Marker-positive Cells Research Articles

Evaluation of Sézary cell marker expression and cell death behaviour upon invitro treatment by flow cytometry in Sézary syndrome patients.

The diagnosis of Sézary syndrome (SS) relies on the identification of blood Sézary cells (SC) by different markers via flow cytometry. Treatment of SS is challenging since its pathogenesis is characterized by cell death resistance rather than hyperproliferation. In this study, we establish an integrated approach that considers both the expression of SC markers and sensitivity to cell death both spontaneously and upon invitro treatment. Peripheral blood mononuclear cells were isolated from 20 SS patients and analysed for the SC markers CD7 and CD26 loss as well as CD158k and PD1 gain. The cells were then treated with different established and experimental therapies invitro and cell death was measured. Spontaneous and therapeutically induced cell death were measured and correlated to cellular marker profiles. In the marker-positive cells, spontaneous cell death sensitivity was reduced. Different treatments invitro managed to specifically induce cell death in the putative CTCL cell populations. Interestingly, a repeated analysis after 3 months of treatment revealed the CTCL cell death sensitivity to be restored by therapy. We propose this novel integrated approach comprising the evaluation of SC marker expression and analysis of cell death sensitivity upon treatment that can also enable a better therapy stratification.

P-054 Paternal age and cryopreservation impact sperm chromatin status as indicated by changes in protamine and histone levels

Abstract Study question Do paternal age and cryopreservation alter sperm chromatin status in normospermic men? Summary answer Paternal age and cryopreservation appear to impact sperm chromatin condensation by altering protamine and histone DNA levels. What is known already Semen parameters do not accurately reflect male fertility and thus are not adequate to assess the impact of patient profile and semen manipulation/conservation on sperm functional quality. DNA protamination leads to sperm chromatin condensation, which is associated with sperm maturity but also appears to function as a defence mechanism against oxidative attack. In the face of contrasting/insufficient previous data, the impact of advanced paternal age (APA) and cryopreservation on sperm chromatin condensation and histone/protamine balance remains unclear. Study design, size, duration Twenty-two normospermic men, 11 of which with advanced paternal age (APA; ≥42) and 11 pre-APA (≤35), provided semen samples by masturbation (1-5 days abstinence). Fresh and cryopreserved/thawed samples from all patients were assessed for chromatin histone staining, while samples from a subgroup of 10 patients (5 pre-APA and 5 APA) were assessed for protamine presence. The effects of age and cryopreservation on the percentage of stained cells were assessed with the Fisher’s exact test. Participants/materials, setting, methods Semen samples were provided by male partners of couples treated at our fertility centre. Sample aliquots were cryopreserved and analyzed after thawing (CryoSperm & Sperm Preparation Medium, Origio). Histone presence was assessed through aniline blue staining and protamine presence was indirectly assessed through staining with the protamine competitor Chromomycin A3 (CMA3); stained cells were visualized by bright field and fluorescence microscopy, respectively. Two hundred sperm cells were evaluated in each sample by the same embryologist. Main results and the role of chance Mean paternal age was 31.5±3.9 and 44.4±2.1 years for pre-APA and APA patient-groups, respectively. Major parameters observed in fresh semen samples were: 2.3±0.7 vs. 3.6±1.6mL (volume); 39±28 vs. 38±12 x10ˆ6/mL (concentration); 7±12 vs. 6±5% (rapid progressive motility); 41±12 vs. 38±13% (slow progressive motility); 12±5 vs. 11±4% (non-progressive motility); 40±17 vs. 45±16% (immotile) and 5±1 vs. 6±2% (normal morphology), for pre-APA and APA, respectively. The percentage of aniline blue (histone marker) positive cells was less than half in fresh samples from APA as compared to pre-APA patients (10.2% and 23.4%, respectively, p < 0.0001). Consistently, the percentage of CMA3 (protamine absence marker) positive cells was less than half in APA as compared to pre-APA patients (6.4% and 16.4%, respectively, p < 0.0001). In both age groups, the percentages of aniline blue and CMA3 positive cells were higher in cryopreserved/thawed samples as compared to their fresh counterparts (aniline blue positive: 35.2% vs. 23.4% and 22.0% vs. 10.2%; CMA3 positive: 27.2% vs. 16.4% and 18.8% vs. 6.4%, in cryopreserved vs. fresh in pre-APA and APA, respectively, p < 0.0001). Limitations, reasons for caution Our study is limited by the potential interference of confounding factors not equally distributed in age groups. The conclusions from this study must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings Our findings suggest that APA increases, while cryopreservation decreases, sperm chromatin condensation, as indicated by changes in DNA histone and protamine abundance. Our study thus sheds light on the impact of paternal age and cryopreservation on sperm quality, providing new valuable parameters for reproductive medicine practice and research. Trial registration number na

GammaGateR: semi-automated marker gating for single-cell multiplexed imaging.

Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. To address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. The R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR.

Restoration of hippocampal adult neurogenesis by CDRI-08 (Bacopa monnieri extract) relates with the recovery of BDNF-TrkB levels in male rats with moderate grade hepatic encephalopathy.

Modulation of in vivo adult neurogenesis (AN) is an evolving concept in managing neurodegenerative diseases. CDRI-08, a bacoside-enriched fraction of Bacopa monnieri, has been demonstrated for its neuroprotective actions, but its effect on AN remains unexplored. This article describes the status of AN by monitoring neuronal stem cells (NSCs) proliferation, differentiation/maturation markers and BDNF-TrkB levels (NSCs signalling players) vs. the level of neurodegeneration and their modulations by CDRI-08 in the hippocampal dentate gyrus (DG) of male rats with moderate grade hepatic encephalopathy (MoHE). For NSC proliferation, 10mg/kg b.w. 5-bromo-2'-deoxyuridine (BrdU) was administered i.p. during the last 3days, and for the NSC differentiation study, it was given during the first 3days to the control, the MoHE (developed by 100 mg/kg b.w. of thioacetamide i.p. up to 10days) and to the MoHE male rats co-treated with 350 mg/kg b.w. CDRI-08. Compared with the control rats, the hippocampus DG region of MoHE rats showed significant decreases in the number of Nestin+/BrdU+ and SOX2+/BrdU+ (proliferating) and DCX+/BrdU+ and NeuN+/BrdU+ (differentiating) NSCs. This was consistent with a similar decline in BDNF+/TrkB+ NSCs. However, all these NSC marker positive cells were observed to be recovered to their control levels, with a concordant restoration of total cell numbers in the DG of the CDRI-08-treated MoHE rats. The findings suggest that the restoration of hippocampal AN by CDRI-08 is consistent with the recovery of BDNF-TrkB-expressing NSCs in the MoHE rat model of neurodegeneration.

Characterization of Angiogenic, Matrix Remodeling, and Antimicrobial Factors in Preterm and Full-Term Human Umbilical Cords.

Little is known about morphogenetic changes in the umbilical cord during the maturation process. Extracellular matrix remodeling, angiogenesis, progenitor activity, and immunomodulation are represented by specific markers; therefore, the aim of this study was to determine the expression of matrix metalloproteinase-2 (MMP2), tissue inhibitor of metalloproteinases-2 (TIMP2), CD34, vascular endothelial growth factor (VEGF), and human β-defensin 2 (HBD2) in preterm and full-term human umbilical cord tissue. Samples of umbilical cord tissue were obtained from 17 patients and divided into two groups: very preterm and moderate preterm birth umbilical cords; late preterm birth and full-term birth umbilical cords. Routine histology examination was conducted. Marker-positive cells were detected using the immunohistochemistry method. The number of positive structures was counted semi-quantitatively using microscopy. Statistical analysis was carried out using the SPSS Statistics 29 program. Extraembryonic mesenchyme cells are the most active cell producers, expressing MMP2, TIMP2, VEGF, and HBD2 at notable levels in preterm and full-term umbilical cord tissue. Statistically significant differences in the expression of CD34, MMP2, and TIMP2 between the two patient groups were found. The expression of VEGF was similar in both patient groups, with the highest number of VEGF-positive cells seen in the extraembryonic mesenchyme. The expression of HBD2 was the highest in the extraembryonic mesenchyme and the amniotic epithelium, where mostly moderate numbers of HBD2-positive cells were detected. Extracellular matrix remodeling in preterm and term umbilical cords is strongly regulated, and tissue factors MMP2 and TIMP2 take part in this process. The expression of VEGF is not affected by the umbilical cord's age; however, individual patient factors can affect the production of VEGF. Numerous CD34-positive cells in the endothelium of the umbilical arteries suggest a significant role of progenitor cells in very preterm and moderate preterm birth umbilical cords. Antimicrobial activity provided by HBD2 is essential and constant in preterm and full-term umbilical cords.

