Triple Immunofluorescence Research Articles

STIM1, ORAI1, and KDM2B in circulating tumor cells (CTCs) isolated from prostate cancer patients.

Introduction: Previous publications have shown that STIM1, ORAI1, and KDM2B, are implicated in Ca2+ signaling and are highly expressed in various cancer subtypes including prostate cancer. They play multiple roles in cancer cell migration, invasion, and metastasis. In the current study we investigated the expression of the above biomarkers in circulating tumor cells from patients with metastatic prostate cancer. Methods: Thirty-two patients were enrolled in this study and CTCs' isolation was performed with Ficoll density gradient. Two different triple immunofluorescence stainings were conducted with the following combination of antibodies: CK/KDM2B/CD45 and CK/STIM1/ORAI1. Slides were analyzed using VyCAP microscopy technology. Results: CTC-positive patients were detected in 41% for (CK/KDM2B/CD45) staining and in 56% for (CK/STIM1/ORAI1) staining. The (CK+/KDM2B+/CD45-) and the (CK+/STIM1+/ORAI1+) were the most frequent phenotypes as they were detected in 85% and 94% of the CTC-positive patients, respectively. Furthermore, the expression of ORAI1 and STIM1 in patients' PBMCs was very low exhibiting them as interesting specific biomarkers for CTC detection. The (CK+/STIM1+/ORAI1+) phenotype was correlated to bone metastasis (p = 0.034), while the (CK+/STIM1+/ORAI1-) to disease relapse (p = 0.049). Discussion: STIM1, ORAI1, and KDM2B were overexpressed in CTCs from patients with metastatic prostate cancer. STIM1 and ORAI1 expression was related to disease recurrence and bone metastasis. Further investigation of these biomarkers in a larger cohort of patients will clarify their clinical significance for prostate cancer patients.

Comprehensive Analysis of CXCR4, JUNB, and PD-L1 Expression in Circulating Tumor Cells (CTCs) from Prostate Cancer Patients.

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance.

Abstract 7502: MCT1 and ΜCT4 on circulating tumor cells isolated from non small cell lung cancer patients

Abstract Background: Lung cancer is the leading cause of cancer-related deaths. Non-small-cell lung cancer (NSCLC) is the most common subtype of lung cancer and is characterized by poor prognosis and survival rate. This is associated with the fact that most patients are diagnosed at advanced stages of the disease and the possibility of recurrence after treatment remains high. Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) play a crucial role in cellular metabolism, as they are responsible for the transport of lactate and other monocarboxylic acids across the plasma membrane. Their function can further regulate cell survival and proliferation, especially under hypoxic conditions. To elucidate the relationship between CTCs and the metabolic aspect of cancer, we aimed to investigate the expression of MCT1 and MCT4 in CTCs derived from NSCLC patients and its potential association with patients’ outcome. Methods: Fifty-three NSCLC patients at baseline were enrolled in the current study. CTCs were isolated using the ISET system and triple immunofluorescence experiments were performed, using CK, MCT1, MCT4, and CD45 antibodies. The expression of MCT1 and MCT4 in CTCs was assessed by confocal laser scanning microscopy and statistical analysis was performed using SPSS software. Results: CTCs were detected in 29 out of 53 (55%) NSCLC patients. CK expression was characterized as high or low, with the predominant phenotypes among CK-positive patients being the CKhigh/MCT1+/CD45-and CKlow/MCT4-/CD45- (63% and 66%, respectively). Whereas the phenotype CKhigh/MCT4+/CD45- was detected in 52% of the patients. The phenotypes with the lowest frequency were the CKlow/MCT4+/CD45- (7%), CKlow/MCT1+/CD45- (15%), and the CKhigh/MCT1-/CD45- (15%). Most of the isolated CTCs had the CKhigh/MCT1+/CD45- and CKlow/MCT4-/CD45- phenotypes with a mean percentage of 48% and 51% respectively, while only a small number of CTCs per patient were CKlow/MCT1+/CD45- or CKlow/MCT4+/CD45- (8%and 5%, respectively). The phenotype CKlow/MCT1-/CD45- was associated with poorer overall survival (OS) [p = 0.005, (15 vs 33 months respectively)], HR=0.351), while the presence of more than 3 CKhigh/MCT4+/CD45- CTCs was related to poorer progression-free survival (PFS) [p = 0.042 (4 vs 17 months respectively)]. Conclusions: MCT1 and MCT4 were expressed in a high proportion of NSCLC patients at baseline and may constitute interesting biomarkers relevant to cancer progression. However, further analysis in a larger cohort of patients is required to clarify their potential significance in clinical practice. Citation Format: Karolina Mangani, Evangelia Pantazaka, Evi Lianidou, Vassilis Gerogoulias, Athanasios Kotsakis, Athina Markou, Galaktia Kallergi. MCT1 and ΜCT4 on circulating tumor cells isolated from non small cell lung cancer patients [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 7502.

