MuS110 and MT110 are BiTE antibodies bispecific for CD3 and EpCAM, which is the most frequently and highly expressed tumor-associated antigen on breast cancer. And pronounced expression of IDO1 has ...also been reported in breast cancer. Our study aimed to investigate whether IDO1 inhibitor D-1MT combing with MuS110/MT110 had synergistic antitumor effects on IDO expressing EpCAM-positive breast cancer cells in vitro and in vivo. Data suggested that the expression of IDO1 on Epcam-positive breast cancer 4T1 and MCF-7 decreased MuS110/MT110 antitumor efficacy by the suppression of T cells activation in vitro. Combining D-1MT with MT110 in IDO+MCF-7 cells, or with MuS110 in IDO+4T1 cells, significantly improved the antitumor efficacy of BiTE antibodies via increasing T cell cytotoxicity and contributing to cytokines releasing. In vivo assay, combination of D-1MT with MT110 in NOD/SCID mice bearing IDOhi MCF-7 xenografts or MuS110 in immune competent BALB/c mice bearing IDOhi 4T1 xenografts suggested the similar synergistic effect. Together, IDO inhibition could reverse the suppression of T cells due to IDO expressing on breast cancer, and improve the antitumor efficacy of EpCAM/CD3-bispecific BiTE antibody.
•IDO1 decreased MuS110/MT110 antitumor efficacy by suppressing T cells activation.•Combining D-1MT with MT110 or MuS110 improved the efficacy of BiTE antibodies•D-1MT increased T cell cytotoxicity and contributed to cytokines releasing.•Combination of D-1MT with MuS110/MT110 in mice suggested the synergistic effect
The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and ...context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo.
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•EPCAM(high) cells from metastatic biopsies can propagate breast cancer in xenografts•Resistance to EMT sustains tumorigenesis and metastatic spread of the human disease•Irreversible EMT results in restrained tumorigenic potential in clonal subsets•ZEB1 and its target GRHL2 control distinct epigenetic paths of EMT susceptibility
Saini et al. show that human breast cancer cells resist EMT induction and maintain high EPCAM expression to propagate the metastatic disease. In contrast, loss of EPCAM defines clones arresting in a mesenchymal state with suppressed tumorigenic features. Clonal levels of ZEB1 and GRHL2 control global epigenetic programs determining EMT resistance.
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein primarily known to mediate homotypic cell contacts in epithelia tissues. Because EpCAM expression is limited to normal and ...malignant epithelia, it has been used as diagnostic marker for the detection of carcinoma cells in mesenchymal organs such as blood, bone marrow or lymph nodes. In particular, the detection and molecular characterization of EpCAM-positive circulating tumor cells (CTCs) in the blood of carcinoma patients has gained considerable interest over the past ten years. EpCAM is primarily considered as an adhesion molecule, but recent studies have shown diverse biological functions including regulation of cell proliferation and cancer stemness. In this review, we summarize the current knowledge on the biological properties of EpCAM with emphasis on mechanisms involved in cancer progression and discuss the clinical implications of these findings for the clinical use of EpCAM as a diagnostic marker.
INTRODUCTIONThis study aims to evaluate diagnostic accuracy of flow cytometry (FCM) in detecting malignant epithelial cells in serous effusions.MATERIALS AND METHODSFlow cytometric assessment of 96 ...serous fluids (86 ascitic, 10 pleural) was performed by using epithelial cell adhesion molecule (EpCAM) (in all 96 fluids) and MUC-1 (in a subgroup of 40 fluids) as epithelial markers and CD45 and CD14 as leucocyte markers. The percentage of EpCAM positivity and MUC-1 positivity was calculated in the CD14 and CD45 dual negative population by selective gating. The findings were then correlated with the defined gold standard criteria.RESULTSThe sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy for EpCAM was found to be 92.06%, 96.96%, 98.31%, 86.48%, and 93.75%, respectively, while that for MUC-1 was 79.16%, 93.75%, 95%, 71.4%, and 85%, respectively. The sensitivity, specificity, PPV, NPV, and diagnostic accuracy for dual positivity for EpCAM and MUC-1 was found to be 83.3%, 100%, 100%, 80%, and 90% respectively. On combining FCM with cytomorphology the sensitivity, specificity, PPV, NPV, and diagnostic accuracy all increased greatly to 95.3%, 100%, 100%, 91.4%, and 96.8%, respectively.CONCLUSIONSThis study highlights the importance of multicolored flow cytometric analysis in detecting epithelial malignancies in effusions specially in cases belonging to the atypia of undetermined significance and suspicious for malignancy categories and in cases with strong clinical suspicion of malignancy with negative fluid cytology. We recommend the combined use of FCM and cytology for this specific subgroup of patients in routine clinical practice for fast and accurate reporting.
