Pattern Of Integrin Expression Research Articles

A Machine Learning-Based Investigation of Integrin Expression Patterns in Cancer and Metastasis.

Integrins, a family of transmembrane receptor proteins, play complex roles in cancer development and metastasis. These roles could be better delineated through machine learning of transcriptomic data to reveal relationships between integrin expression patterns and cancer. We collected publicly available RNA-Seq integrin expression from 8 healthy tissues and their corresponding tumors, along with data from metastatic breast cancer. We then used machine learning methods, including t-SNE visualization and Random Forest classification, to investigate changes in integrin expression patterns. Integrin expression varied across tissues and cancers, and between healthy and cancer samples from the same tissue, enabling the creation of models that classify samples by tissue or disease status. The integrins whose expression was important to these classifiers were identified. For example, ITGA7 was key to classification of breast samples by disease status. Analysis in breast tissue revealed that cancer rewires co-expression for most integrins, but the co-expression relationships of some integrins remain unchanged in healthy and cancer samples. Integrin expression in primary breast tumors differed from their metastases, with liver metastasis notably having reduced expression. Integrin expression patterns vary widely across tissues and are greatly impacted by cancer. Machine learning of these patterns can effectively distinguish samples by tissue or disease status.

Integrin profle of circulating tumour cells in breast cancer patients

Background. Integrins, as adhesion molecules, play a key role in the interaction of cells with the basal membrane and intercellular matrix. Numerous studies demonstrate evidence of increased expression of integrins on tumor cells in different types of cancer. Thus, β3 and αV integrins are associated with stem-like features of tumor cells, and β4 integrin as α6β4 heterodimer provides anchorage-independent survival of malignant mammary epithelial cells. However, all the described functions of integrins have been investigated exclusively on primary tumor cells. The functional significance and expression pattern of integrins on circulating tumor cells (CTCs) remains unclear. The aim of the study was to evaluate the β3, β4 and αvβ5 integrin expression on CTCs and its association with molecular subtype, stage and lymph node metastasis in breast cancer patients Material and Methods. The study included 22 patients with T1–4N0–3M0 invasive ductal breast carcinoma. Venous blood was taken from patients without neoadjuvant chemotherapy (group 1) and after neoadjuvant chemotherapy (group 2) in the volume of 12 ml into vacuum tubes with EDTA. The expression of CTC integrins including stemness features CD44/CD24, CD133 and ALDH1, and epithelial-mesenchymal transition (EMT) (N-cadherin) was evaluated by flow cytometry. Results. CTCs with β3+β4-αvβ5- and β3-β4+αvβ5+ phenotypes and stemness properties were associated with larger tumor size (T4) in breast cancer patients. The β3 integrin expression was associated with more aggressive molecular subtypes of breast cancer. Administration of neoadjuvant chemotherapy did not affect the expression pattern of β3, β4 and αvβ5 integrins in CTCs. Conclusion. In breast cancer, most CTCs expressed β3, β4 and αvβ5 integrins despite the lack of attachment to the basal membrane and intercellular matrix. The expression of the above integrins on CTCs was associated with breast cancer molecular subtype, stage and lymph node metastasis, and therefore its evaluation can be considered as one of the objectives of liquid biopsy study.

Expression Profiles of ITGA8 and VANGL2 Are Altered in Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).

Kidney failures in infants are mostly caused by congenital anomalies of the kidney and urinary tract (CAKUT), which are among the most common congenital birth disorders worldwide when paired with cardiac abnormalities. People with CAKUT often have severe kidney failure as a result of a wide range of abnormalities that can occur alone or in conjunction with other syndromic disorders. In this study, we aimed to investigate the expression pattern of CAKUT candidate genes alpha-8 integrin (ITGA8) and Van Gogh-like 2 (VANGL2) in fetal tissues of healthy and CAKUT-affected kidneys using immunohistochemistry and immunofluorescence. We found that under CAKUT circumstances, the expressions of ITGA8 and VANGL2 are changed. Additionally, we showed that VANGL2 expression is constant during fetal aging, but ITGA8 expression varies. Moreover, compared to normal healthy kidneys (CTRL), ITGA8 is poorly expressed in duplex kidneys (DKs) and dysplastic kidneys (DYS), whereas VANGL2 is substantially expressed in dysplastic kidneys (DYS) and poorly expressed in hypoplastic kidneys (HYP). These results point to VANGL2 and ITGA8 as potential prognostic indicators for CAKUT malformations. Further research is necessary to explore the molecular mechanisms underlying this differential expression of ITGA8 and VANGL2.

