EpCAM Expression Research Articles

Isolation, Ex Vivo Culture, and Stimulation of Tracheal and Nasal Chemosensory Cells.

Brush cells are chemosensory epithelial cells present at most mucosal surfaces.Brush cells are a dominant source of cysteinyl leukotrienes and IL-25 in the airway epithelium and are equipped with the machinery to generate prostaglandins and acetylcholine. Activation of innate type 2 lymphoid cells and dendritic cells triggered by brush cell-derived mediators skew the immune response in the airway to type 2 inflammation that underlies atopic disease such as asthma. This chapter describes an effective method of brush cell isolation from the mouse trachea for transcriptional analysis and from the nasal cavity for transcriptional analysis and ex vivo stimulation.The nasal or tracheal mucosa is first incubated in a dispase solution for easy mechanical separation of the epithelial layer from the underlying submucosa. The detached epithelium is then digested with a papain solution. This method provides high yields of viable brush cells in a single-cell suspension, which can be used for flow cytometric analysis, single-cell sorting, cell culture, and functional assays.In the nose, where brush cells are more abundant, we present two methods of isolation of brush cells: (1) using fluorescent reporter mice that mark brush cells or (2) using a combination of high expression of EpCAM and low expression of CD45 to obtain a population of cells that is enriched for nasal chemosensory brush cells.

Ep-CAM (MOC-31) expression in tooth germ and ameloblastoma.

Background Ep-CAM, a transmembrane glycoprotein expressed in most epithelium in normal conditions, has diverse roles in these tissues, including in cell adhesion, proliferation, differentiation, cell cycle regulation, migration and intracellular signaling. It is also over-expressed in most malignant neoplasia, participating in the initiation, progression, and metastatic dissemination of the tumor. The expression and roles of this protein in oral neoplasia, particularly in odontogenic tumors, remain unestablished. The objective of this study consisted in analyzing the expression of this protein in ameloblastoma and tooth germ.Material and Methods Ep-CAM (MOC-31) expression was evaluated by immunohistochemistry in tooth germs (TG) (n = 16) ameloblastomas (AM) (n = 60) and 2 ameloblastic carcinomas. Sections were visualized in their totality with an optical microscope, and positivity observed in cell membrane and cytoplasm was graded according to the following semi-quantitative scale: Neg, "essentially unstained", for negative sections or staining <5% of cells; + for staining of 5-50% of cells; ++ for staining >50% of cells.Results Most tooth germs expressed MOC-31 (81.3%), strong staining was observed both in the inner epithelium of the enamel organ and in the adjacent stellate reticulum. 16.7% of the AM cases showed MOC-31 expression, the immunoexpression expression was diffuse at the cytoplasmic and membrane level. The only two cases of ameloblastic carcinoma included were strong positive to MOC-31. No correlation was observed between protein expression and gender, age, clinical variants, or histological subtypes.Conclusions Overexpression was found in TG and ameloblastic carcinoma compared to AM; further studies with different experimental strategies are suggested to clarify the biological significance of this finding. Key words:Tooth germ, ameloblastoma, Ep-CAM, MOC-31, immunohistochemistry.

3151 – SINGLE-CELL RNA SEQUENCING ANALYSIS REVEALS ROLE FOR GATA3 IN THE AORTIC ENDOTHELIUM AT THE TIME OF HEMATOPOIETIC STEM CELL EMERGENCE