Increased expression of the P2Y12 receptor is involved in the failure of autogenous arteriovenous fistula caused by stenosis

Objective This study investigated the role of the P2Y12 receptor in autogenous arteriovenous fistula (AVF) failure resulting from stenosis. Methods Stenotic venous tissues and blood samples were obtained from patients with end-stage renal disease (ESRD) together with AVF stenosis, while venous tissues and blood samples were collected from patients with ESRD undergoing initial AVF surgery as controls. Immunohistochemistry and/or immunofluorescence techniques were utilized to assess the expression of P2Y12, transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein 1 (MCP-1), and CD68 in the venous tissues. The expression levels of P2Y12, TGFβ1, and MCP-1 were quantified using quantitative reverse transcription–polymerase chain reaction and western blot analyses. Double and triple immunofluorescence staining was performed to precisely localize the cellular localization of P2Y12 expression. Results Expression levels of P2Y12, TGFβ1, MCP-1, and CD68 were significantly higher in stenotic AVF venous tissues than in the control group tissues. Double and triple immunofluorescence staining of stenotic AVF venous tissues indicated that P2Y12 was predominantly expressed in α-SMA-positive vascular smooth muscle cells (VSMCs) and, to a lesser extent, in CD68-positive macrophages, with limited expression in CD31-positive endothelial cells. Moreover, a subset of macrophage-like VSMCs expressing P2Y12 were observed in both stenotic AVF venous tissues and control venous tissues. Additionally, a higher number of P2Y12 +/TGF-β1+ double-positive cells were identified in stenotic AVF venous tissues than in the control group tissues. Conclusion Increased expression of P2Y12 in stenotic AVF venous tissues of patients with ESRD suggests its potential involvement in the pathogenesis of venous stenosis within AVFs.

Investigating the Role of Tumor-Infiltrating Lymphocytes as Predictors of Lymph Node Metastasis in Deep Submucosal Invasive Colorectal Cancer: A Retrospective Cross-Sectional Study.

The role of tumor-infiltrating T cells (TILs) in colorectal cancer (CRC) and their significance in early-stage CRC remain unknown. We investigated the role of TILs in early-stage CRC, particularly in deep submucosal invasive (T1b) CRC. Sixty patients with CRC (20 each with intramucosal [IM group], submucosal invasive [SM group], and advanced cancer [AD group]) were randomly selected. We examined changes in TILs with tumor invasion and the relationship between TILs and LN metastasis risk. Eighty-four patients with T1b CRC who underwent initial surgical resection with LN dissection or additional surgical resection with LN dissection after endoscopic resection were then selected. TIL phenotype and number were evaluated using triple immunofluorescence for CD4, CD8, and Foxp3. All subtypes were more numerous according to the degree of CRC invasion and more abundant at the invasive front of the tumor (IF) than in the center of the tumor (CT) in the SM and AD groups. The increased Foxp3 cells at the IF and high ratios of Foxp3/CD4 and Foxp3/CD8 positively correlated with LN metastasis. In conclusion, tumor invasion positively correlated with the number of TILs in CRC. The number and ratio of Foxp3 cells at the IF may predict LN metastasis in T1b CRC.

Expression of Endothelin-1, Endothelin Receptor-A, and Endothelin Receptor-B in facial melasma compared to adjacent skin.