P650 Integrin expression patterns of patients with IBD treated with vedolizumab.

Abstract Background Anti-α4β7 integrin monoclinic antibody vedolizumab is an effective treatment for moderate and severe Ulcerative Colitis and Crohn’s Disease. Occasionally vedolizumab-treated patients experience articular extraintestinal manifestations, in the form of arthralgias, which could be attributed to lymphocyte tracking modification caused by vedolizumab. This study aims to describe α4β7 andα4β1 integrin expression on circulating lymphocytes and the effect of vedolizumab on integrin expression, as well asto identify expression patterns associated with arthralgias under vedolizumab. Methods A cross-sectional study was conducted. Peripheral blood was collected in EDTA-coated bottles before a scheduled drug administration. Expression of α4β7 and α4β1 integrins on CD4+T, CD8+T, B and plasma cells was studied by flow cytometry (DX Flex, BC), using CD3, CD4, CD8, CD19,CD138, CD49d (α4), β7, CD29 (β1). Kaluza C software and SPSS23 were used for analysis. Results In total, 40 IBD patients (16 of them treated withvedolizumab) and 8 healthy controls were included. There was a higher percentage of α4β1expressing lymphocytes compared to α4β7+ ones (P<0.001) (figure 1). Vedolizumab-treated patients had lower percentages of integrin-expressing B cells (α4β7 P=0.001 vs controls, α4β7 P=0.052 vs non-vedolizumab IBD, α4β1 P=0.002 vs controls, α4β1 P=0.015 vs non-vedolizumab IBD) and α4β1 plasma cells (P=0.013 vs non-vedolizumab IBD) (figure 2). In patients treated with vedolizumab with arthralgias, α4β7 expression on B-cells was more suppressed, whereas α4β1 expression on plasma cells was less suppressed, compared to those that had not developed arthralgias, but not in a statistically significant level (figure 3). Conclusion Patients treated with vedolizumab presented different patterns of integrin expression on circulating B cells. Manifestation of arthralgias under vedolizumab may be associated with a disruption of α4β7/α4β1 proportion. Funding: This study was funded through a research grant from the Greek IBD study group (EOMIFNE) to G.B. EOMIFNE Grant: 2020EOMIFNEp1

Activated CMPs Act in Concert with the Spleen and Accelerate Hematopoietic Inflation after Transplantation

Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After transplantation, injected HSCs rapidly expand to reconstitute the entire blood system. This early post-transplant "hematopoietic inflation" is a crucial phase in sustaining life. However, the time course of hematopoietic inflation and the spatial transition within the whole body have remained a mystery. The discovery of colonies forming in the spleen early after HSC transplantation in the 1960s has been a strong advocate of the existence of HSCs, but biological role of transient nature of spleen colonies has not been understood. In this study, we elucidated the spatiotemporal dynamics of hematopoietic inflation in the same individual in a murine HSC transplantation model. Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize hematopoietic inflation in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and each progenitor cell has a different hematopoietic inflation pattern. We found that activated common myeloid progenitors (aCMPs) use the spleen as a site for hematopoietic inflation, producing much more cells early after transplantation than normal CMPs. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. In the allogeneic transplantation setting, universal exCMPs could sustain life for a long time by a serial transplantation. Human exCMPs could be easily expanded as well, and the clinical use of human exCMPs is highly anticipated as a supportive therapy after HSCT. Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that aCMPs and the spleen cooperated to contribute to hematopoietic inflation post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract P029: Variable Expression Of Integrin Subunits In The Male And Female Kidneys Suggests A Key Factor Responsible For The Higher Prevalence Of Chronic Kidney Diseases In Women