The endothelial-to-haematopoietic transition (EHT) gives rise to the first murine haematopoietic stem cells (HSCs) in the aorta-gonads-mesonephros (AGM) region around E10.5. While our understanding of the transcriptional networks regulating HSC emergence is ever-increasing, the mechanisms governing the EHT process are still being investigated. The transcription factor Gata3, whose function has previously been characterized in the sympathetic nervous system AGM niche, is expressed directly in the haemogenic endothelium prior to EHT. Previous studies have shown that Gata3-expressing endothelial cells (ECs) have a greater capacity to produce haematopoietic colonies, over Gata3-negative ECs, while endothelial-specific Gata3 knockout leads to a greatly decreased ability to repopulate AGM-transplant recipient mice. Single-cell RNA sequencing analysis of Gata3 knockout ECs sorted from murine AGMs reveals an upregulation of factors involved in endothelial cell fate differentiation, such as Pecam1, and a dysregulation of adhesion molecule genes, including increased expression of Epcam and Vcam1, all of which are likely to disrupt the normal EHT process. Furthermore, downregulation of critical stem cell proliferation regulator Kitl may explain the negative effect of Gata3 knockout on transplanted AGMs. Another key finding has been the identification of cell surface antigens CD200 and CD200r1 as new markers for the enrichment of haemogenic cells via trajectory analysis of Gata3+ endothelial cells differentiating into pre-HSCs. CD200, which is expressed on arterial endothelium, contains the CD41lo cells capable of engrafting after transplantation, while CD200r1 is expressed within the pre-HSC compartment, but only CD200r1-negative cells are able to repopulate. These markers will undoubtedly prove useful in future studies attempting to isolate HSC precursors from murine AGMs. The endothelial-to-haematopoietic transition (EHT) gives rise to the first murine haematopoietic stem cells (HSCs) in the aorta-gonads-mesonephros (AGM) region around E10.5. While our understanding of the transcriptional networks regulating HSC emergence is ever-increasing, the mechanisms governing the EHT process are still being investigated. The transcription factor Gata3, whose function has previously been characterized in the sympathetic nervous system AGM niche, is expressed directly in the haemogenic endothelium prior to EHT. Previous studies have shown that Gata3-expressing endothelial cells (ECs) have a greater capacity to produce haematopoietic colonies, over Gata3-negative ECs, while endothelial-specific Gata3 knockout leads to a greatly decreased ability to repopulate AGM-transplant recipient mice. Single-cell RNA sequencing analysis of Gata3 knockout ECs sorted from murine AGMs reveals an upregulation of factors involved in endothelial cell fate differentiation, such as Pecam1, and a dysregulation of adhesion molecule genes, including increased expression of Epcam and Vcam1, all of which are likely to disrupt the normal EHT process. Furthermore, downregulation of critical stem cell proliferation regulator Kitl may explain the negative effect of Gata3 knockout on transplanted AGMs. Another key finding has been the identification of cell surface antigens CD200 and CD200r1 as new markers for the enrichment of haemogenic cells via trajectory analysis of Gata3+ endothelial cells differentiating into pre-HSCs. CD200, which is expressed on arterial endothelium, contains the CD41lo cells capable of engrafting after transplantation, while CD200r1 is expressed within the pre-HSC compartment, but only CD200r1-negative cells are able to repopulate. These markers will undoubtedly prove useful in future studies attempting to isolate HSC precursors from murine AGMs.

EMT-Related Markers in Serum Exosomes are Potential Diagnostic Biomarkers for Invasive Pituitary Adenomas.

PurposeAssessing the invasiveness of pituitary adenomas (PAs) is critical to making the best surgical and treatment plan. However, it is difficult to determine the invasiveness of pituitary adenomas based on current clinical methods, such as imaging and histological methods. The present article aims to investigate noninvasive methods to discover viable biomarkers for invasive pituitary adenomas and provide a basis for early intervention of pituitary adenomas.MethodsE-cadherin, N-cadherin, Epcam, TGF-β, Smad3, and Smad7 were detected in the tissues and exosomes in 10 cases of invasive PAs and 10 cases of noninvasive PAs by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemical analysis.ResultsCompared with that in the noninvasive group, the expression of N-cad in the exosomes of the invasive group was significantly increased, and the expression of E-cad and Epcam was reduced. In the invasive group, the expression levels of TGF-β1 and Smad3 were reduced. These results were consistent across exosomes and groups. In further cell experiments, the EMT ratio in the SIS3 treatment group, and especially in the TGF-β1 plus SIS3 treatment group (P <0.001), was significantly increased, and the EMT ratio was significantly lower when one-half the dose of TGF-β and SIS3.ConclusionThe results indicate that EMT-related biomarkers in serum exosomes can be potentially used for assessing the invasiveness of pituitary adenoma.

Single-particle analysis of tear fluid reveals abundant presence of tissue factor-exposing extracellular vesicles with strong coagulation activity

Extracellular vesicles (EVs) in easily accessible body fluids have emerged as a promising source for liquid biopsy. Although tear collection is fast, safe, and noninvasive, EVs of tear fluid are less studied and their involvement in physiological and pathological processes is largely unknown. The aim of present study was to analyze and characterize EVs in tear fluid at the single-particle level to reveal the population heterogeneity. A laboratory-built nano-flow cytometer (nFCM) was used to analyze the purity, size distribution, and particle concentration of EVs isolated from unstimulated tears (basal tears) upon double ultracentrifugation (17 min at 100,000×g, 4 °C) via side scattering detection. The expression of CD9, CD63, CD81, CD47, CD45, CD24, and EpCAM was assessed via immunofluorescent detection. The EV concentration in tear fluid was measured to be 1.1 ± 0.7 × 1011 particles/mL, which is approximately 100-fold higher than that of plasma EVs. In particular, it was identified for the first time that tears have strong coagulant activity owing to the abundant presence of tissue factor (TF) on tear EVs. The concentration of TF-exposing EVs (4.4 ± 3.1 × 1010 particles/mL) was found to be approximately 100-fold higher than their counterparts in saliva (4.5 ± 2.1 × 108 particles/mL). We postulate that TF-exposing vesicles in tears might play a role in host defense by promoting clot formation and thus reducing the risk of pathogen invasion. The coagulant activity of tears triggered by TF-exposing EVs could provide a new research perspective for ophthalmic research.