Abstract 2743: Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients

Abstract Introduction: Disseminated cancer cells (DCC) can be detected years before metastatic manifestation in bone marrow of lung cancer patients. Although a consensus protocol has been established, the impact of markers and methods for DCC detection are still not settled. We recently found that EpCAM+ DCC detected by immunofluorescence (IF) outperform the classical immunocytologic (IC) detection by cytokeratin (CK) antibodies regarding prognostic impact. However, it remained unclear whether this difference resulted from biological traits associated with EpCAM+ vs CK+ DCC or from methodological reasons. We therefore performed a side-by-side comparison of IC vs IF for CK and EpCAM. Methods: We prospectively collected bone marrow (BM) aspirates from 56 non-metastasized NSCLC patients that underwent tumor resection with curative intention. After density gradient centrifugation, we analyzed the mononucleated cells by (i) IC using the anti-CK antibody A45-B/B3 and (ii) by IF using the anti-CK antibody A45-B/B3 and the anti-EpCAM antibody HEA-125. IF signals were captured and objectively quantified using ImageJ, whereas IC was evaluated by two independent observers. Marker-positive cells were isolated and subjected to Whole Genome Amplification (WGA) and whole genome sequencing for assessment of copy number alterations (CNA). Survival analysis was performed for progression-free survival (PFS), tumor-specific survival (TSS) and overall-survival (OS) after a median follow-up time of 72.5, 78.7, 78.7 months, respectively. Results: IC and IF revealed the presence of CK+ DCCs in 37.5% and 41.1% of cases, respectively (P=0.34). IF identified EpCAM+ DCCs in 55.4% of patients, resulting in a 63.8% DCC-positivity rate found by IF, when combined with CK. The average number of DCCs per million MNC, the DCC density (DCCD), was 0.92, 0.93 and 1.44 for CKIC and CKIF and for EpCAMIF, respectively. No association was found between CKIC + DCCs and PFS, TSS or OS. In contrast, detection of EpCAMIF + and/or CKIF + DCCs showed a trend towards reduced PFS (P=0.055), TSS (P=0.225) and OS (P=0.155). Subgroup analysis revealed that both EpCAMIF + and CKIF + cells were correlated with worse PFS (P=0.016 and P=0.040, respectively) and TSS (P=0.033 and P=0.008, respectively). CKIF-positivity was also associated with worse OS (P=0.014), whereas EpCAM was not. CKIF + DCCs remained an independent prognostic variable for TSS and OS upon multivariate testing (hazard ratio: 6.45 and 3.31, respectively), while EpCAMIF + was not. The IF-marker-defined cell phenotypes exhibited comparable frequencies and distributions of CNAs across the entire genome (~50% of cells). Conclusions: Detection of CK+ DCC by quantified IF analysis apparently outperforms CK detection by IC in survival analysis. The impact of CK and EpCAM expression levels on outcome are under investigation. Citation Format: Daniel Spitzl, Tobias Mederer, Julius Zank, Severin Gütter, Michael Ried, Hans-Stefan Hofmann, Christoph A. Klein. Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of 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 2743.

Abstract 4932: UltiAnalyzer.AI: An automatic and robust AI-Driven tool for the quantification of multiplex immunofluorescence whole slide images

Abstract Introduction: Whole slide multiplexed immunofluorescence (mIF) facilitates in-depth study of the tumor microenvironment with multi-channel images resulting in an enormous amount of protein expression and spatial data across millions of single cells. Therefore, it has become crucial to process this huge amount of data automatically and reliably. Here we introduce UltiAnalyzer.AI, a software tool that integrates a set of artificial intelligence (AI) models specifically designed for the analysis of whole slide mIF slides, that is fully automatic, highly accurate, highly efficient, and fully scalable. Methods: UltiAnalyzer.AI processes are specialized for 1) tissue segmentation, in which tissue regions are identified using a semantic segmentation AI model on low magnification versions of the DAPI channel, 2) tiling, where slide images are divided into 512x512 overlapping tiles for efficient image processing and lower memory requirements, 3) tumor region segmentation based on a semantic segmentation AI model, 4) nucleus segmentation using the open-source StarDist model, and 5) marker-positive cell detection, in which AI object detection models are employed to classify nuclei as positive or negative in each marker channel using the Faster R-CNN architecture. Here, we take advantage of the full 16-bit dynamic range of images produced using Ultivue InSituPlex® amplification technology, although our approach could be applied to other platforms. Intensity thresholding is no longer necessary, and identification of a positive cell mainly considers the morphology of the cell in the context of its cellular neighborhood. Results: Trained on over 60 mIF slides and validated with manual annotations, UltiAnalyzer.AI has demonstrated strong performance, achieving f1-scores between 0.8 to 0.91 for cell detection, and average correlations of 0.9 for cell counts across 15 markers. Processing time is around 3s per tile, enabling the processing of large slides in under an hour. It has been tested on over 1000 slides, and in a comparative study, it matched a VisioPharm-based quantification with 0.95 correlation in positive counts for 8 markers. Cloud deployment allows for processing thousands of slides simultaneously and in considerably less time. Conclusions: UltiAnalyzer.AI represents a significant advancement in the field of whole-slide mIF analysis, offering high accuracy and scalability using fully automated processes. By utilizing the full dynamic range of Ultivue’s mIF technology and avoiding traditional thresholding methods, the tool also increases robustness and reproducibility. The incorporation of cutting-edge AI technology and scalable cloud computing capabilities positions UltiAnalyzer.AI as a leading-edge solution for mIF analysis in digital pathology. Citation Format: Ruben Cardenes, Douglas Wood, Martin Schulze, Je Lee, Lorenz Rognoni. UltiAnalyzer.AI: An automatic and robust AI-Driven tool for the quantification of multiplex immunofluorescence whole slide images [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 4932.