Clinical and epidemiological studies have highlighted the sex differences in the incidence of chronic kidney diseases. However, the underlying pathological mechanisms contributing to the higher prevalence of chronic kidney disease in women are not well known. The cell’s reciprocal communication to the basement membrane is one of the important factors in maintaining kidney functions and injury responses. The cell-basement membrane communication is mediated by cell surface receptors. Integrins are heterodimeric cell surface receptors that consist of noncovalently associated α and β subunits and are the principal receptor that transduces signaling between the cell and basement membrane. This study investigated the sex difference in the expression pattern of integrin α2, α5, and integrin β1. The kidney cortex from 2 months old C57BL/6 mice (both male and female) was investigated for the expression pattern of integrin using q-RTPCR and immunoblotting. Immunoblotting shows a 35% decrease in integrin β1 protein in females (1.13±0.15) compared to males (1.73±0.36). This was further confirmed by the fold change in mRNA expression using q-RTPCR that shows significantly lower integrin β1 mRNA levels in females compared to males 1.097 ± 0.08 (male) and 0.693± 0.11(female). The expression of collagen receptor, integrin α2 did not show a significant difference in expression between males and females in immunoblotting (0.23±0.14 vs 0.78±0.35) and q-RTPCR (1.07±0.46 vs 1.36±0.47). The receptor for fibronectin, integrin α5 also did not alter between males and females by immunoblotting (1.00±0.28 vs 0.98±0.17) and q-RTPCR (1.05±0.55 vs 1.04±0.38). The cell-specific expression in kidneys is progressing using immune histochemistry and primary cell cultures. Integrin β1 KO mice developed higher fibrosis than WT mice when subjected to injury. This observed lower integrin β1 expression could be an important factor in the modification of cell microenvironment and contributes to the higher chronic kidney disease progression in women that require further investigation.

TGF-β promotes stem-like T cells via enforcing their lymphoid tissue retention.

Stem-like CD8+ T cells sustain the antigen-specific CD8+ T cell response during chronic antigen exposure. However, the signals that control the maintenance and differentiation of these cells are largely unknown. Here, we demonstrated that TGF-β was essential for the optimal maintenance of these cells and inhibited their differentiation into migratory effectors during chronic viral infection. Mechanistically, stem-like CD8+ T cells carried a unique expression pattern of α4 integrins (i.e., α4β1hi and α4β7lo) controlled by TGF-β. In the absence of TGF-β signaling, greatly enhanced expression of migration-related markers, including altered expression of α4 integrins, led to enhanced egress of stem-like CD8+ T cells into circulation accompanied by further differentiation into transitional states. Blocking α4 integrin significantly promoted their lymphoid tissue retention and therefore partially rescued the defective maintenance of Tcf-1+ subset in the absence of TGF-β signaling. Thus, TGF-β promotes the maintenance and inhibits the further differentiation of stem-like T cells at least partially via enforcing their lymphoid tissue residency.

Mast cell tissue heterogeneity and specificity of immune cell recruitment.

Mast cells occupy a unique niche within tissues as long lived perpetrators of IgE mediated hypersensitivity and anaphylaxis, as well as other immune responses. However, mast cells are not identical in different tissues and the impact of this tissue heterogeneity on the interaction with other immune cells and on defined immune responses is still unclear. In this review, we synthesize the characteristics of mast cell heterogeneity in the gut and the skin. Furthermore, we attempt to connect mast cell heterogeneity with functional diversity by exploring differences in mast cell-induced immune cell recruitment in these two model organs. The differential expression of certain receptors on mast cells of different tissues, notably tissue-specific expression patterns of integrins, complement receptors and MRGPRX2, could indicate that tissue environment-dependent factors skew mast cell-immune cell interactions, for example by regulating the expression of these receptors.

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate in vitro models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers via surface chemokine receptors and integrins. Here we applied such a model by investigating CTL adhesion and migration on six types of coated surfaces. We assessed adhesion and motility metrics, the expression of chemokine receptors, and integrins in CTLs of patients with atherosclerosis and healthy donors. Using fibronectin, platelet-poor plasma from patients with atherosclerosis, and conditioned medium from atherosclerotic plaques we revealed the role of substrate in CTL adhesiveness: fibronectin alone and fibronectin combined with platelet-poor plasma and conditioned medium elevated the CTL adhesiveness – in patients the elevation was significantly higher than in healthy donors (p = 0.02, mixed 2-way ANOVA model). This was in line with our finding that the expression levels of integrin-coding mRNAs were elevated in the presence of fibronectin (p < 0.05) and ITGB1, ITGA1, and ITGA4 were specifically upregulated in patients compared to healthy donors (p < 0.01). Our experimental model did not affect the expression levels of mRNAs CCR4, CCR5, and CX3CR1 coding the chemokine receptors that drive T-lymphocyte migration to plaques. Thus, we demonstrated the substrate-dependence of integrin expression and discriminated CTLs from patients and healthy donors by adhesion parameters and integrin expression levels.