Targeting Wnt Signaling in the Tumor Immune Microenvironment to Enhancing EpCAM CAR T-Cell therapy.

Colorectal cancer (CRC) patients are still lacking viable treatments. Chimeric antigen receptor (CAR) T cells have shown promise in hematologic malignancies, but their efficacy in solid tumors has been limited due to the immunosuppressive tumor microenvironment. We found that cancer antigen- EpCAM expression increased in the metastatic stage compared with the primary stage in cancers and the activation of Wnt and TGFβ pathways was positively correlated with EpCAM expression in multiple cancers, including colorectal cancer. We constructed CAR T cells targeting EpCAM that successfully showed selective cytotoxicity in highly EpCAM-expressing cancer cell lines. The combination of EpCAM CAR-T with the Wnt inhibitor-hsBCL9CT-24 displayed synergetic effect against EpCAM-positive colon cells in vitro and also in vivo. A mechanistic study showed that hsBCL9CT-24 treatment could modulate the tumor environment and improve infiltration of T cells, while possibly promoting the effector T cells at the early stages and postponing the exhaustion of CAR T cells at advanced stages. Overall, these results demonstrated that the combination of EpCAM CAR T-cell therapy with the Wnt inhibitor can overcome the limitations of CAR T cells in treating solid tumors.

Anti-cancer adjuvant drug screening via epithelial-mesenchymal transition-related aptamer probe.

Epithelial-mesenchymal transition (EMT) is implicated in the pathological processes of cancer metastasis and drug resistance. Anti-cancer drugs may also potentially lead to EMT, resulting in their reduced therapeutic effect. Therefore, the combination of these anti-cancer drugs with anti-EMT agents has been promoted in clinic. Screening anti-EMT drugs and evaluation of EMT process are highly dependent on EMT biomarkers on cell membrane. At present, the detection of EMT biomarker is mainly by Western blot method, which is time-consuming and complicated. In this work, for effectively screening anti-EMT drugs by evaluation of the EMT process, a type of aptamer probe based on aggregation-induced emission (AIE) was designed. The aptamer SYL3C was employed to target the EMT biomarker EpCAM on cell membrane. Two fluorophores, FAM and tetraphenylethene (TPE, an AIE dye), were modified at the two ends of SYL3C, respectively. This aptamer probe (TPE-SYL3C-FAM) can monitor the EpCAM expression, which can be recovered by anti-EMT drugs. By observation of the change in TPE emission intensity, the anti-EMT effect of drugs can be evaluated. The FAM emission was used as internal reference to reduce environmental interferences. This probe can be potentially used to screen anti-EMT agents as anti-cancer adjuvant drugs with high throughput.

The expression and clinical significance of GPR39 in colon cancer.

Colorectal cancer is the third most common cancer and requires more prognostic biomarkers for precise treatment. GPR39 is a GPCR which can interact with Zn and modulate the colonocytes' survival. The clinical significance of GPR39 in colon cancer has never been reported. In our study, we compared GPR39 expression between colon cancers and tumor-adjacent tissues by retrieving TCGA data and detected the expression of GPR39 in colon cancers with qPCR and immunohistochemistry. The clinical significance of GPR39 was evaluated by analyzing the correlations with clinicopathological factors with the chi-square test. The prognostic significance of GPR39 was estimated with univariate and multivariate analyses. The expression of several other biomarkers including PPARG, EPCAM, and PD-L1 was investigated by re-analyzing TCGA data, qPCR, and IHC. The prognostic value of PPARG, EPCAM, and PD-L1 was also estimated with univariate analysis. In both TCGA database and our 15 colon cancer pairs, GPR39 expression was significantly upregulated in colon cancer tissues. GPR39 was an independent prognostic biomarker in colon cancer for poor prognosis. With TCGA data re-analysis, qPCR, and IHC, we showed that GPR39 expression was significantly correlated with the expression of EPCAM and PD-L1, but not PPARG. EPCAM and PD-L1 were also unfavorable prognostic biomarkers of colon cancer. GPR39 was upregulated in colon cancer tissues compared with tumor-adjacent tissues. GPR39 was an independent prognostic biomarker in colon cancer for poor prognosis. EPCAM and PD-L1 were substantially associated with GPR39 expression, and they were also identified as prognostic biomarkers in colon cancers.

