EpCAM Research Articles

Abstract P4-07-12: Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations

Abstract Background: Sacituzumab govitecan (SG, Trodelvy®) is a human trophoblast cell surface antigen 2 (TROP-2) directed antibody drug conjugate (ADC) coupled to an active form of irinotecan (SN-38) via our novel hydrolyzable linker (CL2A). SG is the only FDA-approved ADC treatment for TNBC patients in the second-line setting. TROP-2 is a transmembrane protein encoded by the tumor-associated calcium signal transducer 2 (TACSTD2) gene and highly expressed in TNBC, an aggressive type of cancer accounting for approximately 15% of all breast cancers. TROP-2 overexpression is associated with poor survival and relapse, but its biological function in TNBC remains poorly understood. Hypothesis/rationale: To better understand TROP-2 and TROP-2-directed ADC biology, we developed and characterized TROP2high vs TROP2low TNBC syngeneic tumors and an SG surrogate directed to murine TROP-2. Experimental design: We established 2 syngeneic TNBC models with differential TROP-2 expression: 4T1 cells were flow sorted into high (>95%) vs low (< 7%) TROP-2 expressors and EMT6 cells were transduced with a murine TACSTD2-encoding lentivirus. Balb/c mice were subcutaneously implanted with 0.5 × 10 E6 TROP-2high, TROP-2low, or parental tumor cells (4T1 or EMT6). Tumor immunophenotyping and transcriptomic analyses were performed 15 and 24 days after implantation. An SG mouse surrogate was engineered to mimic SG, using an anti-TROP-2 antibody (Rab64) that cross-reacts with human and murine TROP-2 covalently attached to SN-38 by the CL2A linker. SG surrogate activity was characterized in vitro and in 4T1 syngeneic models. Results: SG surrogate demonstrated high affinity for human and mouse TROP-2 (KD=1.1 and 1.4 nM, respectively) with SN-38 release rates and PK similar to that of SG. Flow cytometry analysis after bulk cell sorting of 4T1 or lentivirus transduction of EMT6 confirmed high TROP2 expression after at least 3 in vitro passages. Fifteen days after subcutaneous implantation, flow cytometry analysis of tumor single-cell suspensions revealed significant differences in immune infiltrates between 4T1-derived tumor groups (n=5/group; mean percentages in TROP2high vs TROP2low 4T1-derived tumors of cells expressing CD45: 65% vs 10%, P < 0.0001; CD8: 5.5% vs 1%, P = 0.0033; CD4: 22% vs 4%, P = 0.0055; macrophages: 12.5% vs 2.5%, P = 0.0002; myeloid cells: 52% vs 75%, P = 0.0066). In addition, TROP2high 4T1-derived tumors were smaller and had significantly less necrosis than TROP2low and unsorted 4T1-derived tumors 25 days after implantation. Finally, transcriptomics analyses of TROP2high vs TROP2low 4T1-derived tumors demonstrated the association of TACSTD2 expression levels with regulation of distinct molecular pathways. Conclusion: Syngeneic tumors derived from 4T1 cells with differential TROP2 expression levels are associated with differential cellular states and tumor microenvironment composition. In contrast, no significant phenotypic changes were observed in tumors derived from TACSTD2-transduced compared with mock-transduced EMT6 cells. Taken together, these results suggest that expression of the TACSTD2 gene is associated with, but not causative of, different tumor phenotypic states. Additional studies to investigate TROP-2 expression as a correlative marker of patient prognosis and the antitumor immune response are warranted. The effects of in vivo treatment with an SG surrogate on 4T1 tumor growth and immune phenotype will be discussed at the time of the presentation. Citation Format: Chih-Chien Chou, Jordan Kardos, Becky Yang, Jessica Orf, Rutwij Dave, Yurong Lai, Chingwei V. Lee, Giuseppe A. Papalia, Kelli Boyd, Lauri Diehl, Nathalie Scholler. Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-12.

Updates in gynecologic care for individuals with lynch syndrome.

