Cytokeratin Research Articles

Assessing cellular agriculture potential: Population homogeneity and gene expression in cultured bovine mammary epithelial cells

Mammary epithelial cells (MEC) are responsible for milk synthesis in the bovine mammary gland during lactation, and could thereby potentially be used for in-vitro milk components production. Development of such a model, however, requires the MECs to preserve their lactogenic traits following in-vitro growth. Potential impact of growing MEC in the lab following their isolation was studied in relation to their population homogeneity and milk synthesis related gene expressions. Upon isolation, flow cytometry results showed that > 30 % of the obtained cells were positive for cytokeratin 18 (CK18), a marker for the MEC. This proportion increased to 95 % MEC after the first passage. Cell functionality was examined by comparing the expressions of milk synthesis related genes at passage 2 (P2) to passage 6 (P6) following treatment with lactogenic agents. CSN1S1 was found to be expressed significantly higher at P2 compared to P6. In addition, CSN2, CSN3 and PRLR tended to have lower expressions in P6 compared to P2. DGAT1, STAT5A, AKT1, and RPS6KB1 were expressed comparably in both passages. The expressions of LALBA and CSN1S2 were not detectable in any of the samples. Therefore, despite having high levels of CK18 positive cells at later passages, in line with other studies, further passaging affected the expressions of some of the genes related to milk synthesis. A reduced gene expression capacity in the propagated MEC may limit their potential for in-vitro milk components production.

Probiotic supplementation for 24 weeks in patients with non-alcoholic steatohepatitis: the PROBILIVER randomized clinical trial.

Considering the increasing prevalence of non-alcoholic steatohepatitis (NASH) and treatment gaps, this study aimed to evaluate the effect of probiotic supplementation on liver function markers, nutritional status, and clinical parameters. This double-blind, randomized clinical trial (ClinicalTrials.gov ID: NCT0346782) included adult outpatients with biopsy-proven NASH. The intervention consisted of 24 weeks of supplementation with the probiotic mix Lactobacillus acidophilus (1 × 109 CFU) + Lactobacillus rhamnosus (1 × 109 CFU) + Lactobacillus paracasei (1 × 109 CFU) + Bifidobacterium lactis (1 × 109 CFU), or placebo, twice a day. The following parameters were evaluated: demographic and clinical data, transient elastography (FibroScan), liver enzymes, NAFLD fibrosis score, fatty liver index, laboratory assessment, serum concentration of toll-like receptor-4 (sTLR-4) and cytokeratin 18 (CK-18), anthropometric data, dietary intake, and physical activity. Regarding data analysis, the comparison between the groups was based on the delta of the difference of each variable analyzed (value at the end of treatment minus the baseline value) using the t-test for independent samples or the Mann-Whitney U-test. Forty-four patients with NASH completed the trial (51.4 ± 11.6 years). At baseline, 87% of participants had a mild liver fibrosis degree on biopsy, normal values of liver enzymes, transient elastography values consistent with grade 1 fibrosis in both groups, increased waist circumference (WC), a BMI of 30.97 kg/m2, and 76% presented with metabolic syndrome (MetS). After the intervention, no differences were observed between the probiotic and placebo groups in terms of MetS, WC, BMI scores, or liver enzyme levels (p > 0.05 for all). The elastography values remained consistent with grade 1 fibrosis in both groups. Although CK-18 was reduced in both groups, a larger effect size was noted in the probiotic group (D = 1.336). sTLR-4 was also reduced in both groups, with no difference between groups (p = 0.885). Intervention with probiotics in the early stages of NASH demonstrated no significant change in hepatic and clinical parameters. ClinicalTrials.gov, identifier NCT0346782.

An insight into immunohistochemical profile of triple negative breast carcinoma in a tertiary care center