ITGB4 as a novel serum diagnosis biomarker and potential therapeutic target for colorectal cancer.

PurposeTo develop new and effective biomarkers for the diagnosis of colorectal cancer (CRC).Experimental designThe serum expression of ITGB4 (49 CRC and 367 HC) was detected by enzyme‐linked immunosorbent assay (ELISA), and its diagnostic value was analyzed using the receiver operating characteristic (ROC) curve. The sensitivity and specificity of ITGB4 in CRC diagnosis were calculated through statistical analysis. The optimal clinical cutoff value was calculated using the Youden index, and diagnostic efficacy was analyzed in a larger serum sample (98 CRC and 1631 non‐CRC). The expression of ITGB4 was measured by CyTOF (cell experimental technology) at the single‐cell level, and characteristics were analyzed using viSNE and SPADE TREE.ResultsSerum ITGB4 and CEA levels were significantly higher in CRC patients than in HC and non‐CRC patients. The use of serum ITGB4 levels for the diagnosis of CRC has a high sensitivity (79%) but not high specificity when the clinical cutoff value was 0.70 ng/mL. However, the optimal cutoff value was 1.6 ng/mL with 86.2% specificity and 52.0% sensitivity, and the diagnostic efficacy was greatly improved with high specificity (82.0%) and sensitivity (71.4%) when combined with CEA. ITGB4 expression characteristics were measured and related to the expression of EpCAM, Ck8/18, and perforin at the single‐cell level. Single‐cell analysis showed that cell clusters with low expression of CK8/18 and ITGB4 were more sensitive to 5FU and radiotherapy (RT).ConclusionsITGB4 is an effective diagnostic serum biomarker and a potential therapeutic target for CRC.

Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.

P–436 Identification of ovarian cell subpopulations by multicolor flow cytometry and its potential impact on ovarian reconstruction programs

Abstract Study question How could multicolor flow cytometry (MFC) help to identify ovarian subpopulations that could be used for ovarian reconstruction with isolated follicles? Summary answer MFC is useful to identify ovarian cell subpopulations in the ovarian cortex. What is known already Ovarian tissue cryopreservation is a fertility preservation option for women before gonadotoxic chemo- and/or radiotherapy. However, graft of cryopreserved ovarian tissue must be performed with caution in women suffering from malignancies that may metastasize to the ovaries. For this purpose, functional ovarian tissue qualification is essential to identify ovarian cell subpopulations that could be used for ovary reconstruction in combination with isolated follicles. Furthermore, ischemic tissue damage occurring after the graft is currently another important issue to be resolved for successful ovarian reuse. Study design, size, duration We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Then, we used MFC for the identification of different cell subpopulations in the cell suspension thus obtained. Participants/materials, setting, methods Human ovarian tissues from patients undergoing surgery for polycystic ovary syndrome were used in this study. Biopsies of ovarian cortex (fresh or frozen-thawed) were dissociated using an automated dissociation method. We used FVS780 and SYTO13 markers to gate viable ovarian cells by MFC. Variable markers were chosen to differentiate and identify cell subpopulations among the viable ovarian cells. Main results and the role of chance The dissociation yield was on average 1.59 ± 1.58 x 106 and 0.78 ± 0.72 x 106 viable ovarian cells per 100 mg of fresh (n = 17) and frozen-thawed (n = 43) ovarian cortical tissue, respectively. On average, 35.4 ± 13.1% of viable ovarian cells were CD34 + (n = 61, stromal phenotype). Concerning endothelial phenotype, 7.8 ± 5.5% of CD31+ cells (n = 51) and 5.3 ± 3.6% of CD144+ cells (n = 29) were identified among viable ovarian cells. Vimentin marker is found in 25.6 ± 10.8% of viable ovarian cells (n = 23) and CD326 (EpCAM expression) in 0.6 ± 0.8% (n = 16). Finally, pericyte phenotype (CD34-/Vimentin-/CD31-/CD146+/ CD140b+) was identified in 4.6 ± 4.3% of viable ovarian cells (n = 7). Limitations, reasons for caution We do not know how these ovarian cell subpopulations could be a factor associated or not with time for ovarian function recovery in vivo after ovarian tissue graft and the impact of these ovarian cells on the ovarian microenvironment of an artificial ovary. Wider implications of the findings: Functional qualification of ovarian tissue can be performed by MFC. MFC is a promising tool for ovarian cortex qualification before reuse of cryopreserved ovarian tissue. Cell sorting could be used to separate and isolate cell subpopulations and add these cells with isolated follicles in an ovarian reconstruction program. Trial registration number Not applicable