Astrocyte expression of aging-associated markers positively correlates with neurodegeneration in the frontal lobe of the rhesus macaque brain.

As the population over the age of 65 increases, rates of neurodegenerative disorders and dementias will rise - necessitating further research into the cellular and molecular mechanisms that contribute to brain aging. With the critical importance of astrocytes to neuronal health and functioning, we hypothesized that alterations in astrocyte expression of aging-associated markers p16INK4a (p16) and sirtuin 1 (SIRT1) with age would correlate with increased rates of neurodegeneration, as measured by FluoroJade C (FJC) staining. To test this hypothesis, 19 rhesus macaques at the Tulane National Primate Research Center were selected based on the following criteria: archival FFPE CNS tissue available to use, no noted neuropathology, and an age range of 5-30 years. Tissues were cut at 5 μm and stained for GFAP, p16, SIRT1, and FJC, followed by whole-slide imaging and HALO® image analysis for percentage of marker-positive cells and relative intensity of each stain. We found the percentage of p16+ cells increases with age in total cells and astrocytes of the frontal (p = 0.0021, p = 0.0012 respectively) and temporal (p = 0.0226, p = 0.0203 respectively) lobes, as well as the relative intensity of p16 staining (frontal lobe: p = 0.0060; temporal lobe: p = 0.0269). For SIRT1, we found no correlation with age except for an increase in the relative intensity of SIRT1 in the temporal lobe (p = 0.0033). There was an increase in neurodegeneration, as measured by the percentage of FJC+ cells in the frontal lobe with age (p = 0.0057), as well as in the relative intensity of FJC staining in the frontal (p = 0.0030) and parietal (p = 0.0481) lobes. Importantly, increased p16 and SIRT1 expression in astrocytes correlated with increasing neurodegeneration in the frontal lobe (p = 0.0009, p = 0.0095 respectively). Together, these data suggest that age-associated alterations in astrocytes contribute to neurodegeneration and provide a target for mechanistic studies in the future.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Similar Distribution between EPA-containing Phosphatidylcholine and Mesenchymal Stem Marker Positive Cells in the Aortic Wall of Abdominal Aortic Aneurysm Model Rat Fed a Low-EPA Content Diet.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by progressive dilation of the abdominal aorta. Previous studies have suggested that dietary components are closely associated with AAA. Among those dietary components, eicosapentaenoic acid (EPA) is considered to have suppressive effects on AAA. In the AAA wall of AAA model animals bred under EPA-rich condition, the distribution of EPA-containing phosphatidylcholine (EPA-PC) has been reported to be similar to that of the markers of mesenchymal stem cells (MSCs) and M2 macrophages. These data suggest that the suppressive effects of EPA on AAA are related to preferential distribution of specific cells in the aortic wall. However, the distribution of EPA-PC in the AAA wall of AAA model animals fed a diet containing small amounts of EPA, which has not been reported to inhibit AAA, has not yet been explored. In the present study, we visualized the distribution of EPA-PCs in the AAA wall of AAA model animals fed a diet containing small amounts of EPA (1.5% EPA in the fatty acid composition) to elucidate the vasoprotective effects of EPA. Positive areas for markers of MSCs were significantly higher in the region where EPA-PC was abundant compared to the regions where EPA-PC was weakly detected, but not for markers of M2 macrophages, matrix metalloproteinase (MMP)-2, and MMP-9. The distribution of MSC markers was similar to that of EPA-PC but not that of M2 macrophages and MMPs. These data suggest preferential incorporation of EPA into MSCs under the conditions used in this study. The incorporation of EPA into certain cells may differ according to dietary conditions, which affect the development of AAA.

Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated Device.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

Behavioral effects of adult male mice induced by low-level acetamiprid, imidacloprid, and nicotine exposure in early-life.

Acetamiprid (ACE) and imidacloprid (IMI), the neonicotinoid chemicals, are widely used as pesticides because of their rapid insecticidal activity. Although these neonicotinoids exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are largely unclear. This study investigated the effects of low-level, chronic neonicotinoids exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to an acceptable daily intake level of neonicotinoids in drinking water during the prenatal and postnatal periods. Additionally, we also exposed mice to nicotine (NIC) as a positive control. We then examined the effects on the central nervous system in adult male offspring. In the IMI and NIC exposure groups, we detected behavior that displayed impairment in learning and memory. Furthermore, immunohistochemical analysis revealed a decrease in SOX2 (as a neural stem cell marker) and GFAP (as an astrocyte marker) positive cells of the hippocampal dentate gyrus in the IMI and NIC exposure groups compared to the control group. These results suggest that exposure to neonicotinoids at low levels in early life affects neural circuit base formation and post-maturation behavior. Therefore, in the central nervous system of male mice, the effects of low-level, chronic neonicotinoids exposure during the perinatal period were different from the expected effects of neonicotinoids exposure in mature animals.

Less demand on stem cell marker-positive cancer cells may characterize metastasis of colon cancer.

CD44 and CD133 are stem cell markers in colorectal cancer (CRC). CD44 has distinctive isoforms with different oncological properties like total CD44 (CD44T) and variant CD44 (CD44V). Clinical significance of such markers remains elusive. Sixty colon cancer were examined for CD44T/CD44V and CD133 at mRNA level in a quantitative PCR, and clarified for their association with clinicopathological factors. (1) Both CD44T and CD44V showed higher expression in primary colon tumors than in non-cancerous mucosas (p<0.0001), while CD133 was expressed even in non-cancerous mucosa and rather decreased in the tumors (p = 0.048). (2) CD44V expression was significantly associated with CD44T expression (R = 0.62, p<0.0001), while they were not correlated to CD133 at all in the primary tumors. (3) CD44V/CD44T expressions were significantly higher in right colon cancer than in left colon cancer (p = 0.035/p = 0.012, respectively), while CD133 expression were not (p = 0.20). (4) In primary tumors, unexpectedly, CD44V/CD44T/CD133 mRNA expressions were not correlated with aggressive phenotypes, but CD44V/CD44T rather significantly with less aggressive lymph node metastasis/distant metastasis (p = 0.040/p = 0.039, respectively). Moreover, both CD44V and CD133 expressions were significantly decreased in liver metastasis as compared to primary tumors (p = 0.0005 and p = 0.0006, respectively). Our transcript expression analysis of cancer stem cell markers did not conclude that their expression could represent aggressive phenotypes of primary and metastatic tumors, and rather represented less demand on stem cell marker-positive cancer cells.