Although melasma is highly prevalent, its pathogenesis is not yet fully understood. In the skin, endothelin-1 (ET-1) is primarily produced by keratinocytes in response to UVB exposure, which is mediated by an increase in IL-1α or reactive oxygen species. ET-1 plays a role in melanogenesis by binding to specific receptor B (ERB) or receptor A (ERA). However, the expression of ET-1, ERA, and ERB in melasma has not been systematically investigated. The objective of this study was to evaluate the expression of ET-1, ERA, and ERB in facial melasma compared to the adjacent unaffected skin. Cross-sectional study, with 40 skin samples (20: facial melasma; 20: adjacent unaffected skin) from women with facial melasma without treatment for 30 days except for sunscreen. A triple staining immunofluorescence technique was performed for anti-vimentin, DAPI, plus one of the following antibodies: (a) anti-ET1, (b) anti-ERA; (c) anti-ERB. Interfollicular areas on the slides of each topography (melasma; unaffected skin) were photographed in triplicate under confocal laser microscopy. The mean staining intensities of the image histograms (0-255 pixels intensity) were estimated for different types of cells (suprabasal keratinocytes, basal layer, and upper dermis) and were blindly compared between topographies. The mean (SD) age of the participants was 44.9 (9.2). The expression of ET-1 was increased in the whole epidermis with melasma when compared to the adjacent skin, being 32.8% (CI95% 14.7%-52.6%) higher in the spinous layer (p=0.013), 30.4% (CI95% 13.7%-47.9%) higher in the basal layer (p=0.014), and 29.7% (CI95% 11.4%-49.7%) higher in the melanocytes (p=0.006). There was no noticeable expression of ET-1 within the cells on the upper dermis. Neither ERA nor ERB resulted in differential epidermal expression between melasma and unaffected skin (p≥0.1). ET-1 is expressed more intensely on the epidermis from the skin with facial melasma compared to the unaffected adjacent skin.

294-LB: Altered a-Cell Phenotype in Pancreata Is Associated with Increased Peri-islet Collagen Ceposition

Cystic fibrosis (CF)-related diabetes (CFRD) affects up to 50% of adults with CF, adding to treatment burden and impacting overall wellbeing. Major changes are observed in pancreas morphology with acinar tissue replaced by fibrosis and/or fat. Islets are still present even in advanced disease and may cluster together. It is believed CFRD is caused by impaired insulin secretion and loss of β-cell mass, however, detailed pathogenesis remains unknown. We investigated post-mortem pancreatic tissue from 19 donors with CF (age range: premature-27 years) and 10 donors without CF (6-65 years). CF pattern of injury (fibrotic/lipoatrophic) was assessed by routine histology. Sirius Red/Fast Green stain was used to assess fibrosis. Artificial intelligence analysis was performed to quantify intra-islet and peri-islet collagen distribution in 16 cases (7 CF, 9 control). Triple immunofluorescence staining for insulin, glucagon, and vimentin followed by confocal microscopy was used to image up to 50 islets per donor. Glucagon and vimentin-positive (glu/vim+) α-cells were observed in both CF and non-CF pancreata (mean (range) 32 (0-90)% of islets). The presence of this altered α-cell phenotype positively correlated with age in all donors (R=0.41, p<0.05). Presence of glu/vim+ α-cells correlated with peri-islet collagen density in analysed CF and control cases (glu/vim+ islets 44 (0-82)%) (R=0.60, p<0.05). In control pancreata alone glu/vim+ cells were seen in 1-82% (mean 41%) islets and correlated positively with intra-islet collagen density (R=0.76, p<0.05). Interestingly, two CF cases with confirmed diabetes showed low prevalence of this altered phenotype (2% of islets) with 13 vs 40% fibrosis in the whole section. Our data provide further evidence for exocrine-endocrine signalling in the human pancreas and suggest that presence of vimentin-expressing α-cells may be driven by increased collagen deposition in the islet microenvironment during aging and in CFRD pathogenesis. Disclosure N. Kattner: None. S. Richardson: None. Y. Al-selwi: None. D. Tiniakos: None. J. A. Shaw: Advisory Panel; Betalin Therapeutics, Provention Bio, Inc., Consultant; Mogrify. Funding Cystic Fibrosis Trust (SRC019)