Introduction: Triple Negative Breast Cancer (TNBC) is a type of breast cancer that fails to express Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER2) in Immunohistochemical analysis. TNBC has a unique molecular profile, aggressive nature and lack of efficient targeted therapies. The aim of the study was to evaluate the expression of basal markers like Epidermal Growth Factor Receptor (EGFR) and Cytokeratin 5/6 (CK 5/6) and to correlate their expression with the clinico-pathological parameters, thus enabling the identification of the aggressive basal like phenotype of TNBC in resource-constrained settings. Methodology: The study was conducted at a South Indian tertiary care centre for 2 years. Data such as age of presentation, tumour measurements, margins and necrosis, numbers, levels and sizes of lymph nodes were collected. Data on histological type and grade, in situ component, lymphovascular and perineural invasion, stromal reaction and lymph node status were recorded. Primary Immunohistochemical studies were performed for the markers ER,PR and HER2.Patients with negative expression for all the three markers were identified. IHC studies for EGFR and Cytokeratin 5/6(CK5/6) were done on tissue blocks and the expression of these two markers were equated with the clinico-pathological parameters. Results: Among the 94 cases, 44 cases (46.8%) expressed CK5/6 and 46 (48.9%) expressed EGFR. A plausible association was noted between the expression of the two markers with age of the patient, tumour size, histological grade and tumour necrosis. Conclusion: Combination of the time honoured gross and histopathological examinations along with the application of basal markers like CK 5/6 and EGFR, helps in identifying the basal-like phenotype with dismal clinical outcome among the triple-negative breast cancers in resource-constrained settings. An insight into such an aggressive tumour, provided by these markers may be critical in designing personalized treatment strategies for such patients.

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 5378: Spatially resolved human leukocyte antigen (HLA) expression correlates with motif neoepitope expression and durable response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC)

Abstract Background: We previously showed electrostatic interactions enhance binding between “motif” neoepitopes and HLA and predict advanced NSCLC ICB benefit, suggesting optimal neoantigen presentation drives effective anti-tumor immune response (Cummings 2020). However, not all cases with motif neoepitopes exhibit favorable ICB outcomes. Methods: As proof-of-concept, participants from our published NSCLC ICB cohort with available pre-treatment archival tissue were selected based on HLA supertype B44, predicted motif neoepitopes, and best response of progressive disease (PD) vs partial/complete response (PR/CR). Whole exome sequencing, HLA typing and neoepitope prediction were previously performed (Cummings 2020). GeoMx slides were prepared, hybridized with RNA probes and stained with antibodies to cytokeratin (CK), nuclear material and CD45 per manufacture instructions (Geiss 2008). Antibody to CD8 (Novus) was optimized at 1:50 dilution. Twelve regions of interest (ROI) were selected per sample including the following compartments: CK+CD8+, CK+CD8-, CK-CD8+ and CK-CD8- (negative control). Each ROI included ~150 nuclei selected using GeoMx Digital Spatial Profiler. Sequencing was performed on Illumina Novaseq 6000 (50bpx2) demultiplexed using Illumina Bcl2fastq v2.19.1.403. Raw reads were normalized and compared using paired student’s T-tests with Tukey’s correction via R v4.1. Neoepitope (non/motif) comparisons used matched numbers per sample selected by highest expression. Results: Seven participants (3 PD, 3 PR, 1 CR) were included, each exhibiting 1-7 motif neoepitopes without HLA loss of heterozygosity. 84 ROI included 24 CK+CD8+, 24 CK+CD8-, 24 CK-CD8+ and 12 CK-CD8- with at least one ROI per category per participant. PD samples had fewer CD8+ ROI (12 vs 36) and lower CXCL9 (p=0.011) and CD274 (p=0.014). HLA-A/B/C expression followed a gradient with highest expression in CK+CD8+ ROI with limited detection in CK-CD8- ROI. Among HLA, HLA-B had highest median expression (A: 226, B: 650, C: 202; p<0.001). In CK+ ROI, motif neoepitope expression was detectable for all participants but not statistically different from non-motif expression (p=0.23). HLA-A/B/C all exhibited lower expression in PD CK+ ROI (p<0.001). Conclusion: HLA expression may modify prediction of clinically meaningful neoepitopes and response to ICB. Validation in a larger, prospective cohort is planned. Citation Format: Lucas Goldman, Sarah Gorbatov, Collin Shen, Alexa Berezowitz, Edward Garon, Amy Lauren Cummings. Spatially resolved human leukocyte antigen (HLA) expression correlates with motif neoepitope expression and durable response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5378.