P1418: EXPANDED COMMON MYELOID PROGENITORS ACT IN CONCERT WITH THE SPLEEN AND SUPPORT EARLY HEMATOPOIETIC RECOVERY

Background: Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After intravenous injection, HSCs are thought to home directly toward the bone marrow niche, and their homing mechanisms and interactions with the microenvironment have been intensively studied. However, the time course of the transplanted HSCs to reconstitute the entire blood system and the spatial transition within the whole body have remained a mystery. Aims: We aim to elucidate the spatiotemporal dynamics of hematopoiesis in the same individual in a murine HSC transplantation model. Methods: Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize HSC migration in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Results: Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and we found that common myeloid progenitors (CMPs) use the spleen as a site for hematopoiesis, producing many cells early after transplantation. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. Image:Summary/Conclusion: Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that CMPs and the spleen cooperated to contribute to early hematopoiesis post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery.

Abstract IA024: Tumor exosome and exomere biomarkers for early cancer detection

Abstract In 1889, Stephen Paget first proposed that organ distribution of metastases is a non-random event, yet metastatic organotropism remains one of the greatest mysteries in cancer biology. A growing number of studies demonstrate that tumor-derived microvesicles, referred to as exosomes, may alter the tumor microenvironment at future sites of metastasis promoting pre-metastatic niche formation, and thus creating a favorable “soil” for incoming metastatic “seeds.” However, by what mechanism, and whether their role is significant in tumor progression remains unknown. We have recently demonstrated that exosomes released by lung-, liver- and brain-tropic tumor cells preferentially fuse with resident cells at their future metastatic sites, such as fibroblasts and epithelial cells in the lung, Kupffer cells in the liver, and endothelial cells in the brain. We found that tumor-derived exosome homing to organ-specific cell types prepares the pre-metastatic niche. Moreover, treatment with exosomes derived from lung-tropic models can redirect to the lung the metastatic distribution of cells that normally lack the capacity to form lung metastasis. Proteomic profiling of exosomes revealed distinct integrin expression patterns associated with each organ-specific metastatic site. Whereas exosomal α6β4 integrins were associated with lung metastasis, exosomal integrin αvβ5 was linked to liver metastasis and αvβ3 to brain metastasis. Targeting α6β4 and αvβ5 integrins decreased exosome uptake and metastasis in the lung and liver, respectively. Moreover, our clinical data indicate that integrin expression profiles in circulating plasma exosomes from cancer patients could be used to predict organ-specific metastasis. To gain a more comprehensive understanding of the relationship between exosomal protein cargo and tumor progression, we have built one of the largest exosome proteomics databases, including human and murine normal, pre-cancer and cancer exosomes from a variety sources, from cell lines to plasma and other bodily fluids. This dataset has allowed us to extend the list of markers that can be used to identify and isolate exosomes from human samples. When comparing the protein cargo of blood plasma exosomes isolated from normal subjects versus cancer patients with early stage pancreatic, colorectal, breast and lung cancer, we were able to define a cancer-specific proteomic signature that distinguishes cancer exosomes. Using paired samples from tumor tissue and plasma of the same cancer patients with breast, lung or pancreatic cancer, we were able to derive cancer type-specific exosomal signatures, which can be used as biomarkers to identify and classify tumors of unknown origin. Therefore, proteomic profiling of exosomes has great clinical potential as a non-invasive, rapid liquid biopsy that could aid in tumor detection classification. Citation Format: Ayuko Hoshino, Han Sang Kim, Linda Bojmar, Kofi Ennu Gyan, Haiying Zhang, Irina Matei, David Lyden. Tumor exosome and exomere biomarkers for early cancer detection [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA024.