MP42-19 BILAYERED AUTOLOGOUS ADIPOSE-DERIVED MESENCHYMAL CELL-GELATIN SHEETS RECONSTRUCT URETERAL TISSUES IN RABBITS

You have accessJournal of UrologyCME1 Apr 2023MP42-19 BILAYERED AUTOLOGOUS ADIPOSE-DERIVED MESENCHYMAL CELL-GELATIN SHEETS RECONSTRUCT URETERAL TISSUES IN RABBITS Noriyuki Ogawa, Tetsuya Imamura, Tomonori Minagawa, Teruyuki Ogawa, and Osamu Ishizuka Noriyuki OgawaNoriyuki Ogawa More articles by this author , Tetsuya ImamuraTetsuya Imamura More articles by this author , Tomonori MinagawaTomonori Minagawa More articles by this author , Teruyuki OgawaTeruyuki Ogawa More articles by this author , and Osamu IshizukaOsamu Ishizuka More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003280.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Treatments of ureteral defects or strictures due to disease or trauma require reconstruction by urological surgery. However, those surgeries have some limitations, such as reoperation, or depending on ureteral length. Tissue engineering techniques would provide novel approaches to avoid the limitations. In this study, we fabricated bilayered autologous adipose-derived mesenchymal cell (AMC)-gelatin sheets and transplanted them into rabbits to replace surgically excised ureteral segments. METHODS: AMCs harvested from abdominal adipose tissues of female New Zealand White rabbits were cultured on collagen-coated dishes and labeled with a red fluorescent dye, PKH26, for later identification. Monolayers of AMCs that were cultured on temperature-responsive dishes were harvested by a gelatin hydrogel sheet. Two AMC-gelatin sheets were then overlaid together with the cell sides juxtaposed, forming a bilayered AMC-gelatin sheet. Following each partial ureterectomy of approximately 1 cm, the bilayered autologous AMC-gelatin sheet was transplanted, joining the proximal and distal ends of the remaining the ureter (experimental group; n=9). Control animals were conducted with a bilayered acellular-gelatin sheet (control group; n=9). RESULTS: At 4 and 8 weeks after transplantation, the proximal regions of ureters of control groups exhibited flexures and dilations compared with experimental groups. Both the control and experimental reconstructed ureteral walls had smooth muscle layers; however, those in the experimental reconstructed ureteral walls were significantly thicker and better organized than those in the control groups. In addition, the lumina of the 8-week reconstructed ureteral tissues in experimental groups did not show histological strictures as seen in the control ureters. Some AMCs differentiated into smooth muscle marker-positive cells. The experimental ureteral walls contained smooth muscle cells derived from the PKH26-labeled AMCs and others that were derived through migration and differentiation of cells from the remaining proximal and distal ends of the original ureter. CONCLUSIONS: These results indicated that the bilayered AMC-gelatin sheets could replace and/or reconstruct ureter tissues. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e575 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Noriyuki Ogawa More articles by this author Tetsuya Imamura More articles by this author Tomonori Minagawa More articles by this author Teruyuki Ogawa More articles by this author Osamu Ishizuka More articles by this author Expand All Advertisement PDF downloadLoading ...

Clinicopathologic and genetic characterization of angiofibroma of soft tissue: a study of 12 cases including two cases with AHRR::NCOA3 gene fusion.

Angiofibroma of soft tissue (AFST) is a benign tumour characterised by prominent arborizing blood vessels throughout the lesion. Approximately two-thirds of AFST cases were reported to have AHRR::NCOA2 fusion, and only two cases have been reported to have other gene fusions: GTF2I::NCOA2 or GAB1::ABL1. Although AFST is included in fibroblastic and myofibroblastic tumours in the World Health Organization's 2020 classification, histiocytic markers, especially CD163, have been reported to be positive in almost all examined cases, and it still remains the possibility of a fibrohistiocytic nature of the tumour. Therefore, we aimed to clarify the genetic and pathological spectrum of AFST and identify whether histiocytic marker-positive cells were true neoplastic cells. We evaluated 12 AFST cases, which included 10 cases with AHRR::NCOA2 and two with AHRR::NCOA3 fusions. Pathologically, nuclear palisading, which has not been reported in AFST, was detected in two cases. Furthermore, one tumour resected by additional wide resection revealed severe infiltrative growth. Immunohistochemical analysis indicated varying levels of desmin-positive cells in nine cases, whereas CD163- and CD68-positive cells were diffusely distributed in all 12 cases. We also performed double immunofluorescence staining and immunofluorescence in situ hybridisation in four resected cases with >10% desmin-positive tumour cells. The results suggested that the CD163-positive cells differed from desmin-positive cells with AHRR::NCOA2 fusion in all four cases. Our findings suggested that AHRR::NCOA3 could be the second most frequent fusion gene, and histiocytic marker-positive cells are not genuine neoplastic cells in AFST.

Autologous Bilayered Adipose-Derived Mesenchymal Cell-Gelatin Sheets Reconstruct Ureters in Rabbits.