#5466 CSF-1R SYSTEM ACTIVATES PARIETAL EPITHELIAL CELLS LEADING TO FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is the most common glomerular cause leading to end-stage kidney disease. Actual treatment of primary FSGS by immunosuppressive agents presents inconsistent results. Thus, new innovative strategies different from those used to date by taking into consideration podocyte renewal and maintenance or even strategies complementary to immunosuppression are needed. In FSGS, parietal epithelial cells (PECs) switch to an activated phenotype (aPECs) in response to podocyte damage promoting glomerulosclerosis. Colony-stimulating factor (CSF-1) is a hematopoietic growth factor that acts via its specific receptor, the tyrosine kinase receptor CSF-1R. CSF-1 has been detected in sera and renal biopsies from patients with different renal complications, including FSGS, attributing their role and presence to macrophage but not to glomerular cells. The potential cellular and molecular mechanisms involved are also unknown. In this work, we evaluated the implication of CSF-1/CSF-1R axis in the pathogenesis of FSGS and its potential as new pharmacological target point by using specific CSF-1R inhibitors. Specifically, we focused on the modulation of PECS activation by de novo production of CD44 and the preservation of podocyte loss. Method We evaluated the role of the CSF-1/CSF-1R axis as a driver of glomerular damage in FSGS in adriamycin-induced nephropathy (ADR) in mice, the main experimental model to study human FSGS. To this end, we treated or not ADR-animals with CSF-1R specific inhibitors, GW2580 or Ki20227 (n = 5-7 group). We determined the expression and localization of CSF-1R in the glomerulus in tissue by triple immunofluorescence (WT-1, SSeCks and CSF-1R) and their relevance in glomerulosclerosis (PAS and pro-fibrotic genes), the determination of de novo CD44 formation and its correlation with ERK1/2 pathway by immunohistochemistry (IH). We detected podocyte in the glomerulus by WT-1 IH and the localization of aPECS in the glomerular tuff by Claudin-1 and SSeCkS markers. Finally, we used isolated human kidney progenitor cells with and without CSF-1 treatment (n = 6/group) to identify potential key interactors of CSF-1 by RNAseq. We validated genes of interest in the FSGS experimental model. Results We observed a constitutively expression of CSF-1R in the glomerulus of control mice that significantly increased in ADR-treated mice, specifically in podocytes (WT1) and PECs (SSeCks), confirmed with mRNA expression. ADR-treated mice showed important events of sclerotic glomerulus and pro-fibrotic genes as collagen that was significantly reversed by the treatment with CSF-1R inhibitors (p<0.001). Results reflected a CSF-1R inhibitor-dependent renal function recovery with the use of the inhibitors with a reduction of proteinuria and an increase of glomerular filtration rate (p<0.001). We found a positive CD44 staining both in Bowman's capsule and inner the tuff of ADR-treated mice, accompanied by an increase of ERK1/2 activation. CSF-1R inhibitors significantly reduced the percentage of glomeruli with de novo CD44 production. Remarkably, podocyte depletion was also preserved with very similar levels to non-treated animals (p<0.001). For RNAseq results, 227 differentially expressed genes (considering a criterion of a probability of differential expression >0.9 and a |M| > 1) were subjected to enrichment analysis. The top significant gene ontology terms were mainly involved with interferon-induced genes. Genes involved in this pathway were validated in the FSGS model, showing an increase in mice treated with ADR compared to controls and alleviated with CSF-1R inhibitors (p<0.001). Conclusion In this study we propose a novel therapeutic strategy to FSGS-associated pathology based on the inhibition of CSF1-R activity having an impact on reducing aPECs, glomerulosclerosis, proteinuria, improving renal function and preserving the podocyte-progenitor phenotype, thus, in podocyte preservation against damage.

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1–2) COPD patients compared to severe-very severe (GOLD 3–4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1–2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

A postulated model for photoimmunopathogenesis of chronic actinic dermatitis around adaptive immunity, including Th17 cells, Tregs, TRMs, cytotoxic T cells, and/or common-γ chain receptor+ cells.

The pathogenesis of chronic actinic dermatitis (CAD) is more complicated than other photodermatoses. However, the relationship between the clinical severity of CAD and the offending photocontact or contact allergens or both, and the correlations of CAD immunopathogenesis with the immunoregulatory molecules involved in adaptive immunity are yet to be investigated. We performed phototesting with broad-spectrum ultraviolet (UV) B, UVA, and visible light to establish the presence of photosensitivity in 121 patients with CAD, together with photopatch and contact patch testing. Nine patients with CAD were selected according to their clinical severity score for CAD (CSS-CAD), and triple direct immunofluorescence analysis was performed with paraffin-embedded skin biopsy samples. As CSS-CAD was closely correlated with the multiplicity of photo(contact) allergens, particularly photoallergens, three or more photoallergens were detected in the severe CAD group (52.5%); less in the moderate group (32.8%); and only one in the mild group (14.8%; P=.025). In the groups showing greater severity of disease, the absolute numbers of IFN-γ+ , IL-17+ , CD4+, CD8+, common-γ chain receptor (common-γCR)+ , and CD69+ tissue-resident memory cells increased on average; there was also an increase in the CD4+/CD8+ cell ratio, with the more severely affected groups. However, the levels of TNF-α+ and FoxP3+ regulatory T (Treg) cells and the mean IL-17/IFN-γ cell ratio decreased in the more severely affected CSS-CAD subgroups. Based on the clinical analysis and immunopathogenic results, avoidance of excessive sun exposure, and topical and systemic blocking agents for photo(contact) allergens are recommended. Additionally, conventional immunomodulators and emerging agents including JAK-STAT inhibitors may be administered for CAD treatment in the future.