Abstract 2303: Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes

Abstract Background: Circulating tumor cell clusters (CTCCs) are aggregated groups of tumor cells that detached from primary tumors and circulate the bloodstream. However, while Circulating Tumor Cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined, with only a few descriptive investigations evaluating their clinical utility. Adding to CTCC complexity is that various subtypes exist, including homotypic clusters consisting of a homogenous population of tumor cells and heterotypic CTCCs made of CTCs with immune/stromal white blood cells (WBCs). Furthermore, CTCs can undergo Epithelial-Mesenchymal Transition (EMT), a process characterized by loss of epithelial traits and upregulation of mesenchymal traits, and can form clustered EMTs (CEMTs). Moreover, CTCs can fuse with macrophages forming Tumor Macrophage Hybrid Cells, aka Cancer-Associated Macrophage Like cells (CAMLs) when in circulation. We enumerated all these types of cells and complex aggregates in the peripheral blood of patients with metastatic breast cancer (mBC). Methods: We enumerated the 6 populations from patient samples enrolled in IRB aproved prospective pilot studies. Whole peripheral blood (7.5mL) was filtered and stained with cytokeratin (CK) & CD45/CD14 to identify CTCs. CTCs were defined as an intact DAPI nuclei and strong filamentous CK. Homotypic CTCCs were defined as ≥2 CTCs attached together. Heterotypic CTCCs were defined as ≥1 CTC attached to ≥1 WBC. EMTs were defined as having DAPI nuclei and weak non-filamentous CK. CEMTS were defined as ≥2 EMTs. CAMLs were defined as having an enlarged polynucleated DAPI, and positive for CD45/CD14 or non-filamentous CK. Median progression free survival (mPFS) and median overall survival (mOS) over 24 months (m) were evaluated. Results: We evaluated the blood of 79 mBC patients. Single CTCs were found in 57% of patients (n=34/79), homotypic CTCCs 15% (n=12/79), heterotypic CTCCs 66% (n=27/79), EMTs 56% (n=44/79), CEMTs 23% (n=18/79) and CAMLs 97% (n=77/79). Over 24 ms, patierns with detectable heterotypic CTCCs & homotypic CTCCs had the shortest response times at mPFS=2.8 & mOS=5.8 ms, or mPFS=2.1 & mOS=4.3 ms, respectively. This was followed by CTCs mPFS=3.7 & mOS=13.7 ms, EMTs mPFS=4.3 & mOS=14.4 ms, CAMLs mPFS=4.3 & mOS=14.4 ms, and CEMT mPFS=4.7 & mOS=13.2 ms. Additionally, CTCCs were rare in HER2+ patients at 4.4% (n=1/23), but no associations to other hormone statuses were found. Conclusion: CTCCs appear to represent an array of subtypes with different biological and clinical meanings. Here we enumerated CTCC subtypes in the blood of mBC patients and stratified the various types to clinical outcomes. These data suggest CTCCs are clinically important and further understanding of their biology in the context of tumor pathogenesis is needed. Citation Format: Daniel L. Adams, Carolina Reduzzi, Aravind Aryasomayajula, R. Katherine Alpaugh, Saranya Chumsri, Cha-Mei Tang, Giuseppe Del Priore, William V. Williams, Massimo Cristofanilli. Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes [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 2303.

Abstract 4248: Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development

Abstract Introduction: Approximately 30% of advanced bladder cancer (BCa) tumors exhibit MTAP gene deletion, which has been associated with luminal subtype BCa, poor response to chemotherapy, and shorter progression to metastatic disease (De Souza 2023). MTAP deletion has been proposed as a potential biomarker for response to pemetrexed and an emerging class of PRMT5 and MAT2A inhibitors in development for MTAP-deleted tumors. Here, we describe the first patient-derived 3D model of BCa with MTAP deletion and its translational potential as a drug sensitivity platform. Methods: A patient (pt) with metastatic BCa harboring MTAP deletion underwent a pelvic lymph node biopsy. A fresh tumor sample was processed under collagenase/dispase solution for 30 minutes and filtered through a 70-um cell strainer. Cells were cultures in low attachment plates with MammoCult™ Basal Medium (Stem Cell Technologies). After 5 passages and 3D spheroid formation, immunostaining for cytokeratin (CK) 5 and 6, GATA3, CK20, and Ki67 staining were performed to confirm the urothelial origin. The MTAP-del was confirmed by a western blot of the organoid specimen and compared to other BLCa cell lines (UMUC, RT4, J82, and 5637). The viability assays were performed using the CellTiter-Glo® 3D Cell kit. Next-generation sequencing (NGS) of primary BCa tumor revealed deletion of MTAP, CDKN2A, CDKN2B, and mutations in p53, TERT, and KDM6A. Results: The patient organoid sample was positive for the luminal CK5/6 markers and negative for GATA3. Ki67 and CK20 were primarily expressed in the nucleus of the same sample. The organoid retained morphologic and immunohistochemical features of the patient’s primary bladder tumor, showing the same squamous differentiation and similar Ki-67 proliferative index. The organoid displayed no evidence of the MTAP protein by western blot (similar findings with the MTAP-del cell lines UMUC and RT4). The organoid showed sensitivity to pemetrexed [pemetrexed IC50=0.12 uM (other MTAP-del BLCa IC50: 0.1-0.23 uM)] and resistance to gemcitabine and cisplatin [Gem IC50=55 nM (other BLCa IC50: 1-29 nM), Cis IC50=90.5 uM (other BLCa IC50: 2-17 uM). Conclusions: We describe the first patient-derived MTAP-deleted BCa organoid recapitulating the features of the primary tumor. The organoid displayed sensitivity to pemetrexed as described with MTAP-deleted BCa. The organoid represents a unique platform for testing novel therapeutic strategies for MTAP-deleted BCa. Citation Format: Maximilian Schwermann, Benedito Carneiro, Shaolei Lu, Andre De Souza, Matthew Hadfield, Anthony Mega, Galina Lagos, Sari Khaleel, Dragan Golijanin, Sheldon Holder, Praveen Srinivasan, William MacDonald, Andrew George, Lanlan Zhou, Leiqing Zhang, Wafik El-Deiry. Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development [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 4248.