O-191 Assessing the use of tumour-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex tissue fragments before autotransplantation

Abstract Study question Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ovarian tissue? Summary answer Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis. What is known already Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity. Study design, size, duration Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging. Participants/materials, setting, methods Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles. Main results and the role of chance Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue. Limitations, reasons for caution The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo. Wider implications of the findings Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers. Trial registration number not applicable

Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec1.

Simple SummaryMetastasis-targeting therapy might improve outcomes in oligometastatic prostate cancer. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40–60% of prostate cancer cases and might be used as a target for specific delivery of toxins and drugs. Radionuclide molecular imaging could enable non-invasive detection of EpCAM and stratification of patients for targeted therapy. Designed ankyrin repeat proteins (DARPins) are scaffold proteins, which can be selected for specific binding to different targets. The DARPin Ec1 binds strongly to EpCAM. To determine an optimal design of Ec1-based probes, we labeled Ec1 at two different positions with four different nuclides (68Ga, 111In, 57Co and 125I) and investigated the impact on Ec1 biodistribution. We found that the C-terminus is the best position for labeling and that 111In and 125I provide the best imaging contrast. This study might be helpful for scientists developing imaging probes based on scaffold proteins.The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40–60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy. The study aimed to investigate the influence of radiolabel position (N- or C-terminal) and composition on the targeting and imaging properties of Ec1. Two variants, having an N- or C-terminal cysteine, were produced, site-specifically conjugated to a DOTA chelator and labeled with cobalt-57, gallium-68 or indium-111. Site-specific radioiodination was performed using ((4-hydroxyphenyl)-ethyl)maleimide (HPEM). Biodistribution of eight radiolabeled Ec1-probes was measured in nude mice bearing PCa DU145 xenografts. In all cases, positioning of a label at the C-terminus provided the best tumor-to-organ ratios. The non-residualizing [125I]I-HPEM label provided the highest tumor-to-muscle and tumor-to-bone ratios and is more suitable for EpCAM imaging in early-stage PCa. Among the radiometals, indium-111 provided the highest tumor-to-blood, tumor-to-lung and tumor-to-liver ratios and could be used at late-stage PCa. In conclusion, label position and composition are important for the DARPin Ec1.

Abstract 1823: Directing T cells using multivalent, bispecific chemically self-assembling nanorings (CSANs) to target brain tumors overexpressing EGFR and B7H3 antigens

Abstract Introduction: CNS solid tumors have few therapeutic options and disease progression is rapid especially in pediatrics. Current standard-of-care strategies with radiation and surgery typically result in severe neuroendocrine and cognitive deficiencies. Therapies that target select antigens overexpressed on only the malignant cells would alleviate some of these off-target side-effects. The Wagner lab has developed a non-genetic approach to facilitate selective cell-cell interactions using Chemically Self-Assembled Nanorings (CSANs). We have shown that functionalizing the CSAN construct with a cancer antigen-targeting protein and a T-cell targeting single chain antibody fragment (antiCD3 scfv) forms a multivalent, bispecific nanoring with the ability to traffic T-cells to the tumor. We therefore hypothesize that targeting overexpressed cancer antigens on medulloblastoma and/or glioblastoma using our technology would address many of the concerns associated with permanent genetic engineering such as CAR T cells. Methods/Results: B7H3 (CD276) is a checkpoint molecule that is overexpressed on a wide variety of solid tumor types. For this study, we developed a new bispecific CSAN able to effectively direct human T cells to B7H3 overexpressing medulloblastoma. The Wagner lab is also currently able to produce CSANs that facilitate T cell interactions with many other solid tumor antigens such as EGFR and EpCAM. Therefore, we characterized the expression of EGFR, B7H3, and EpCAM on ONS76 and Daoy medulloblastoma and U87 glioblastoma in 2D and 3D models in vitro. We show that treating a B7H3+/EGFR+ allograft of ONS76 medulloblastoma with antiB7H3-CD3 or antiEGFR-CD3 CSANs resulted in ONS76 cytotoxicity and increased T cell lytic activity. We are currently analyzing the efficacy of our CSANs against orthotopically injected medulloblastoma in immunodeficient mice. We plan to probe the ways in which targeting these specific antigens induces changes in their expression within the tumor and identify potential modes of immune escape mechanisms such as antigen expression loss. Conclusion: We show that bispecific CSANs are able to non-genetically direct T cells to selectively eradicate cancer cells overexpressing the targeted antigen. The conclusion of in vivo orthotopic models of medulloblastoma are required to demonstrate the ability of the CSANs to effectively cross the BBB and the efficacy of specifically targeting the checkpoint molecule B7H3. Citation Format: Ellie Mews, Pauline Jackson, Ozgun Kilic, Justine Delgado, Mahathi Patchava, David Largaespada, Carston R. Wagner. Directing T cells using multivalent, bispecific chemically self-assembling nanorings (CSANs) to target brain tumors overexpressing EGFR and B7H3 antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1823.