Repair of ureteral defects or strictures due to disease or trauma is usually dependent upon surgery that often requires either reoperation or an alternative treatment. By taking advantage of tissue engineering and regenerative techniques, it may be possible to define new approaches to ureteral repair. In this study, we fabricated autologous bilayered adipose-derived mesenchymal cell (AMC)-gelatin sheets and transplanted them into rabbits to replace surgically excised ureteral segments. AMCs harvested from abdominal adipose tissues of female New Zealand white rabbits were cultured on collagen-coated dishes and labeled with PKH26, a red fluorescent dye, for later identification. Monolayers of the cultured PKH26-labeled AMCs were detached and applied to gelatin hydrogel sheets. Two gelatin sheets were then united with the AMC monolayers apposed together, forming a bilayered AMC-gelatin sheet. Following each partial ureterectomy, a bilayered autologous AMC-gelatin sheet was transplanted, joining the proximal and distal ends of the remaining ureter (n = 9). Control animals underwent the same procedure except that the transplant was achieved with a bilayered acellular-gelatin sheet (n = 9). At 4 and 8 weeks after transplantation, the proximal regions of ureters treated with the control bilayered acellular-gelatin sheets exhibited flexures and dilations, which are not characteristic of unoperated ureters. In contrast, the bilayered AMC-gelatin sheet-transplanted rabbits did not have ureteral flexures or dilations. About midway between the proximal and distal ends, both the control and experimental reconstructed ureteral walls had smooth muscle layers; however, those in the experimental reconstructed ureteral walls were significantly thicker and better organized than those in the control reconstructed ureteral walls. Some AMCs differentiated into smooth muscle marker-positive cells. The experimental ureteral walls contained smooth muscle cells derived from the PKH26-labeled AMCs and others that were derived through migration and differentiation of cells from the remaining proximal and distal ends of the original ureter. In addition, the lumina of the 8-week reconstructed ureteral tissues in experimental rabbits did not show histological strictures as seen in the control ureters. These results suggest that the bilayered AMC-gelatin sheets have the potential to replace defective tissues and/or reconstruct damaged ureters. Impact Statement To reconstruct ureter tissues following partial ureterectomy, we fabricated bilayered adipose-derived mesenchymal cell (AMC)-gelatin sheets based on cell sheet engineering principles. The bilayered AMC-gelatin sheets were transplanted into rabbits to replace a surgically excised ureteral segment. At 4 and 8 weeks after, the ureters that received bilayered AMC-gelatin sheets did not exhibit severe flexures, dilations, or strictures. The experimental ureteral walls had smooth muscle marker-positive cells that were differentiated from the AMCs, and similar cells were present in the adjacent intact ureteral tissues. Therefore, the bilayered AMC-gelatin sheets have the potential to reconstruct ureters damaged through disease or trauma.

Immune cells in mesothelioma microenvironment simplistic marker of response to nivolumab plus ipilimumab?

IntroductionMalignant pleural mesothelioma (MPM) is a malignant disease of the pleura which recently can be treated with immune checkpoint inhibitors (ICI). To optimize this treatment, a better understanding of the tumor micro environment is needed. We investigated subgroups of immune cells in subsequent tumor biopsies of patients treated with ICI. MethodsBiopsies from MPM patients included in two clinical ICI trials (nivolumab alone and an ipilimumab/nivolumab combination) were examined. At baseline and after 6 weeks of treatment, pleural biopsies were taken to examine the tumor microenvironment (CD20+, CD4+, CD8+, FoxP3+ and PD-1+ ). Cell density was defined as the number of marker positive cells per mm2. Radiological responses were evaluated as partial response, stable disease or progressive disease according to modified RECIST criteria. ResultsThirty-four and 36 patients were included in the nivolumab and ipiliumumab/nivolumab trial respectively. In the nivolumab trial, no significant differences in cell densities were seen in baseline biopsies of patients with partial response versus progressive disease. In contrast, in the ipilimumab/nivolumab trial, a higher cell density of CD4+, CD8+, FoxP3+ and PD-1+ cells at baseline was significantly correlated with partial responses. On-treatment biopsies of both trials did not show significant changes when compared to baseline biopsies. ConclusionBiopsies from patients responding to nivolumab plus ipilimumab treatment show a significant higher cell density of CD4+, CD8+, FoxP3+ and PD-1+ cells, without a change after 6 weeks of treatment. This observation is a first step in exploring the tumor microenvironment as predictor of response in ICI treatment in MPM.

Identification of microRNAs related with neural germ layer lineage-specific progenitors during reprogramming.

Differentiated cells can be reprogrammed to embryonic stem cell-like cells called induced pluripotent stem cells (iPSCs), in which the natural developmental differentiation process is reversed. It is unclear whether the multi-lineage cells can be isolated and identified during reprogramming. In the current study, we detected the expression of lineage markers, isolated neural lineages, and identified the related microRNAs during iPSC formation. Our results demonstrated that a neuroectoderm appeared earlier than mesoderm and definitive endoderm before forming colonies when mouse embryonic fibroblasts were subjected to iPSC formation using transcription factors (TFs). On day 3, the cells expressed Sox1 and Nestin and had ultrastructure consistent with the transition to identity neural germ layer lineage. Fluorescence-activated cell sorting analysis revealed a peak (40%) in neural progenitor marker-positive cells. When subsequently cultured in a neural precursor cell medium, these cells proliferated slowly, became round and aggregated, generating into neurons and glia. Genome-wide microRNA (miRNA) analysis identified 45 differentially regulated miRNAs. Molecular network analysis demonstrated that these miRNAs validated 6,047 experimental mRNA targets. The GO functional annotation analysis of mRNA targets revealed that most genes were related to neurogenesis, such as growth cone, neuronal cell body, neuron projection, and cell junction synapse. The network of protein-protein interactions was observed, which demonstrated that key nodes of neural lineage reprogramming-associated targets were Sall1, Foxa2, Nf2, Ctnnb1, Shh, and Bmpr1a. Therefore, these data suggested that TFs can drive the reprogramming of somatic cells towards a pluripotent state via neuroectoderm. Moreover, the neural lineage reprogramming system can address how miRNAs influence their target sites.