Dynamic monitoring of PD-L1 and Ki67 in circulating tumor cells of metastatic non-small cell lung cancer patients treated with pembrolizumab.

Programmed cell death protein ligand-1 (PD-L1) expression in non-small cell lung cancer (NSCLC) tumors guides treatment selection. PD-L1 expression in circulating tumor cells (CTCs) may provide further information. We have explored PD-L1 and marker of proliferation Ki-67 (Ki67; also known as MKI67) in CTCs in longitudinal samples of 47 advanced NSCLC patients receiving pembrolizumab. A triple immunofluorescence, against cytokeratin, PD-L1, and Ki67, was performed on peripheral blood mononuclear cells, at baseline, post-first cycle, post-third, and primary resistance (PMR). Patients displaying PMR (progression at first evaluation) were classified as progressive disease (PD) and those with clinical benefit as disease control (DC). CTCs were categorized as PD-L1high/low/medium/negative and Ki67+ or Ki67- . CTC evaluation revealed a significant increase in the PD-L1low CTC rate at PMR compared to baseline (2.5% at baseline vs. 36.5% at PMR), whereas a reduction in the PD-L1high CTC rate was observed (31.5% vs. 0%, respectively). Investigation of CTC status between PD and DC patients showed that PD patients more frequently increased total and PD-L1low CTCs after first cycle compared to DC (83% of PD vs. 37% of DC and 67% of PD vs. 8% of DC, respectively). Progression-free survival (PFS) was longer in patients with decreased total and PD-L1low CTCs after first cycle compared to those with increased CTCs (median PFS: not reached vs. 2 months). PD-L1+ patients presenting a high Ki67 index (% Ki67+ CTCs > 30%) before treatment had a shorter PFS compared to those with a low Ki67 (≤ 30%), and overall survival (OS) was shorter in PD-L1+ patients harboring Ki67+ CTCs compared to those not presenting (median OS: 11.8 months vs. 33.1 months, respectively). In sequential samples of patients with a durable benefit, a low Ki67 index was observed. Our results suggest that monitoring PD-L1 and Ki67 expression in CTCs of NSCLC patients treated with pembrolizumab may be predictive for pembrolizumab efficacy.

Weighted gene co-expression network reveals driver genes contributing to phenotypes of anaplastic thyroid carcinoma and immune checkpoint identification for therapeutic targets.

Anaplastic thyroid carcinoma (ATC) is a rare but extremely malignant tumor, with a rapid growth rate and early metastasis thus leading to poor survival of patients. The molecular mechanisms underlying these aggressive traits of ATC remain unknown, which impedes the substantial progress in treatment to prolong ATC patient survival. We applied weighted gene co-expression network analysis (WGCNA) to identify ATC-specific modules. The Metascape web and R package clusterProfiler were employed to perform enrichment analysis. Combined with differentially expressed gene analysis, we screened out the most potential driver genes and validated them using receiver operator characteristic (ROC) analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry (IHC), and triple immunofluorescence staining. A gene expression matrix covering 75 normal samples, 83 papillary thyroid carcinoma (PTC), 26 follicular thyroid carcinoma (FTC), 19 poor-differentiated thyroid carcinoma (PDTC), and 41 ATC tissue samples were integrated, based on which we detected three most potential ATC-specific modules and found that hub genes of these modules were enriched in distinct biological signals. Hub genes in the turquoise module were mainly enriched in mitotic cell cycle, tube morphogenesis, and cell differentiation, hub genes in the magenta module were mainly clustered in the extracellular matrix organization, positive regulation of cell motility, and regulation of Wnt signaling pathway, while hub genes in the blue module primarily participated in the inflammatory response, innate immune response, and adaptive immune response. We showed that 9 top genes, 8 transcription factors (TFs), and 4 immune checkpoint genes (ICGs) were differentially expressed in ATC compared to other thyroid samples and had high diagnostic values for ATC, among which, 9 novel ATC-specific genes (ADAM12, RNASE2, CASP5, KIAA1524, E2F7, MYBL1, SRPX2, HAVCR2, and TDO2) were validated with our clinical samples. Furthermore, we illustrated that ADAM12, RNASE2, and HAVCR2 were predominantly present in the cytoplasm. Our study identified a set of novel ATC-specific genes that were mainly related to cell proliferation, invasion, metastasis, and immunosuppression, which might throw light on molecular mechanisms underlying aggressive phenotypes of ATC and provide promisingly diagnostic biomarkers and therapeutic targets.