Abstract 586: Expression of CD68 on CKpos/CD45pos circulating cells in breast cancer patients: An additional hint supporting the fusion hybrid theory

Abstract Background: Cells expressing both cytokeratyins (CK) and CD45 (dual-positive, DPcells), circulating in the blood of patients with solid tumors, have been reported for different cancers. Their origin is not clear yet, but one hypothesis is that they are hybrids, deriving from the fusion of tumor cells and macrophages. Recently, we have demonstrated that DPcells can have aberrant genomes, and that, in metastatic breast cancer, they are associated with shorter progression free survival. However, a direct proof of their origin from tumor/macrophage fusion is lacking. Here, we investigated the expression of a macrophage marker (CD68) on DPcells in breast cancer patients. Methods: Blood samples collected from BC patients were analyzed using the CellSearch platform, which allowed for the evaluation of the expression of CK, CD45 and CD68 on circulating cells enriched for EpCAM expression. Based on the marker expression, cells were classified as circulating tumor cells (CTCs, CKpos/CD45neg), and DPcells (CKpos/CD45pos). Results: Twenty-six blood samples were collected from 26 patients with luminal, HER2+, and triple negative BC (n = 13, 5, and 8, respectively). Of these, 6 had non-metastatic (NM) BC (i.e. early breast cancer or locally advanced breast cancer), and 20 had metastatic BC (MBC). Overall, 43 DPcells were identified in 14 (54%) samples (3/6 from NMBC and 11/20 from MBC patients). CD68 expression was detected on 26 DPcells (60%) from 13 samples. In the majority of cases (7/13 samples), CD68pos and CD68neg DPcells were detected together, whereas, in 5 samples, CD68pos DPcells only were present. CD68neg DPcells alone were detected in a single sample. Among the 20 MBC cases, 11 were classified as stage IVaggressive and 9 as stage IVindolent (i.e. with ≥ 5 CTCs and &amp;lt; 5 CTCs, respectively). CD68pos DPcells were present in 5/11 stage IVaggressive and in 5/9 stage IVindolent patients, suggesting that they are independent from CTC-based classification. As expected, CD68 was not detected on the majority of CTCs (796 out of 841), but, interestingly, 5% of CTCs were CD68pos. Conclusions and future perspectives: These preliminary results suggest that DPcells can derive from heterotypic cell fusion between tumor cells and macrophages. This would provide a new perspective on the unique interaction between tumor cells and the microenvironment and the potential role of these new cell entity in promoting disease progression. However, many questions about this subpopulation of circulating cells remain unanswered and future studies are needed to better characterize DPcells at both phenotypic and genomic level, to understand their biological and clinical relevance. Citation Format: Carolina Reduzzi, Paolo D'Amico, Youbin Zhang, Vera Cappelletti, Massimo Cristofanilli. Expression of CD68 on CKpos/CD45pos circulating cells in breast cancer patients: An additional hint supporting the fusion hybrid theory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 586.