Integrin-mediated adhesion and mechanosensing in the mammary gland

The mammary gland is dynamically remodelled during its postnatal development and the reproductive cycles. This inherent plasticity has been suggested to increase the susceptibility of the organ to carcinogenesis. Morphological changes in the mammary epithelium involve cell proliferation, differentiation, apoptosis, and migration which, in turn, are affected by cell adhesion to the extracellular matrix (ECM). Integrin adhesion receptors function in the sensing of the biochemical composition, patterning and mechanical properties of the ECM surrounding the cells, and strongly influence cell fate. This review aims to summarize the existing literature on how different aspects of integrin-mediated adhesion and mechanosensing, including ECM composition; stiffness and topography; integrin expression patterns; focal adhesion assembly; dynamic regulation of the actin cytoskeleton; and nuclear mechanotransduction affect mammary gland development, function and homeostasis. As the mechanical properties of a complex tissue environment are challenging to replicate in vitro, emphasis has been placed on studies conducted in vivo or using organoid models. Outright, these studies indicate that mechanosensing also contributes to the regulation of mammary gland morphogenesis in multiple ways.

Persistance of Inflammatory Phenotype of Neutrophils in Sickle Cell Disease Children Despite Chronic Exchange Transfusion Program: A Cause of the Progression of Cerebral Vasculopathy?

Sickle cell disease (SCD) is a severe hemoglobinopathy due to the production of abnormal hemoglobin S (HbS). Although red blood cell (RBC) dysfunction is the major contributor to disease, several studies highlighted the important role of polymorphonuclear neutrophils (PMNs), both during acute and chronic complications. One of the most severe complication of SCD is ischemic stroke due to large cerebral artery occlusion. In 1998, the Stroke Prevention (STOP) trial demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in SCD children with cerebral vasculopathy (CV). However, there is a wide heterogeneity in the course of CV in patients receiving chronic transfusions, since only about half of them improved their CV under transfusion program, while 25% are only stabilized and 29% continue to get worse despite a percentage of HbS permanently below 30%. The aim of our study is to investigate the impact of transfusion programs on neutrophils activation and ageing, in order to identify if inflammation could contribute to the persistence of SCD complications despite red cell transfusion. We performed a prospective study including 58 homozygous SCD children and 10 healthy donors. Of these, 12 had no specific treatment, 11 were on Hydroxyurea (HU) treatment, 21 were on an exchange transfusion program, and 14 were on both an exchange transfusion program and HU treatment for an average of 4.9 years due to persistent CV. Monthly exchange transfusion are carried out either by erythrapheresis or by manual exchanges, consisting of the continuous bleeding of whole blood compensated by simultaneous transfusion of packed red blood cells. Neutrophils were isolated from fresh blood samples before exchange transfusion session and labelled with 8 markers specific of adhesion, activation and ageing. We quantified by flow cytometry the expression of 3 integrins (CD18, CD11a, CD11b), 3 ageing markers (CD182, CD184, CD62L) and 2 adhesion molecules (CD162 and CD66a). We also measured the plasmatic level of elastase, which reflects the NETose activity of PMNs As previously reported, we observed a high leukocytosis and an activated profile of PMNs in the 12 non-transfused SCD patients compared to healthy controls (Figure 1), characterized by an overexpression of the integrin CD18/CD11b (p=0,03) and CD18/CD11a (p=0,02), a higher level of circulating aged PMNs CD184 high/CD62Llow (p=0,04), a higher expression of CD162 (p=0,01) and CD66a (0,01) as well as a higher plasmatic level of elastase (p=0.01). Interestingly, in the PMNs of the 21 patients receiving monthly exchange transfusion, we found an identical expression pattern of integrins, selectins, ageing markers and elastase level compared to those of the PMNs from non-transfused patients. Furthermore, we also observed a persistence of high neutrophilic leukocytosis. This activation pattern was the same for patients on manual exchange or erythrapheresis, even with a tendency towards a more inflammatory profile in patients on erythrapheresis (Figure 1). In the PMNs from the 11 patients receiving HU compared to untreated SCD patients, we found an expected decrease in high leukocytosis and membrane integrin expression CD18/CD11b and CD18/CD11a. The addition of HU therapy in 14 patients in exchange transfusion program allows to alleviate neutrophilic leukocytosis and membrane integrin expression. Our study shows for the first time that replacing sickle RBCs with healthy RBCs is not sufficient to reverse the pathological phenotype of PMNs in SCD. A persistence of the PMNs activation pattern is observed both despite erythrapheresis, where plasma and white blood cells go back to the patient, and in manual exchanges, where the patient is bled from a large volume of whole blood. Given the major role of inflammation in endothelial damage and vasculopathy in SCD, our data could explain the incomplete efficacy of transfusion exchange programs to treat CV. This raises the question to systematically combine anti-inflammatory and anti-white blood cell adhesion treatments such as Hydroxyurea or P-Selectin inhibitors for these patients. Disclosures No relevant conflicts of interest to declare.