Transient receptor potential melastatin-7 in the rat dorsal root ganglion

AimsTransient receptor potential melastatin-7 (TRPM7) is a selective cation permeable channel which plays important roles in cellular and developmental biology such as cell proliferation, survival, differentiation and migration. This channel is also known to be necessary for transmitter release in the peripheral nervous system. In this study, immunohistochemistry for TRPM7 was conducted in the rat lumbar dorsal root ganglion (DRG). MethodsTriple immunofluorescence methods were used to demonstrate distribution of TRPM7 and its relationship to other TRP channels in the DRG. Retrograde tracing and double immunofluorescence methods were also performed to know peripheral targets of DRG neurons containing TRPM7 and TRP vanilloid 1 (TRPV1). In addition, transection of the sciatic nerve was conducted to demonstrate an effect of the nerve injury on TRPM7expression in the DRG. ResultsTRPM7-immunoreactivity was expressed by 53.9% of sensory neurons in the 4th lumbar DRG. TRPM7-immunoreactive (-IR) DRG neurons mostly had small (<600 µm²) and medium-sized (600–1200 µm²) cell bodies. By triple and double immunofluorescence methods, approximately 70% of TRPM7-IR DRG neurons contained TRPV1-immunoreactivity. Although the number of DRG neurons co-expressing TRPM7 and TRPM8 was small in the DRG, almost all of TRPM8-IR DRG neurons co-expressed TRPM7-immunoreactivity. By combination of retrograde tracing method and immunohistochemistry, TRPM7 was expressed by half of DRG neurons innervating the plantar skin (61.9%) and gastrocnemius muscle (51.2%), and 79.6% of DRG neurons innervating the periosteum. Co-expression of TRPM7 and TRPV1 among periosteum DRG neurons (75.7%) was more abundant than among cutaneous (53.2%) and muscular (40.4%) DRG neurons. DRG neurons which co-expressed these ion channels in the periosteum had smaller cell bodies compared to the skin and muscle. In addition, the sciatic nerve transection decreased the number of TRPM7-IR neurons in the DRG (approximately 60% reduction). The RT-qPCR analysis also demonstrated reduction of TRPM7 mRNA in the injured DRG. ConclusionThe present study suggests that TRPM7 is mainly located in small nociceptors in the DRG. The content of TRPM7 in DRG neurons is probably different among their peripheral targets. TRPM7 in DRG neurons may be able to respond to noxious stimulation from their peripheral tissues. The nerve injury can decrease the level of TRPM7 mRNA and protein in DRG neurons.