Abstract 6561: Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer

Abstract Background: Ovarian Cancer (OC) is one of the leading causes of death among gynecological tumors with an insufficient understanding of OC disease evolution. Since tumor tissue is not always available, blood allows the investigation of single circulating tumor cells (sCTC) over time. We combined immunomagnetic enrichment and image-based sCTC sorting for phenotypic and genotypic analysis of sCTCs in the follow-up of OC to provide insights into sCTC heterogeneity and their malignant character. Patients and Methods: 10 ml blood of high-grade serous OC patients (pts) at the time of primary diagnosis (n=42), after chemotherapy (n=23) and after Avastin (n=11) was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® using CD45 and CD235a antibodies coupled to MACS® MicroBeads. For image-based sCTC sorting (DEPArray®), an immunofluorescence protocol staining for Hoechst, cytokeratin (CK), the folate receptor alpha (FRa), Vimentin (Vim) and CD45 was applied. Whole genome amplification (WGA) was performed in recovered single cells, comprising cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number alteration (CNA) analysis. Ampli1TM low-pass analysis was used to analyze single cell WGA products. 21 primary tumors (PT) were assessed for whole-genome CNA and the Ampli 1TM OncoSeek panel was used for deep sequencing of sCTCs. Results: Phenotypic assessment revealed FRa-positive cells in 28/42 (67%) pts, CK-positive cells in 32/42 (76%) pts and CK-FRa-double-positive cells in 15/42 (36%) pts, respectively, with a significant (p= 0.0205) reduction of cells after chemotherapy only documented for FRa-positive cells. Interestingly, cells with a high nuclear staining but no expression of target antigens were found in 37/42 pts (88%) and significantly (p=0.002) expanded during treatment. Genotyping revealed that only 7% (19/289) of single cells deriving from 13 different pts were aberrant. Whole genome CNA analysis in all pre-treatment sCTCs revealed enriched CNAs in chromosomes 2, 7 and 12. In post-treatment sCTCs, CNA dynamics highlighted the adaptive potential of the homologous recombination pathway and suggested further investigation of MAPK signaling in resistant OC. Low-pass CNA comparison between sCTCs and the PT was feasible in nine pts, 2/9 with persistent PT characteristics in sCTCs at different sampling times. TP53 variants were the most frequently detected ones, persistent CNA in the CDK4 oncogene and emerging CNA in the ALK oncogene were found in sCTCs from two pts. Conclusion: We identified sCTCs in the follow-up of OC and demonstrated interpatient as well as intrapatient heterogeneity of sCTCs. Matched sCTCs and PT data showed similar CNAs to a certain amount. Our low percentage of aberrant cells suggests that higher patient numbers are needed to confirm our findings to understand disease progression. Citation Format: Sabine Kasimir-Bauer, Nikolas H. Stoecklein, Rui P. Neves, Sven T. Liffers, Jan D. Kuhlmann, Pauline Wimberger, Paul Buderath, Rainer Kimmig, Carolin Salmon. Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer [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 6561.