Lynch syndrome is an autosomal dominant hereditary cancer syndrome caused by germline pathogenic variants (PVs) in DNA mismatch repair genes (MLH1, MSH2, PMS2, MSH6) or the EPCAM gene. It is estimated to affect 1 in 300 individuals and confers a lifetime risk of cancer of 10-90%, depending on the specific variant and type of cancer. Lynch syndrome is the most common cause of inherited colorectal cancer, but for women, endometrial cancer is more likely to be the sentinel cancer. There is also evidence that certain PVs causing Lynch syndrome confer an increased risk of ovarian cancer, while the risk of ovarian cancer in others is not well defined. Given this, it is essential for the practicing gynecologist and gynecologic oncologist to remain up to date on the latest techniques in identification and diagnosis of individuals with Lynch syndrome as well as evidence-based screening and risk reduction recommendations for those impacted. Furthermore, as the landscape of gynecologic cancer treatment shifts towards treatment based on molecular classification of tumors, knowledge of targeted therapies well-suited for mismatch repair deficient Lynch tumors will be crucial. The objective of this review is to highlight recent updates in the literature regarding identification and management of individuals with Lynch syndrome as it pertains to endometrial and ovarian cancers to allow gynecologic providers the opportunity to both prevent and identify Lynch-associated cancers earlier, thereby reducing the morbidity and mortality of the syndrome.

Abstract P4-08-18: Engineered Toxin Bodies Specific for TROP2 Positive Cancers

Abstract Engineered Toxin Bodies Specific for TROP2 Positive Cancers Authors: Garrett L. Cornelison, Ileana Pedraza, Kendra Garrison, Elizabeth M. Kapeel, Channing Pletka, Abdul Khan, Jessica Momb, Rebecca Martin, Adam Bartos, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson Molecular Templates, Austin, TX Lanier Biotherapeutics, Bogart, GA Molecular Templates produces next generation immunotoxins called Engineered toxin bodies (ETBs). ETBs are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forced internalization, undergoing retrograde translocation to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes resulting in the inhibition of protein synthesis and induction of apoptosis. In addition, Molecular Templates has expanded the ETB platform to include Antigen Seeding Technology (AST) to generate ETBs with the ability to deliver foreign protein antigen to targeted populations of tumor cells. This mechanism of action allows for the intracellular processing of antigen and subsequent surface MHC-I presentation required for activation of a re-directed T lymphocyte response and the capacity to restore a functional immune clearance program against the tumor. Three ETBs are in clinical development (MT-5111 targeting HER2, MT-0169 targeting CD38, and AST enabled MT-6402 targeting PD-L1). The novel mechanisms of action have potential benefit in different indications including in the relapsed setting, when disease has progressed after chemotherapies and other targeted therapies, and additionally may be able to combine with standard of care. ETBs are being developed that target other cell surface receptors expressed on solid tumors including tumor-associated calcium signal transducer 2 (TROP2). TROP2 is a clinically validated target in metastatic triple-negative breast cancer (mTNBC) and other cancers such as metastatic urothelial carcinoma (mUC) using antibody drug conjugate (ADC) therapies such as sacituzumab govitecan (Trodelvy®). In vitro, ETBs targeting TROP2 specifically and directly kill tumor cells expressing TROP2 with picomolar activity. ETBs can bind to TROP2 in the presence of the Trodelvy parent monoclonal antibody, sacituzumab, and ETBs retain potency on TROP2 positive cell lines in the presence of clinically relevant concentrations of sacituzumab. AST enabled Trop2 targeted ETBs retain direct cell killing activity and can deliver multiple viral antigens to induce cytokine secretion and T-cell mediated killing in a co-culture assay of TROP2 target cells with antigen matched HLA type and antigen specific T-cells. In vivo, TROP2 targeted ETBs demonstrate good tolerability in a murine HCC1806 triple-negative breast cancer xenograft model and significantly reduce tumor burden relative to vehicle control. These pre-clinical in vitro and in vivo data suggest AST enabled Trop2 targeted ETBs have the potential to deplete Trop2 positive malignancies through multiple unique mechanisms of action. Citation Format: Garrett L. Cornelison, Ileana Pedraza, Kendra Garrison, Elizabeth M. Kapeel, Channing Pletka, Abdul Khan, Jessica Momb, Rebecca Martin, Adam Bartos, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson. Engineered Toxin Bodies Specific for TROP2 Positive Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-18.