Abstract 1710: A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo

Abstract Tumor-Specific Immuno-Gene (T-SIGn) cancer gene therapy vectors, which are based on the oncolytic adenovirus enadenotucirev, carry a gene cassette encoding combinations of human transgenes under control of the virus major late promoter (MLP) which restricts expression to tumor cells. These vectors can be dosed intravenously for delivery to tumors and selective expression of therapeutic proteins locally within the tumor microenvironment. Because these viruses only infect human tumor cells, in vivo studies are limited to human tumor xenografts in immunodeficient mice. Recently, we have optimized a 3D microfluidic system for evaluating response to immunotherapies using patient-derived organotypic tumor spheroids (PDOTS). PDOTS contain stromal as well as autologous myeloid and lymphoid cell populations and respond to PD-1 blockade in short-term ex vivo culture. As an alternative preclinical approach for evaluating activity and mechanistic underpinnings of T-SIGn viruses expressing human immunomodulatory transgenes, we have been developing the PDOTS ex vivo culture model. Here we report on the visualization of oncolytic virus uptake, replication in tumor cells and activation of T cells with a T-SIGn virus encoding an EpCAM-targeted bispecific T-cell activator (NG-611) within NSCLC PDOTS. Surgical NSCLC cases from St. Elizabeth's Medical Center, collected under an IRB approved protocol, were processed in PDOTS and assessed for EpCAM expression and number of T cells. Five different samples were treated with NG-611 (mixed with a GFP expressing vector) at concentrations ranging from 2x109 to 2x106 vp/mL final volume of culture. Virus infection was shown after 3 and 5 days of culture by dose-dependent expression of GFP reporter transgene driven by a CMV promoter, and virus replication in tumor cells was confirmed by using GFP expressed under the virus MLP promoter. Upregulation of IFNγ and Granzyme B in culture media indicated a strong activation of T-cells via expression of the bispecific T-cell activator. Cytotoxic T-cell activation also led to dose-dependent cell killing in NSCLC spheroids as shown by life/death analysis. Having established this proof of concept for functionality of T-SIGn viruses in PDOTS, ongoing studies are evaluating the immunomodulatory activities of a series of T-SIGn viruses expressing human IL-12 with or without other cytokine/chemokines. Citation Format: Moataz Noureddine, Katy West, Elizabeth Isaacson, Sylvia Alarcon, Christopher S. Lathan, John Wain, David A. Barbie, Cloud P. Paweletz, Elena Ivanova, Brian R. Champion. A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1710.

EpCam is required for maintaining the integrity of the biliary epithelium

Tufting enteropathy (TE) is a rare congenital disorder often caused by mutations in the gene encoding epithelial cell adhesion molecule (EpCam). The disease leads to diarrhoea, intestinal failure and dependence on total parenteral nutrition (TPN). These patients often have liver impairments, but the pathology and mechanism of the damage are not well understood. We evaluated liver biopsies from TE patients to understand the pathophysiology. We identified three patients with TE who underwent liver biopsy. Two normal controls and 45 patients on TPN secondary to short gut syndrome were selected for comparison (five were age- and TPN duration matched to the TE patients). We found that all TE patients showed a complete loss of EpCam expression in enterocytes and biliary epithelial cells, while the normal and TPN groups show basolateral expression. Histologically TE patients showed ductopenia, which was not seen in control groups. E-cadherin and β-catenin are normally located along the lateral membrane of biliary epithelial cells. However, they were relocated to the apical membrane in TE patients, indicating a defect in the apical-basal polarity of cholangiocytes. We examined hepatic reparative cells and found near absence of hepatic progenitor cells and intermediate hepatobiliary cells with mild reactive ductular cells in TE patients. Our findings show that TE is associated with disrupted polarity of cholangiocyte and ductopenia. We demonstrate for the first time a role of EpCam in the maintenance of integrity of biliary epithelium. We also provided evidence for a disrupted development of hepatic reparative cells.

Design, synthesis, and antitumor activity of PLGA nanoparticles incorporating a discovered benzimidazole derivative as EZH2 inhibitor

PurposeTargeting enhancer of zeste homolog 2 (EZH2) can represent a hopeful strategy for oncotherapy. Also, the use of PLGA-based nanoparticles as a novel and rate-controlling carrier system was of our concern. MethodsBenzimidazole derivatives were synthesized, and their structures were clarified. In vitro antitumor activity was evaluated. Then, a modeling study was performed to investigate the ability of the most active compounds to recognize EZH2 active sites. Compound 30 (Drug) was selected to conduct pre-formulation studies and then it was incorporated into polymeric PLGA nanoparticles (NPs). NPs were then fully characterized to select an optimized formula (NP4) that subjected to further evaluation regarding antitumor activity and protein expression levels of EZH2 and EpCAM. ResultsThe results showed the antitumor activity of some synthesized derivatives. Docking outcomes demonstrated that Compound 30 was able to identify EZH2 active sites. NP4 exhibited promising findings and proved to keep the antitumor activity of Compound 30. HEPG-2 was the most sensitive for both Drug and NP4. Protein analysis indicated that Drug and NP4 had targeted EZH2 and the downstream signaling pathway leading to the decline of EpCAM expression. ConclusionsTargeting EZH2 by Compound 30 has potential use in the treatment of cancer especially hepatocellular carcinoma.