Abstract 4231: A biobank of bladder cancer organoids as a platform for screening new therapeutic approaches

Abstract In patients with bladder cancer (BC) immune therapies have recently demonstrated an improvement for overall survival rates compared to chemotherapy alone. However, several challenges persist and it remains an unmet need to develop novel therapeutic strategies. Organoids have emerged as a promising animal-free tool revealing patients’ heterogeneity and allowing large preclinical therapeutic screening. Urosphere developed a BC organoids biobank with phenotypic, pharmacological and molecular characterization. The aim of this study was to demonstrate the relevance of our organoids models and their use for validation of therapeutic approaches such as targeted or CAR-T cell therapies using EGFR as target. Molecular characterization of the organoids was performed by exome and whole transcriptome sequencing. Cell architecture and differentiation were assessed by labeling cytokeratins (CK) 5, 17 and 20, and uroplakin 3 (UPK3). Proliferation and tumor status were validated with Ki67 and GATA3, respectively. To evaluate targeted-therapy, organoids were treated with erlotinib for 5 days and cell viability was measured with CellTiter-Glo3D® (CTG) assay. To validate CAR-T cell approach, organoids were co-cultured with anti-EGFR or control anti-CD19 CAR-T cells at 4 effector:target ratios. Cell viability was measured with CTG and Caspase-Glo® 3/7 3D assays. Pro-inflammatory and granzyme B release were analyzed by Luminex® assay. Phenotypically, organoids showed proliferating structures whose tumor status was confirmed by GATA3 expression. Cellular heterogeneity present in original tumors was also found, with differential expression of CKs showing the presence of basal (CK5/CD17) and/or differentiated superficial (CK20/UPK3) cells. Organoids from 2 models with high transcriptomic EGFR expression were selected and protein expression was confirmed by immunofluorescence. For targeted therapy, erlotinib presented significant efficacy in both models with a better activity in the model presenting the highest EGFR expression. After 48h of co-culture with anti-EGFR CAR-T cells, a decrease of cell viability associated with an increase of apoptosis was observed. After 72h of co-culture, an increase in the secretion of pro-inflammatory cytokines (IFN-γ and TNF-α) and granzyme B by anti-EGFR CAR-T cells was also observed. In co-cultures with organoids higher expressing EGFR, CAR-T cells activation appeared higher compared to those co-cultured with organoids lower expressing EGFR. Organoids reproduce characteristics of the tumor from which they are derived, making them a predictive preclinical model. In an EGFR targeting approach, the use of organoids to evaluate both targeted- and CAR-T cell therapies was validated with concordant results. This robust translational model is fully transposable to other therapeutic approaches, whether in a traditional culture model or in a co-culture system. Citation Format: Emilie Decaup, Claire Béraud, Anne Sophie Bajeot, Xavier Gamé, Nicolas Monjotin, Pascal Rischmann, Philippe Lluel, Mathieu Roumiguié. A biobank of bladder cancer organoids as a platform for screening new therapeutic approaches [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 4231.

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [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 3706.

Comparative Characterization of Human Meibomian Glands, Free Sebaceous Glands, and Hair-Associated Sebaceous Glands Based on Biomarkers, Analysis of Secretion Composition, and Gland Morphology.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography-mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell-cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell-cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Noninvasive single-cell-based prenatal genetic testing: Aproof of concept clinical study.

To clinically assess a cell-based noninvasive prenatal genetic test using sequence-based copy number analysis of single trophoblasts from maternal blood. Blood was obtained from 401 (243+158) individuals (8-22weeks) and shipped overnight. Red cells were lysed, and nucleated cells stained for cytokeratin (CK) and CD45 and enriched for positive CK staining. Automated scanning was used to identify and pick single CK+ /CD45- trophoblasts which were subjected to next-generation sequencing. Blood was obtained from 243 pregnancies scheduled for CVS or amniocentesis. Luna results were normal for 160 singletons while 15 cases were abnormal (14 aneuploidy and one monozygotic twin with Williams syndrome deletion). The deletion was confirmed in both fetuses. Placental mosaicism occurred in 7 of 236 (3.0%) Luna cases and in 3 of 188 (1.6%) CVS cases (total 4.6%). No scorable trophoblasts were recovered in 32 of 236 usable samples. Additionally, 158 low-risk pregnancies not undergoing CVS/amniocentesis showed normal results in 133 cases. Seven had aneuploidy results, and there were three likely pathogenic deletions/duplications, including one15q11-q13 deletion. Although the sample size is modest and statistically accurate measures of test performance are not possible, the Luna test detected aneuploidy and deletions/duplications based on concordance with CVS/amniocentesis.