Exosomes (50-150 nm) play significant biological functions in intercellular communication and transportation of diverse biomolecules, including proteins and nucleic acids. In particular, malignant ...exosomes have received a great deal of attention as possible indicators for cancer detection and treatment. To swiftly and precisely identify malignant exosomes from normal exosomes in diverse bodily fluids, we developed polydiacetylene (PDA)-based aptasensors with distinct optical features exhibiting color shift in response to biological recognition. To identify epithelial cell adhesion molecules (EpCAM) overexpressed on the surface of malignant exosomes, anti-EpCAM aptamer-conjugated diacetylene monomer (TCDA-Apt) was synthesized and used to create anti-EpCAM aptamer-conjugated PDA (anti-EpCAM Apt-PDA) vesicles. In just 15 min following the reaction with malignant exosomes, the anti-EpCAM Apt-PDA vesicles underwent a visible color change from blue to purple. They showed high specificity to EpCAM-positive malignant exosomes over non-malignant exosomes, bovine serum albumin (BSA), and fibrinogen. Moreover, its effectiveness in the point-of-care (POC) detection of malignant exosomes was evaluated using human sera. Therefore, our PDA-based aptasensors have tremendous potential for on-site cancer diagnosis.
EpCAM is a transmembrane glycoprotein that functions as an epithelial marker in endometrial tissues. However, the correlation between EpCAM and endometrial carcinoma (EC) is not clear.
This study ...investigated the association between EpCAM and EC. Immunohistochemistry staining and bioinformatics analysis disclosed the clinical importance of low EpCAM expression. The migratory ability of cells expressing low EpCAM levels was studied in transwell invasion assays in vitro and an orthotopic intra-uterine tumor injection model in vivo. The Connectivity MAP was used to identify drugs that effectively inhibit cells with low EpCAM expression.
According to immunohistochemistry analysis results, low EpCAM expression was associated with an advanced stage and lymph node metastasis in patients with endometrioid EC, and high EpCAM expression favored survival. EpCAM silencing promoted cell invasion, and EpCAM re-expression in EpCAM-silenced EC cells attenuated their invasiveness. EpCAM suppression in an orthotopic uterine implantation model promoted the lymph node metastasis of EC cells. According to quantitative PCR and promoter reporter analyses, estrogen receptor alpha signaling regulated EpCAM expression by enhancing its promoter activity. As shown in the Connectivity MAP analysis, transamin inhibited the invasiveness of EpCAM-silenced EC cells.
The loss of EpCAM may increase the malignancy of EC, and these findings provide new insights into the prognostic role of EpCAM in patients with EC.
•Downregulation of EpCAM favors a poor prognosis and cancer cell invasion of EC.•ERα-induced EpCAM expression suppresses the dissemination of EC cells.•Transamin is a potential inhibitor of highly invasive EC cells.
Assemblies of nanomaterials for biological applications in living cells have attracted much attention. Herein, graphene oxide (GO)–gold nanoparticle (Au NP) assemblies are driven by a splint DNA ...strand, which is designed with two regions at both ends that are complementary with the DNA sequence anchored on the surface of the GO and the Au NPs. In the presence of microRNA (miR)‐21 and epithelial cell‐adhesion molecule (EpCAM), the hybridization of miR‐21 with a molecular probe leads to the separation of 6‐fluorescein‐phosphoramidite‐modified Au NPs from GO, resulting in a decrease in the Raman signal, while EpCAM recognition reduces circular dichroism (CD) signals. The CD signals reverse from negative in original assemblies into positive when reacted with cells, which correlates with two enantiomer geometries. The EpCAM detection has a good linear range of 8.47–74.78 pg mL−1 and a limit of detection (LOD) of 3.63 pg mL−1, whereas miR‐21 detection displays an outstanding linear range of 0.07–13.68 amol ng−1RNA and LOD of 0.03 amol ng−1RNA. All the results are in good agreement with those of the Raman and confocal bioimaging. The strategy opens up an avenue to allow the highly accurate and reliable diagnosis (dual targets) of clinic diseases.