Development and Validation of a Digital Analysis Method to Quantify CD3-immunostained T Lymphocytes in Whole Slide Images of Crohn's Disease Biopsies.

The T-lymphocyte-mediated inflammation in Crohn's disease can be assessed by quantifying CD3-positive T-lymphocyte counts in colonic sections. We developed and validated a process to reliably quantify immunohistochemical marker-positive cells in a high-throughput setting using whole slide images (WSIs) of CD3-immunostained colonic and ileal tissue sections. In regions of interest (ROIs) and/or whole tissue sections of 40 WSIs from 36 patients with Crohn's disease, CD3-positive cells were quantified by an expert gastrointestinal pathologist (gold standard) and by image analysis algorithms developed with software from 3 independent vendors. Semiautomated quantification of CD3-positive cell counts estimated in 1 ROI per section were accurate when compared with manual analysis (Pearson correlation coefficient, 0.877 to 0.925). Biological variability was acceptable in digitally determined CD3-positive cell measures between 2 to 5 ROIs annotated on the same tissue section (coefficient of variation <25%). Results from computer-aided analysis of CD3-positive T lymphocytes in a whole tissue section and the average of results from 2 to 5 ROIs per tissue section lacked reliability (overestimation or underestimation and systematic bias), suggesting that absolute quantification of CD3-positive T lymphocytes in a whole tissue section may be more accurate. Semiautomated image analysis in WSIs demonstrated reproducible CD3-positive cell measures across 3 independent algorithms. A computer-aided digital image analysis method was developed and validated to quantify CD3-positive T lymphocytes in colonic and ileal biopsy sections from patients with Crohn's disease. Results support consideration of this digital analysis method for use in future Crohn's disease clinical studies.

Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma.

Multiplex immunofluorescence staining enables the simultaneous detection of multiple immune markers in a single tissue section, and is a useful tool for the identification of specific cell populations within the tumour microenvironment. However, this technology has rarely been validated against standard clinical immunohistology, which is a barrier for its integration into clinical practice. This study sought to validate and investigate the accuracy, precision and reproducibility of a multiplex immunofluorescence compared with immunohistochemistry (IHC), including tissue staining, imaging and analysis, in characterising the expression of immune and melanoma markers in both the tumour and its microenvironment. Traditional chromogenic IHC, single-plex immunofluorescence and multiplex immunofluorescence were each performed on serial tissue sections of a formalin-fixed paraffin-embedded (FFPE) tissue microarray containing metastatic melanoma specimens from 67 patients. The panel included the immune cell markers CD8, CD68, CD16, the immune checkpoint PD-L1, and melanoma tumour marker SOX10. Slides were stained with the Opal™ 7 colour Kit (Akoya Biosciences) on the intelliPATH autostainer (Biocare Medical) and imaged using the Vectra 3.0.5 microscope. Marker expression was quantified using Halo v.3.2.181 (Indica Labs). Comparison of the IHC and single-plex immunofluorescence revealed highly significant positive correlations between the cell densities of CD8, CD68, CD16, PD-L1 and SOX10 marker positive cells (Spearman’s rho = 0.927 to 0.750, p < 0.0001). Highly significant correlations were also observed for all markers between single-plex immunofluorescence and multiplex immunofluorescence staining (Spearman’s rho >0.9, p < 0.0001). Finally, correlation analysis of the three multiplex replicates revealed a high degree of reproducibility between slides (Spearman’s rho >0.940, p < 0.0001). Together, these data highlight the reliability and validity of multiplex immunofluorescence in accurately profiling the tumour and its associated microenvironment using FFPE metastatic melanoma specimens. This validated multiplex panel can be utilised for research evaluating melanoma and its microenvironment, such as studies performed to predict patient response or resistance to immunotherapies.