A comprehensive exploration of diverse skin cell types in the limb of the desert tortoise (Testudo graeca) through light, transmission, scanning electron microscopy, and immunofluorescence techniques

The Greek tortoise, inhabiting harsh desert environments, provides a compelling case for investigating skin adaptations to extreme conditions. We have utilized light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescence analysis to describe the structure of the arid-adapted limb skin in the Greek tortoise. Our aim was to identify the cell types that reflect the skin adaptation of this tortoise to arid conditions. Utilizing seven antibodies, we localized and elucidated the functions of various skin cells, shedding light on how the tortoise adapts to adverse environmental conditions. Our findings unveiled numerous scales on the limbs, varying in size and color, acting as protective armor against abrasions, bites, and other potential threats in their rocky habitats. The epidermis comprises four layers: stratum basalis, stratum spinosum, peri-corneous layer, and stratum corneum. Cytokeratin 14 (CK14) was explicitly detected in the basal layer of the epidermis, suggesting a role in maintaining epidermal integrity and cellular function. Langerhans cells were observed between epidermal cells filled with ribosomes and Birbeck granules. Numerous dendritic-shaped Langerhans cells revealed through E-Cadherin signify strong immunity in tortoises' skin. Melanophores were identified using the Melan-A antibody, labeling the cytoplasm, and the SOX10 antibody, labeling the nucleus, providing comprehensive insights into melanophores morphology and distribution. Two types of melanophores were found: dendritic below the stratum basalis of the epidermis and clustered oval melanophores in the deep dermal layer. Varied melanophores distribution resulted in a spotted skin pattern, potentially offering adaptive camouflage and protection against environmental challenges. Numerous myofibroblasts were discerned through alpha-smooth actin (α-SMA) expression, indicating that the Greek tortoise's skin possesses a robust tissue repair and remodeling capacity. B-cell lymphocytes detected via CD20 immunostaining exhibited sporadic distribution in the dermis, concentrating in lymphoid aggregates and around vessels, implying potential roles in local immune responses and inflammation modulation. Employing Tom20 to identify skin cells with abundant mitochondria revealed a notable presence in melanophores and the basal layer of the epidermis, suggesting high metabolic activity in these cell types and potentially influencing cellular functions. These findings contribute to our comprehension of tortoise skin anatomy and physiology, offering insights into the remarkable adaptations of this species finely tuned to their specific environmental habitats.

Sulforaphane Inhibits Adhesion and Migration of Cisplatin- and Gemcitabine-Resistant Bladder Cancer Cells In Vitro.

Only 20% of patients with muscle-invasive bladder carcinoma respond to cisplatin-based chemotherapy. Since the natural phytochemical sulforaphane (SFN) exhibits antitumor properties, its influence on the adhesive and migratory properties of cisplatin- and gemcitabine-sensitive and cisplatin- and gemcitabine-resistant RT4, RT112, T24, and TCCSUP bladder cancer cells was evaluated. Mechanisms behind the SFN influence were explored by assessing levels of the integrin adhesion receptors β1 (total and activated) and β4 and their functional relevance. To evaluate cell differentiation processes, E- and N-cadherin, vimentin and cytokeratin (CK) 8/18 expression were examined. SFN down-regulated bladder cancer cell adhesion with cell line and resistance-specific differences. Different responses to SFN were reflected in integrin expression that depended on the cell line and presence of resistance. Chemotactic movement of RT112, T24, and TCCSUP (RT4 did not migrate) was markedly blocked by SFN in both chemo-sensitive and chemo-resistant cells. Integrin-blocking studies indicated β1 and β4 as chemotaxis regulators. N-cadherin was diminished by SFN, particularly in sensitive and resistant T24 and RT112 cells, whereas E-cadherin was increased in RT112 cells (not detectable in RT4 and TCCSup cells). Alterations in vimentin and CK8/18 were also apparent, though not the same in all cell lines. SFN exposure resulted in translocation of E-cadherin (RT112), N-cadherin (RT112, T24), and vimentin (T24). SFN down-regulated adhesion and migration in chemo-sensitive and chemo-resistant bladder cancer cells by acting on integrin β1 and β4 expression and inducing the mesenchymal-epithelial translocation of cadherins and vimentin. SFN does, therefore, possess potential to improve bladder cancer therapy.