Controllable chiral graphene oxide–gold assemblies are fabricated, which display dual strong optical signals with characteristic plasmonic circular dichroism (CD) at 521 nm and surface‐enhanced Raman scattering (SERS) at 1711.29 cm−1. Based on dual signals of plasmonic CD and SERS, for the first time, the quantitative monitoring of EpCAM and miR‐21 in living cells is achieved. Significantly, it is found that the CD signals reverse from negative in the original assemblies into positive when reacted with cell‐related biological components.
Typically, anticancer CD8
pos
T cells occur at low frequencies and become increasingly impaired in the tumor micro environment. In contrast, antiviral CD8
pos
T cells display a much higher ...polyclonality, frequency, and functionality. In particular, cytomegalovirus (CMV) infection induces high numbers of 'inflationary' CD8
pos
T cells that remain lifelong abundantly present in CMV-seropositive subjects. Importantly, these so-called inflationary anti-CMV T cells increase with age, maintain a ready-to-go state, populate tumors, and do not become exhausted or senescent. Given these favorable attributes, we devised a novel series of recombinant Fab-peptide-HLA-I fusion proteins and coined them 'ReTARGs'. A ReTARG fusion protein consists of a high-affinity Fab antibody fragment directed to carcinoma-associated cell surface antigen EpCAM (or EGFR), fused in tandem with soluble HLA-I molecule/β2-microglobulin, genetically equipped with an immunodominant peptide derived from CMV proteins pp65 (or IE-1). Decoration with EpCAM-ReTARG
pp65
rendered EpCAM-expressing primary patient-derived carcinoma cells highly sensitive to selective elimination by cognate anti-CMV CD8
pos
T cells. Importantly, this treatment did not induce excessive levels of proinflammatory T cell-secreted IFNγ. In contrast, analogous treatment with equimolar amounts of EpCAM/CD3-directed bispecific T-cell engager solitomab resulted in a massive release of IFNγ, a feature commonly associated with adverse cytokine-release syndrome. Combinatorial treatment with EpCAM-ReTARG
pp65
and EGFR-ReTARG
IE-1
strongly potentiated selective cancer cell elimination owing to the concerted action of the corresponding cognate anti-CMV CD8
pos
T cell clones. In conclusion, ReTARG fusion proteins may be useful as an alternative or complementary form of targeted cancer immunotherapy for 'cold' solid cancers.
Circulating tumor cells (CTCs) are precursors of the metastatic cascade, which is responsible for 90% of all cancer-related deaths. CTCs arise from solid tumors and travel through the bloodstream and ...lymphatic system. Developments in the isolation and analysis of CTCs promise potential biomarker assays for detection and monitoring of cancer through a minimally invasive procedure. Despite this, the precise role of CTCs in metastasis remains poorly characterized, mainly due to the low density of CTCs (1-10 CTCs per 10 mL of blood) present in patient blood and the lack of robust methods for their isolation in a standard laboratory setting. CellSearch is currently the only FDA-approved CTC enrichment protocol, but limitations of this EpCAM-based method include cost, availability, and the use of a single surface marker for capture. To address these limitations, we have optimized an existing method, MetaCell, which exploits the differences in size of CTCs compared to other blood cells for CTC enrichment from blood. MetaCell contains a membrane with 8 μm pores, and blood is filtered through this kit by capillary action and CTCs, which are typically larger than the pores and remain on top of the membrane, while most leukocytes pass through the pores. The membrane along with these CTCs can be detached and transferred to 6-well plates for culturing or directly used for characterization. Here, we provide a detailed protocol for enrichment of CTCs using the filtration device MetaCell and a procedure for characterization of CTCs by immunohistochemical staining.
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