Abstract 1966: Dr

Abstract Background: According to recent publications of our group, JUNB and CXCR4 were overexpressed in CTCs and DTCs isolated from breast cancer patients. This expression was related with patients’ clinical outcome. Current study investigates for the first time, the expression of JUNB and CXCR4 in CTCs from patients with Non-Small Cell Lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC). Methods: Forty four patients were enrolled in this study (30 NSCLC and 14 SCLC patients, before the initiation of 1st line treatment). ISET system was used for NSCLC patients' samples preparation, while the CTCs from SCLC patients were isolated using Ficoll density gradient. Triple immunofluorescence experiments were performed, using CK, JUNB, and CXCR4 antibodies. Results conducted using confocal laser scanning microscopy for NSCLC samples and the VyCAP system for SCLC samples. Results: Sixteen out of 30 NSCLC patients (53.33%), were positive for CTCs, while all SCLC patients harbored CK-positive cells. Most common phenotypes in CK-positive NSCLC patients were the [(CK+/JUNB+/CXCR4+): 50% (8/16patients)], [(CK+/JUNB+/CXCR4-): 43.75%, (7/16)] and the [(CK+/JUNB-/CXCR4-): 37.5% (6/16)]. Less frequent phenotype was the [(CK+/JUNB-/CXCR4+): 6.25%, (1/16)]. Analysis of the mean isolated CTCs/patient revealed that most of the isolated CTCs belonged to the (CK+/JUNB+/CXCR4+) phenotype (42.19%), while less frequent were the (CK+/JUNB+/CXCR4-): 33.13%, (CK+/JUNB-/CXCR4-): 18.44% and the (CK+/JUNB-/CXCR4+): 6.25% phenotypes. Survival analysis revealed that the presence of (CK+/JUNB+/CXCR4+) was related to poorer OS (cox regression: p=0.008) and PFS (Log Rank, p=0.014) All SCLC patients had detectable CTCs in their blood with 13 out of 14 of them having the (CK+/JUNB-/CXCR4-) phenotype (92.86%). The rest of CTC’s phenotypes (CK+/JUNB+/CXCR4+), (CK+/JUNB+/CXCR4-) and (CK+/JUNB-/CXCR4+) where found in 10 out of 14 patients (71.43%) each. Examination of the mean percentage of the total isolated CTCs/patient indicated that the (CK+/JUNB-/CXCR4-) phenotype was the most frequent (49.71%), while the percentages for the rest phenotypes were [(CK+/JUNB+/CXCR4-): 25.94%], [(CK+/JUNB-/CXCR4+): 12.65%] and (CK+/JUNB+/CXCR4+): 11.70%]. Conclusion: JUNB and CXCR4 were upregulated in CTCs from NSCLC and SCLC patients. However, in NSCLC the most frequent phenotype (CK+/JUNB+/CXCR4+) was also related to patients’ outcome, underlying the key role of these molecules in metastatic dissemination. Further examination will determine the role of this expression in all lung cancer subtypes. Acknowledge: This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2ΕΔΚ-01562). Citation Format: Argyro Roumeliotou, Evangelia Pantazaka, Anastasia Xagara, Thomas Makatsoris, Angelos Koutras, Vassilis Georgoulias, Athanasios Kotsakis, Galaktia Kallergi. Dr [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 1966.

SOAT1: A Suitable Target for Therapy in High-Grade Astrocytic Glioma?

Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.

Effect of Osimertinib on CTCs and ctDNA in EGFR Mutant Non-Small Cell Lung Cancer Patients: The Prognostic Relevance of Liquid Biopsy.

Simple SummaryOsimertinib has become the standard of care for the first-line treatment of EGFR-mutant NSCLC patients. The aim of this current translational research study was to assess the clinical relevance of liquid biopsy in 47 patients receiving osimertinib. Effects on circulating tumor cells (CTCs) and plasma-DNA (ctDNA) were investigated before, after one treatment cycle, and at the end of treatment. ctDNA and CTCs decreased after one treatment cycle, but increased at the end of treatment. The detection of ctDNA before and after one treatment cycle was associated with shorter progression-free and overall survivals (PFS and OS), whereas ctDNA clearance after one treatment cycle resulted in a significantly longer PFS and OS. ctDNA at baseline emerged as an independent predictor of shorter PFS. Thus, changes in liquid biopsy status (CTCs, ctDNA) during osimertinib treatment can be used as a tool for treatment efficacy.Introduction: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. Methods: Forty-seven EGFR-mutant NSCLC patients who had progressed on prior first- or second-generation EGFR inhibitors were enrolled in the study and treated with osimertinib, irrespective of the presence of the T790M mutation in the primary tumor or the plasma. Peripheral blood was collected at baseline (n = 47), post-Cycle 1 (n = 47), and at the end of treatment (EOT; n = 39). CTCs were evaluated in 32 patients at the same time points (n = 32, n = 27, and n = 21, respectively) and phenotypic characterization was performed using triple immunofluorescence staining (CK/VIM/CD45). Results: Osimertinib resulted in an ORR of 34% (2 CR) and a DCR of 76.6%. The median PFS and OS values were 7.5 (range, 0.8–52.8) and 15.1 (range, 2.1–52.8) months, respectively. ctDNA was detected in 61.7%, 27.7%, and 61.5% of patients at baseline, post-Cycle 1, and EOT, respectively. CTCs (CK+/CD45-) were detected in 68.8%, 48.1%, and 61.9% of patients at the three time points, respectively. CTCs expressing both epithelial and mesenchymal markers (CK+/VIM+/CD45-) were detected in 56.3% and 29.6% of patients at baseline and post-Cycle 1, respectively. The detection of ctDNA at baseline and post-Cycle 1 was associated with shorter PFS and OS, whereas the ctDNA clearance post-Cycle 1 resulted in a significantly longer PFS and OS. Multivariate analysis revealed that male sex and the detection of ctDNA at baseline were independent predictors of shorter PFS (HR: 2.6, 95% C.I.: 1.2–5.5, p = 0.015 and HR: 3.0, 95% C.I.: 1.3–6.9; p = 0.009, respectively). Conclusions: The decrease in both CTCs and ctDNA occurring early during osimertinib treatment is predictive of better outcome, implying that liquid biopsy monitoring may be a valuable tool for the assessment of treatment efficacy.