Relationship between circulating tumor cells and ZEB1 expression in tumor cells in colorectal cancer.

e15509 Background: Intravasation and circulation of tumor cells involves active invasion of cells with an enhanced migration potential as a result of the epithelial-mesenchymal transition (EMT). The ZEB1 protein is one of the key regulators of this process. Our purpose was to evaluate the association between the amount of circulating tumor cells (CTCs) in the peripheral blood of patients with different stages of colorectal cancer and the expression of ZEB1 by tumor cells. Methods: The study included 299 patients (aged 42-86 years, mean age 64.2±1.7) with stage II-IV CRC T1-4N0-2M0-1; histologically verified G1-G3 adenocarcinoma in all patients. The numbers of CTCs were measured in the peripheral blood before surgery using the Veridex CellSearch system (Janssen). CTCs were registered taking into account morphological characteristics and expression of epithelial cell adhesion markers EpCAM, CD45, cytokeratins 8,18,19. The blood sample was evaluated according to the following criteria: 0 CTCs, 1-3 CTCs, and more than 3 CTCs. Tissues of surgically removed tumors were studied with IHC analysis using rabbit polyclonal anti-ZEB1 antibodies (Biorbyt Ltd.) diluted 1:200 and the Reveal Polyvalent HRP-DAB Detection System. The percentage and the intensity of staining were assessed: 0, 1+ weak, 2+ moderate, 3+ strong. ZEB1 expression was considered positive when staining was detected in more than 10% (cut-off) tumor cells with intensities of 2+ and 3+. Statistical analysis of results was performed in the Statistica 13.0 program (StatSoftInc., USA). Results: From 1 to 402 CTCs were determined in 62.9% cases (in 188 of 299 patients); CTCs were not registered in 37.1% cases (111 of 299). Positive ZEB1 expression was observed in 80.6% (241 of 299 patients), while the negative one was much more rare – 19.4% (58 of 299 patients). The rates of CTC detection significantly increased with the positive ZEB1+ expression, compared to the negative expression (75.1% vs. 12.1%). CTCs &gt;3 were detected in the blood in 39.0% in ZEB1+ tumors, but not in ZEB1- tumors. 1-3 CTCs were observed 3 times more often in ZEB1+ tumors (36.1% vs. 12.1; p≤0.05). Conclusions: Statistically significant association was revealed between the epithelial-mesenchymal transition marker ZEB1 expression by tumor cells and the amount of CTCs in the peripheral blood (p &lt; 0.001).

Peripheral blood levels of tumor-infiltrating lymphocytes (TILs) and circulating tumor cells (CTCs) in patients with colorectal cancer.

e15510 Background: Tumor is a structure where malignant cells interact, among others, with cells of the adaptive and innate immune systems largely determining the tumor microenvironment. The role of TILs in the disease prognosis has been shown. The amount of CTCs is one of the factors that significantly affect the risk and rate of metastasis. The purpose of the study was to assess an association between TILs and CTCs in the peripheral blood of patients with various stages of colorectal cancer (CRC). Methods: The study included 299 patients (aged 42-86 years, mean age 64.2±1.7) with stage II-IV CRC T1-4N0-2M0-1. TILs were identified in tumor material after standard histological processing. Lymphocytes were counted per 100 epithelial cells in 5 fields of view at a magnification of x400; the result was expressed as a percentage ( &lt; 5% weak, 5-30% moderate, &gt;30% strong). The numbers of CTCs were measured in the peripheral blood using the Veridex CellSearch system (Janssen), taking into account the expression of epithelial cell adhesion markers EpCAM, cytokeratins 8,18,19 and absence of the CD45 expression. The blood sample was evaluated according to the following criteria: 0 CTCs, 1-3 CTCs, and more than 3 CTCs. Statistical analysis of results was performed in the Statistica 13.0 program (StatSoftInc., USA). Results: Weak lymphocytic infiltration was detected in 32.8%, moderate - in 48.1%, strong - in 19.1% of cases. CTCs were detected in 62.9% cases (in 188 of 299 patients). The percentages of patients with 1-3 CTCs and with &gt;3 CTCs were equal - 50% (94 of 188). CTCs were not registered in 37.1% cases (111 of 299). The absence of CTCs was noted equally often in moderate and strong lymphocytic infiltration – 43.7% and 43.8%. The presence of tumor cells in the peripheral blood was most often detected in weak lymphocytic infiltration, being 1.4 times more frequent than in moderate and strong infiltration (76.5% vs. 56.3% and 56.2%) (p = 0.019, c2= 11.890). Conclusions: The study demonstrated a significant relationship between the level of CTCs and the intensity of lymphocytic infiltration in the tumor (p≤0.05), which can be used as a new prognostic approach.