effects of ECM on cell behaviour evidenced in vitro, could be supported by clinically based data acquired from analysis of tissue samples. Based on current approaches of clinical immunohistochemical analyses, impact of ECM components on tumour cell behaviour is vague. Except of traditionally considered limitations, other reasons may include lack of stratification of analyzed cases based on clinicopathologic parameters, inclusion of patients treated postoperatively by different treatments or neglecting complexity of interactions among tumour constituents. Nevertheless, reliable immunohistochemical studies represent a source of crucial information for design of tumour models comprising ECM corresponding to real clinical situation. Knowledge gathered from such immunohistochemical studies combined with achievements in tissue engineering hold promise for reversal of the unfavourable trends in the current translational oncologic research.

Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity.

Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility.

APE1-mediated DNA damage repair provides survival advantage for esophageal adenocarcinoma cells in response to acidic bile salts.

Chronic Gastroesophageal Reflux Disease (GERD) is the main risk factor for the development of Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Accordingly, EAC cells are subjected to high levels of oxidative stress and subsequent DNA damage. In this study, we investigated the expression and role of Apurinic/apyrimidinic endonuclease 1 (APE1) protein in promoting cancer cell survival by counteracting the lethal effects of acidic bile salts (ABS)-induced DNA damage. Immunohistochemistry analysis of human tissue samples demonstrated overexpression of APE1 in more than half of EACs (70 of 130), as compared to normal esophagus and non-dysplastic BE samples (P < 0.01). To mimic in vivo conditions, we treated in vitro cell models with a cocktail of ABS. The knockdown of endogenous APE1 in EAC FLO-1 cells significantly increased oxidative DNA damage (P < 0.01) and DNA single- and double-strand breaks (P < 0.01), whereas overexpression of APE1 in EAC OE33 cells reversed these effects. Annexin V/PI staining indicated that the APE1 expression in OE33 cells protects against ABS-induced apoptosis. In contrast, knockdown of endogenous APE1 in FLO-1 cells increased apoptosis under the same conditions. Mechanistic investigations indicated that the pro-survival function of APE1 was associated with the regulation of stress response c-Jun N-terminal protein kinase (JNK) and p38 kinases. Pharmacological inhibition of APE1 base excision repair (BER) function decreased cell survival and enhanced activation of JNK and p38 kinases by ABS. Our findings suggest that constitutive overexpression of APE1 in EAC may be an adaptive pro-survival mechanism that protects against the genotoxic lethal effects of bile reflux episodes.

Abstract 2088: Kindlin-3 enhances breast cancer metastasis through upregulation of Twist-mediated tumor angiogenesis

Abstract The FERM domain containing protein, Kindlin-3, has been recognized as a major regulator of integrin function in hematopoietic cells but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in kindlin-3 mRNA compared to non-neoplastic tissue by quantitative polymerize chain reaction. At the protein level, Kindlin-3 was found to be expressed abundantly in breast cancer tissue, and its expression increases with the advancing stage of tumor development. Mining of the Oncomine database (www.oncomine.com) confirmed this association: Kindlin-3 was in the top 3 % of gene products elevated in breast cancer (p&amp;lt; 10-12). When Kindlin-3 was over-expressed in a breast cancer cell line, primary tumor growth and lung metastasis increased by 2.5- and 3-fold, respectively when implanted into mice compared to cells expressing vector alone. Mechanistically, the Kindlin-cells displayed a 2.2-fold increase in Vascular Endothelial Growth Factor (VEGF) secretion and B1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor which promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production and knockdown of B1 integrins diminished Twist and VEGF production by Kindlin-3 cells while non-targeting si-RNA had no effect on expression of these gene products. The activation of this signaling cascade resulted in the enhancement of breast cancer cell invasion, and tumor angiogenesis and metastasis. Hence, our studies established the pathway in which cross-talk between Kindlin-3 and B1 integrins activates the nuclear translocation of TWIST, which leads to enhanced VEGF-A production and secretion, macrophage infiltration as well as activation of the Epithelial-to-mesenchymal-transition (EMT) program. The activation of this signaling cascade culminates in enhanced breast cancer cell migration, invasion and tumor angiogenesis, all of which are required for tumor progression and metastasis. In summary, by analysis of human tissue samples and in vitro and in vivo studies, we have identified a novel role for Kindlin-3 in the progression of breast cancer. To our knowledge, this is the first demonstration of the presence and implication of Kindlin-3 in breast cancer or any cancer. Citation Format: Khalid Sossey-Alaoui, Elzbieta Pluskota, Gangarao Davuluri, Katarzyna Bialkowska, Mitali Das, Daniel Lindner, Edward F. Plow. Kindlin-3 enhances breast cancer metastasis through upregulation of Twist-mediated tumor angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2088. doi:10.1158/1538-7445.AM2014-2088