Cytokeratin-18 as a Non-invasive Biomarker and its Association with Biochemical Parameters in the Diagnosis of Non-alcoholic Fatty Liver Disease

Objective: Nonalcoholic fatty liver disease (NAFLD) is one of the chronic silent diseases in which its therapeutic options and noninvasive markers of disease activity and severity remain limited. We aimed in this study to assess cytokeratin-18 (CK18) as a new non-invasive biomarker to distinguish between NAFLD stages and its correlation with some biochemical parameters.
 Methods: A case control study was conducted on a sample of 90 subjects aged 12-79 years, categorized into three groups (nonalcoholic steatohepatitis "NASH", steatosis and controls). CK18, fasting blood glucose (FBG), lipid profile parameters, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine were determined. Low destiny lipoprotein-cholesterol (LDL-C), and body mass index (BMI) were calculated in addition to performing complete blood count (CBC).
 Results: The results indicate that the mean level of serum CK18 in NASH cases was significantly higher than steatosis and control groups. CK18 has a positive correlation with triglycerides (TG) total cholesterol (TC), ALT, AST, FBG, urea, creatinine, age, BMI, and LDL-C, and a negative correlation with high density lipoprotein-cholesterol HDL-C. Finally, ROC curve showed that the sensitivity of CK18 test is 77.1% and specificity is 96.6%. The cut-off value for CK18 test is 161 U/L.
 Conclusions: In this study, a significant relationship was observed between CK18, hepatic enzymes, and NAFLD degrees. CK18 has good accuracy, sensitivity, and specificity in diagnosing NASH.

Establishment of a chicken intestinal organoid culture system to assess deoxynivalenol-induced damage of the intestinal barrier function

BackgroundDeoxynivalenol (DON) is a mycotoxin that has received recognition worldwide because of its ability to cause growth delay, nutrient malabsorption, weight loss, emesis, and a reduction of feed intake in livestock. Since DON-contaminated feedstuff is absorbed in the gastrointestinal tract, we used chicken organoids to assess the DON-induced dysfunction of the small intestine.ResultsWe established a culture system using chicken organoids and characterized the organoids at passages 1 and 10. We confirmed the mRNA expression levels of various cell markers in the organoids, such as KI67, leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), mucin 2 (MUC2), chromogranin A (CHGA), cytokeratin 19 (CK19), lysozyme (LYZ), and microtubule-associated doublecortin-like kinase 1 (DCLK1), and compared the results to those of the small intestine. Our results showed that the organoids displayed functional similarities in permeability compared to the small intestine. DON damaged the tight junctions of the organoids, which resulted in increased permeability.ConclusionsOur organoid culture displayed topological, genetic, and functional similarities with the small intestine cells. Based on these similarities, we confirmed that DON causes small intestine dysfunction. Chicken organoids offer a practical model for the research of harmful substances.

Assessing the clinical and biochemical efficacy of alpha-lipoic acid in patients with non-alcoholic fatty liver disease: A randomized placebo-controlled clinical trial

Given the importance of inflammatory and metabolic markers in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), this study was designed to evaluate the effects of alpha-lipoic acid (ALA) supplementation on inflammatory and serum levels of fetuin-A, sirtuin1 (SIRT-1), cytokeratin 18 (CK-18), and hepatic steatosis in patients with NAFLD. In a double-blind, placebo-controlled, randomized clinical trial, 50 patients with NAFLD were randomized to receive daily supplementation with either two capsules of ALA (each capsule containing 600 mg ALA plus 400 mg/day of vitamin E) or two placebo capsules (two placebo capsules plus 400 mg/day of vitamin E) for 12 weeks. Significant reductions in homeostasis model assessment for insulin resistance (HOMA-IR) score and serum levels of insulin and fetuin-A were observed in the ALA group compared to the placebo group (all P < 0.05). ALA supplementation was significantly more effective than placebo in reducing hepatic steatosis by at least one grade.

ATOH1, TFAP2B, and CEACAM6 as Immunohistochemical Markers to Distinguish Merkel Cell Carcinoma and Small Cell Lung Cancer.