Clathrin adapters AP-1 and GGA2 support expression of epidermal growth factor receptor for cell growth

The role of Golgi/endosome-localized clathrin adapters in the maintenance of steady-state cell surface epidermal growth factor receptor (EGFR) is not well known. Here, we show that EGFR associates preferentially with both AP-1 and GGA2 in vitro. AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation. Triple immunofluorescence microscopy and proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurred more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes. Biochemical recycling assay revealed that the depletion of AP-1 or GGA2 significantly suppressed EGFR recycling to the plasma membrane regardless of the EGF stimulation. Depletion of AP-1 or GGA2 also reduced cell contents of other tyrosine kinases, MET and ErbB4, and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model. Moreover, AP-1 was expressed in endosomes at higher levels in some cancer tissues. Collectively, these results suggest that AP-1 and GGA2 function in recycling endosomes to retrieve endocytosed EGFR, thereby sustaining its cell surface expression and, consequently, cancer cell growth.

Molecular-Morphological Relationships of the Scaffold Protein FKBP51 and Inflammatory Processes in Knee Osteoarthritis.

Knee osteoarthritis (OA) is one of the most prevalent chronic conditions affecting the adult population. OA is no longer thought to come from a purely biomechanical origin but rather one that has been increasingly recognized to include a persistent low-grade inflammatory component. Intra-articular corticosteroid injections (IACSI) have become a widely used method for treating pain in patients with OA as an effective symptomatic treatment. However, as the disease progresses, IACSI become ineffective. FKBP51 is a regulatory protein of the glucocorticoid receptor function and have been shown to be dysregulated in several pathological scenario’s including chronic inflammation. Despite of these facts, to our knowledge, there are no previous studies of the expression and possible role of FKBP51 in OA. We investigated by double and triple immunofluorescence confocal microscopy the cellular and subcellular expression of FKBP51 and its relations with inflammation factors in osteoarthritic knee joint tissues: specifically, in the tibial plateau knee cartilage, Hoffa’s fat pad and suprapatellar synovial tissue of the knee. Our results show co-expression of FKBP51 with TNF-α, IL-6, CD31 and CD34 in OA chondrocytes, synovial membrane cells and adipocytes in Hoffa’s fat pad. FKBP51 is also abundant in nerve fibers within the fat pad. Co-expression of FKBP51 protein with these markers may be indicative of its contribution to inflammatory processes and associated chronic pain in OA.

Morphological assessment of GABA and glutamate inputs to GnRH neurons in intact female mice using expansion microscopy.

The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin-releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro-oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45µm of the primary dendrite (approximately 0.19 synapsesµm-1 ) with relatively few synapses on the GnRH neuron soma or beyond 45µm of the dendrite (0.05-0.08 synapsesµm-1 ). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co-labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro-oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro-oestrous GnRH surge.

LB709 Therapeutic effect of γδTregs cells in Alopecia Areata

Regulatory γδ T cells (γδTregs) may exert therapeutic effects under some experimental autoimmune disease (AID) conditions. This encouraged us to explore whether this this also applies to alopecia areata (AA), one of the most common human AIDs. Triple immunofluorescence (IF) staining was performed to determine the distribution of γδTregs in human skin presence of γδTregs on skin sections of AA patients. Autologous circulating human γδ Tregs were expanded and either co-cultured with organ-cultured, stressed human scalp hair follicles (HFs) ex vivo or injected into experimentally induced AA lesions on human scalp skin xenotransplants on SCID mice.