Enhanced expression of αVβ3 integrin in villus and extravillous trophoblasts of placenta accreta.

Placenta accreta is one of the most serious complications in obstetrics and gynecology. Villous trophoblasts (VT) and extravillous trophoblasts (EVT) play a central role in normal placentation. Placenta accreta is characterized by abnormal invasion of EVT cells through the uterine layers, due to changes in several parameters, including adhesion proteins. Although αvβ3 integrin is a central adhesion molecule, participating in multiple invasive pathological conditions including cancer, data on placenta accreta are lacking. To study the expression pattern of αvβ3 integrin in placenta accreta in comparison with normal placentas. We collected tissue samples from placentas defined as percreta, the most severe presentation of placenta accreta and from normal control placentas (n = 10 each). The samples underwent protein extractions for analyses of αvβ3 expression by Western blots (WB) and a parallel tissue assessment by immunohistochemistry (IHC). WB results indicated significantly elevated αvβ3 integrin expression in the percreta samples compared to normal placentas. These elevated levels were mainly contributed by EVT cells, as demonstrated by IHC. αvβ3 integrin demonstrated a classical membranal expression in the VT cells, whereas a uniformly distributed expression was documented in the EVT cells. These patterns of the αvβ3 integrin localization were similar in both accreta and normal placental samples. Enhanced αvβ3 integrin expression, mainly in extra villous trophoblasts of placenta percreta, implies for a role of this adhesion molecule in pathological placentation.

Quantitative and qualitative analysis of integrin subtype expression in melanocytes and melanoma cells

Objectives: Changes in the integrin expression pattern have been associated with the malignant transformation of melanocytes suggesting that integrins may be potential biomarkers as well as molecular targets for individualized therapy. Since there is a lack of comprehensive qualitative and quantitative expression data, we characterized the integrin expression profile in normal and malignant human cells of the melanocytic lineage.Methods: Seven melanoma cell lines as well as normal human melanocytes were investigated in western blots including recombinant integrin subunits for quantification.Results: Expression patterns were heterogeneous. In melanoma, overexpression of α4, α6, αL, β5, and β6 was found. Integrins α7, α9, and β4 were overexpressed in a subset of the melanoma cell lines. Overexpression was defined as a lack of expression in melanocytes but expression in more than half (4) of the melanoma lines. 1.9 to 6.7 × 106 integrin molecules (about 0.3% of total cellular protein) were estimated to be expressed per cell. Expression of integrin αE at the protein level was found in melanoma and melanocytes, to the best of our knowledge, for the first time. Integrins αM and β2 were not detected.Conclusion: Integrins α4, α6, αL, β5, and β6 appear to be overexpressed in melanoma cells. These subunits may serve as biomarkers and/or therapeutic targets.

Tumor cell-derived exosomes home to their cells of origin and can be used as Trojan horses to deliver cancer drugs.