Targeted EpCAM-binding for the development of potent and effective anticancer proteins

Protein-based cancer therapies are considered an alternative to conventional anticancer regimens, providing multifunctional properties while showing low toxicity. However, its widespread use is limited by absorption and instability issues, resulting in higher dosage requirements and a prolonged onset of bioactivity to elicit the desired response. Here, we developed a non-invasive antitumor treatment using designed ankyrin repeat protein (DARPin)-anticancer protein-conjugate that specifically targets the cancer biomarker, epithelial cell adhesion molecule (EpCAM). The DARPin-anticancer proteins bind to EpCAM-positive cancer cells and improve the in vitro anticancer efficacy by over 100-folds within 24 h, where the DARPin-tagged human lactoferrin fragment (drtHLF4) IC50 value is within the nanomolar range. Orally administered drtHLF4 was readily absorbed into the systemic flow of the HT-29 cancer murine model, exerting its anticancer effect on other tumors in the host body. Orally administered drtHFL4 cleared HT29-colorectal tumors using a single dose, whereas intratumoral injection cleared HT29-subcutaneous tumors within three doses. This approach addresses the limitations of other protein-based anticancer treatments by providing a non-invasive anticancer therapy with improved potency and tumor-specificity.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value

BackgroundCirculating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking.MethodsWe developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment.ResultsBoth antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information.ConclusionsOur data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material—although they may be prognostically less relevant than EpCAM high-expressing CTCs—and have particular benefit if no CTCs are detected using EpCAM-dependent technologies.

Surface-Initiated Synthesis of Cell-Specific Glycopolymers Using Live Mammalian Cells as Templates.

Molecular recognition is an important process in life activities where specificity is the key. However, the method to gain specificity are often complex and time-consuming. Herein, a novel, versatile, and effective way is developed to obtain cell-specific glycosurfaces by surface-initiated Cu-mediated reversible deactivation radical polymerization(Cu-RDRP)in an open to air fashion. Mammalian cells are used for the first time as live templates to realize cell-sugar monomer-aptation-polymerization which can produce cell-specific glycosurfaces. Both epithelial cell adhesion molecule (EpCAM) positive cells L929 and EpCAM negative cells Hela as models are used to acquire two cell-specific glycosurfaces, which can distinguish template-cells from others. The strategy is effective and convenient without the need of fixative pretreatment of cells. It is found that the specific capture does not rely on EpCAM antibodies, and the specificity is related to the composition and chain sequence of the glycopolymer brushes rather than surface morphology. In addition, these glycosurfaces keep the ability to identify the target cells after ten regenerative treatments, which provides another advantage for practical applications.

Whole-genome sequencing identified novel mutations in a Chinese family with lynch syndrome.

Lynch syndrome (LS) is caused by a germline mutation in one of the mismatch repair genes (MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. The definition of Lynch syndrome is based on clinical, pathological, and genetic findings. Therefore, the identification of susceptibility genes is essential for accurate risk assessment and tailored screening programs in LS monitoring. In this study, LS was diagnosed clinically in a Chinese family using Amsterdam II criteria. To further explore the molecular characteristics of this LS family, we performed whole genome sequencing (WGS) to 16 members in this family and summarized the unique mutational profiles within this family. We also used Sanger sequencing technology and immunohistochemistry (IHC) to verify some of the mutations identified in the WGS analysis. We showed that mutations in mismatch repair (MMR) related genes, as well as pathways including DNA replication, base excision repair, nucleotide excision repair, and homologous recombination were enhanced in this family. Two specific variants, MSH2 (p.S860X) and FSHR (p.I265V) were identified in all five members with LS phenotypes in this family. The MSH2 (p.S860X) variant is the first reported variant in a Chinese LS family. This mutation would result in a truncated protein. Theoretically, these patients might benefit from PD-1 (Programmed death 1) immune checkpoint blockade therapy. The patients who received nivolumab in combination with docetaxel treatments are currently in good health. Our findings extend the mutation spectrum of genes associated with LS in MLH2 and FSHR, which is essential for future screening and genetic diagnosis of LS.

Functionalized Fluorescent Nanostructures Generated from Self-Assembly of a Cationic Tripeptide Direct Cell-Selective Chemotherapeutic Drug Delivery.