Merkel cell carcinoma (MCC) and small cell lung cancer (SCLC) can be histologically similar. Immunohistochemistry (IHC) for cytokeratin 20 (CK20) and thyroid transcription factor 1 (TTF-1) are commonly used to differentiate MCC from SCLC; however, these markers have limited sensitivity and specificity. To identify new diagnostic markers, we performed differential gene expression analysis on transcriptome data from MCC and SCLC tumors. Candidate markers included atonal BHLH transcription factor 1 (ATOH1) and transcription factor AP-2β (TFAP2B) for MCC, as well as carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) for SCLC. Immunostaining for CK20, TTF-1, and new candidate markers was performed on 43 MCC and 59 SCLC samples. All three MCC markers were sensitive and specific, with CK20 and ATOH1 staining 43/43 (100%) MCC and 0/59 (0%) SCLC cases and TFAP2B staining 40/43 (93%) MCC and 0/59 (0%) SCLC cases. TTF-1 stained 47/59 (80%) SCLC and 1/43 (2%) MCC cases. CEACAM6 stained 49/59 (83%) SCLC and 0/43 (0%) MCC cases. Combining CEACAM6 and TTF-1 increased SCLC detection sensitivity to 93% and specificity to 98%. These data suggest that ATOH1, TFAP2B, and CEACAM6 should be explored as markers to differentiate MCC and SCLC.

Analysis of clinicopathologic and imaging features of dual-phenotype hepatocellular carcinoma

Dual-phenotype hepatocellular carcinoma (DPHCC) is a new subtype of hepatocellular carcinoma (HCC). This study aimed to investigate the relationship between the computerized tomography scan (CT) imaging and clinicopathologic features of DPHCC. The CT imaging and clinicopathologic data of 97 HCC cases who underwent radical resection were collected retrospectively. The CT imaging feature was evaluated by the ratio of the average CT value of tumor to liver (TLR) in the plain scan, arterial, portal vein and delayed phases. The association between CT imaging and clinicopathologic features was analyzed using the t-test or chi-square test. Univariate and multivariate recurrence-free survival (RFS) analysis and overall survival (OS) were performed. The positive rates of cytokeratin 7 (CK7) and CK19 were 35.1% and 20.6% respectively. The positive rate of CK19 was significantly higher in cases with age < 47 years (P = 0.005), tumor diameter > 4 cm (P = 0.016) or AFP ≥ 400 ng/ml (P = 0.007). The TLR in the portal vein phase was significantly lower in CK19 positive group (P = 0.024). The recurrence risk was significantly higher in cases with CK19 positive (HR: 2.17, 95% CI 1.16 to 4.04, P = 0.013), tumor diameter > 4 cm (HR: 2.05, 95% CI 1.11 to 3.78, P = 0.019), AFP ≥ 400 ng/ml (HR: 2.50, 95% CI 1.37 to 4.54, P = 0.002) or CA199 ≥ 37 U/ml (HR: 2.23, 95% CI 1.12 to 4.42, P = 0.020). However, imaging features, pathological subtype, CK7 or CK19 expression were not significantly related to HCC OS in the univariate and multivariate analysis (all P > 0.05). The expression of CK19 may be associated with the enhancement feature of the portal vein phase CT image, and CK19 positive may suggest a worse RFS.

Discrepancies Between Morphological and Immunohistochemical Classifications Are Associated With Prognoses and Subtypes of Lung Cancer.

Immunohistochemical (IHC) staining has been routinely used to distinguish adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the lungs; however, it is challenging to obtain an accurate diagnosis, especially for cases with discrepancies between IHC and hematoxylin and eosin (H&E) staining results. This study aimed to clarify the clinicopathological characteristics of these discrepant cases. Tissue microarray specimens from 321 patients with ADC and SCC were used for H&E and IHC staining of thyroid transcription factor 1 (TTF-1), Napsin A, cytokeratin 5/6 (CK5/6), p40, and p63. The pathological diagnosis was made based on (1) H&E, (2) IHC, and (3) both H&E and IHC results. Discrepant cases were defined as those with different diagnoses based on the H&E and IHC results. A total of 32 (10%) discrepant cases were identified. ADC (3.9%) showed fewer discrepant cases than SCC (51%). Discrepant cases of ADC had a significantly higher proportion of poorly differentiated tumors and subtypes of solid and invasive mucinous ADC, and they also had shorter overall and disease-free survival than concordant cases. Solid and invasive mucinous ADC cases showed low positivity for TTF-1 (84% and 40%, respectively) and Napsin A (88% and 80%, respectively), and invasive mucinous ADC cases showed high positivity for CK5/6 (80%). The sensitivity and specificity of TTF-1+Napsin A for ADC were 91% and 83%, respectively, whereas those of CK5/6+p40 for SCC cases were 90% and 96%, respectively. Discrepant cases of ADC are associated with solid and invasive mucinous subtypes and shorter survival.