Cancer is the second leading cause of death worldwide and patients are in urgent need of therapies that can effectively target cancer with minimal off-target side effects. Exosomes are extracellular nano-shuttles that facilitate intercellular communication between cells and organs. It has been established that tumor-derived exosomes contain a similar protein and lipid composition to that of the cells that secrete them, indicating that exosomes might be uniquely employed as carriers for anti-cancer therapeutics.Methods: We isolated exosomes from two cancer cell lines, then co-cultured each type of cancer cells with these two kinds of exosomes and quantified exosome. HT1080 or Hela exosomes were systemically injected to Nude mice bearing a subcutaneous HT1080 tumor to investigate their cancer-homing behavior. Moreover, cancer cell-derived exosomes were engineered to carry Doxil (a common chemotherapy drug), known as D-exo, were used to detect their target and therapeutic efficacy as anti-cancer drugs. Exosome proteome array analysis were used to reveal the mechanism underly this phenomenon.Results: Exosomes derived from cancer cells fuse preferentially with their parent cancer cells, in vitro. Systemically injected tumor-derived exosomes home to their original tumor tissues. Moreover, compared to Doxil alone, the drug-loaded exosomes showed enhanced therapeutic retention in tumor tissues and eradicated them more effectively in nude mice. Exosome proteome array analysis revealed distinct integrin expression patterns, which might shed light on the underlying mechanisms that explain the exosomal cancer-homing behavior.Conclusion: Here we demonstrate that the exosomes' ability to target the parent cancer is a phenomenon that opens up new ways to devise targeted therapies to deliver anti-tumor drugs.

Integrin and transcriptomic profiles identify a distinctive synovial CD8+ T cell subpopulation in spondyloarthritis

ObjectivesCurrent evidence suggests that immune events in the gut may impact joint inflammation in ankylosing spondylitis (AS) but the expression of gut-related trafficking molecules in the inflammed joint is poorly...

Abstract 114: Aberrant integrin alpha v and alpha 5 expression patterns in prostate adenocarcinomas and bone-metastases from prostate cancer are consistent with a bone-colonizing phenotype

Abstract Background: Fibronectin-binding αv and α5 integrins mediate homing, adhesive and survival interactions of prostate cancer cells with bone-marrow mesenchymal stromal cells. Monospecific αv integrin antibody therapy slowed progression of bone metastases but failed to impact overall mortality in prostate cancer. Cross-regulation between αv and α5 integrin is a potential source of adaptive resistance to monospecific blockade of either integrin. We assessed the patterns of expression of these partner integrins in bone metastases and the transition from the physiological gland to the malignant phenotype. Methods Formalin-fixed paraffin-embedded (FFPE) radical prostatectomy samples (n=25) from patients with a ≥ Gleason grade 4 component and decalcified FFPE samples of prostate cancer bone metastases (n=10) were obtained from institutional tissue biorepository. Optimized immunohistochemistry methods developed in prostate cancer cell suspensions were applied to assess expression patterns of αv and α5 integrins in benign and malignant glandular elements in primary tumors and bone specimens. Results Integrin αv was universally expressed in the physiological basal layer of benign prostate glands (n=25;100%) but not in the luminal epithelium. With loss of the basal layer in malignant transition, αv expression was recapitulated in 100% of malignant glandular epithelium in distinct patterns including epithelial membranous (24/25;96%), luminal membranous (6/25; 24%), punctate cytoplasmic (14/25;56%), intense foci of membranous staining (single cells or clusters surrounded by αv-negative tumor) (10/25;40%), and rim stromal patterns (14/25;56%). Luminal membranous and rim stromal αv expression patterns were striking in tumors with cribriform morphology. Furthermore, integrin αv was identified in all evaluable bone metastatic samples (7/7:100%). By contrast, integrin α5 was identified infrequently in the physiological basal layer (1/10;10%), was not expressed in malignant glandular epithelium of primary tumors but was paradoxically expressed in malignant epithelium in bone metastases (5/7:71%). Conclusion Integrin αv expression is universally and exclusively found in the physiological basal layer of the normal gland that harbors stem-functions and is recapitulated in high frequency in prostatic adenocarcinomas in diverse patterns suggestive of distinct biological functions that may contribute to disease progression. Co-expression and enrichment of integrins αv and α5 in osseous metastases is supportive of a proposed cooperative role of these fibronectin-binding integrins toward bone colonization. Given the adaptive cross-regulation we have observed with targeting of either integrin, combined αv and α5 integrin targeting in prostate cancer bone metastases may be required for effective therapy. Citation Format: Brendan Connell, Pavel Kopach, Wenying Ren, Raghav Joshi, Steven Naber, Paul Mathew. Aberrant integrin alpha v and alpha 5 expression patterns in prostate adenocarcinomas and bone-metastases from prostate cancer are consistent with a bone-colonizing phenotype [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 114.

Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide.

Ovalbumin (OVA) is the most abundant protein in egg whites that is unnecessary in the egg yolk-based food industry. The development of OVA-based functional materials is of great interest. Collagen is a major component of the extracellular matrix. In this study, an OVA fragment, the OVA secretory signal peptide (OVA SP), was loaded in collagen gels, which were used as a cell scaffold for various types of cells. We examined the effect of OVA SP loaded in collagen gels to cell properties. The peptide was initially bound to the collagen fibers and then released from the gel. Our results indicate that the released OVA SP suppressed the integrin-mediated cell adhesion and focal adhesion formation. However, the adhesion of NIH3T3 cells was not suppressed by treatment with a chelating agentand an anti-β1 antibody. Our resultssuggest that OVA SP nonspecifically interacts with cell surface proteins. The adhesion of various cell types on collagen gels were changed by the addition of OVA SP, depending on their integrin expression pattern. Additionally, the differentiation of MC3T3-E1 osteoblastic cells was promoted on the OVA SP-loaded collagen gels. This suggests that OVA SP may modulate both the differentiation and the adhesion of cells cultured on the collagen gels.

Detection of beta1 integrin activation in chondrocytes

Purpose: Integrins are heterodimeric transmembrane receptors linking the actin cytoskeleton to the ECM. Their activity is regulated via an allosteric switch between low- and high-affinity states, induced by effector binding to the cytoplasmic tail of the α- and β-subunits or by ligand binding to the ectodomain. β1 integrin activity in chondrocytes has been shown to increase during osteoarthritis leading to increased catabolism, however β1 integrin signalling is required during chondrogenic differentiation. Integrin signalling is involved in mechanisms of cell survival, growth, differentiation and matrix remodelling, however the exact mechanisms by which β1 integrin signalling may interact with other homeostatic signalling pathways in chondrocytes remains unknown. Here, we aim to examine the expression pattern of β1 integrins within their activated state in chondrocytes. Methods: Costal chondrocytes obtained from 8 to 12-week-old male wild type (WT) mice were cultured under standard conditions. β1 integrin within an active conformation was detected by immunofluorescence staining using a Purified Rat Anti-Mouse CD29 Clone 9EG7 antibody. Activation levels were compared in chondrocytes plated for 24 hours on either fibronectin, collagen type II or uncoated glass coverslips. Chondrocytes in non-coated wells were stimulated in a time course with 1 mM Mn2+ to act as a positive control. To further investigate β1 integrin activation as part of attachment behaviour, chondrocytes were added to fibronectin coated and uncoated coverslips for 1 hour, 6 hours and 24 hours before β1 integrin activation was assessed by immunocytochemistry. Integrin-specific downstream signalling was analysed by immunoblotting of p-FAK/FAK and PKC in lysates of treated chondrocytes. Results: β1 integrins within an active conformation could be detected in wild type murine chondrocytes in vitro. The 9EG7 epitope was detected firstly at low levels in chondrocytes grown in standard conditions, however activation levels were found to increase significantly when chondrocytes were cultured on either fibronectin or type II collagen coatings. Mn2+ added to chondrocytes cultured in uncoated wells as a positive control led to increased levels of activated β1 integrin. Activated integrin β1 in chondrocytes grown on fibronectin and type II collagen exhibited a specific activation pattern, which was not found in chondrocytes grown on uncoated surfaces. Immunoblotting demonstrated that activation of β1 integrins via manganese or through interaction with RGD sequence containing ECM substrates led to increased phosphorylation of the focal adhesion kinase and protein kinase C. Conclusions: β1 integrins within an active conformation were able to be detected in in vitro cultured chondrocytes using immunocytochemistry for the 9EG7 epitope of β1 integrin. The activated state was increased both by Mn2+ and when chondrocytes were grown on either fibronectin or type II collagen substrates. This detection of β1 integrin activation state allows for the further investigation of integrin signalling within the context of interactions with homeostatic growth factor and chemokine signalling pathways.