Nanodrug delivery systems (NDDs) capable of conveying chemotherapeutics directly into malignant cells without harming healthy ones are of significant interest in the field of cancer therapy. However, the development of nanostructures with the requisite biocompatibility, inherent optical properties, cellular penetration ability, encapsulation capability, and target selectivity has remained elusive. In an effort to develop cell-selective NDDs, we have synthesized a cationic tripeptide Boc-Arg-Trp-Phe-OMe (PA1), which self-assembles into well-ordered spheres in 100% aqueous medium. The inherent fluorescence properties of the peptide PA1 were shifted from the ultraviolet to the visible region by the self-assembly. These fluorescent nanostructures are proteolytically stable, photostable, and biocompatible, with characteristic blue fluorescence signals that permit us to monitor their intracellular entry in real time. We also demonstrate that these tripeptide spherical structures (TPSS) have the capacity to entrap the chemotherapeutic drug doxorubicin (Dox), shuttle the encapsulated drug within cancerous cells, and initiate the DNA damage signaling cascade, which culminates in apoptosis. Next, we functionalized the TPSS with an epithelial-cell-specific epithelial cell adhesion molecule aptamer. Aptamer-conjugated PA1 (PA1-Apt) facilitated efficient Dox delivery into the breast cancer epithelial cell line MCF7, resulting in cell death. However, cells of the human cardiomyocyte cell line AC16 were resistant to the cell killing actions of PA1-Apt. Together, these data demonstrate that not only can the self-assembly of cationic tripeptides like PA1 be exploited for efficient drug encapsulation and delivery but their unique chemistry also allows for functional modifications, which can improve the selectivity of these versatile NDDs.

ROCKETS - a novel one-for-all toolbox for light sheet microscopy in drug discovery.

Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

Feasibility of Co-Targeting HER3 and EpCAM Using Seribantumab and DARPin-Toxin Fusion in a Pancreatic Cancer Xenograft Model.

Pancreatic cancer (PC) is one of the most aggressive malignancies. A combination of targeted therapies could increase the therapeutic efficacy in tumors with heterogeneous target expression. Overexpression of the human epidermal growth factor receptor type 3 (HER3) and the epithelial cell adhesion molecule (EpCAM) in up to 40% and 30% of PCs, respectively, is associated with poor prognosis and highlights the relevance of these targets. Designed ankyrin repeat protein (DARPin) Ec1 fused with the low immunogenic bacterial toxin LoPE provides specific and potent cytotoxicity against EpCAM-expressing cancer cells. Here, we investigated whether the co-targeting of HER3 using the monoclonal antibody seribantumab (MM-121) and of EpCAM using Ec1-LoPE would improve the therapeutic efficacy in comparison to the individual agents. Radiolabeled 99mTc(CO)3-Ec1-LoPE showed specific binding with rapid internalization in EpCAM-expressing PC cells. MM-121 did not interfere with the binding of Ec1-LoPE to EpCAM. Evaluation of cytotoxicity indicated synergism between Ec1-LoPE and MM-121 in vitro. An experimental therapy study using Ec1-LoPE and MM-121 in mice bearing EpCAM- and HER3-expressing BxPC3 xenografts demonstrated the feasibility of the therapy. Further development of the co-targeting approach using HER3 and EpCAM could therefore be justified.

Epitope Mapping of an Anti-EpCAM Monoclonal Antibody (EpMab-37) Using the Alanine Scanning Method.

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.

Dual-Aptamer-Assisted Ratiometric SERS Biosensor for Ultrasensitive and Precise Identification of Breast Cancer Exosomes.

Due to the heterogeneity of breast cancer, its early accurate diagnosis remains a challenge. Exosomes carry abundant genetic materials and proteins and are ideal biomarkers for early cancer detection. Herein, a ratiometric surface-enhanced Raman scattering (SERS) biosensor for exosome detection was constructed using a regularly arranged Au@Ag nanoparticles/graphene oxide (Au@Ag NPs/GO) substrate with 4-nitrothiophenol (4-NTP) molecules as an internal standard. Aptamers of two overexpressed proteins (epithelial cell adhesion molecule and human epidermal growth factor receptor 2) were linked by a short complementary DNA with rhodamine X modified at the 3'-terminal to form V-shaped double-stranded DNA, which attached to the surface of Au@Ag NPs/GO substrate for the selective recognition of breast cancer cell-derived exosomes. In the presence of exosomes, a competitive reaction occurred, resulting in the formation of the V-shaped double-stranded DNA/exosomes complex, and the V-shaped double-stranded DNA separated from the SERS substrate. The SERS signal of rhodamine X on the V-shaped double-stranded DNA decreased with the concentration of exosomes increasing, whereas the SERS signal of 4-NTP on the substrate remained stable. The ratiometric SERS strategy provides huge electromagnetic enhancement and abundant DNA adsorbing sites on the GO layer, achieving a wide detection range of 2.7 × 102 to 2.7 × 108 particles/mL and an ultralow limit of detection down to 1.5 × 102 particles/mL, without the requirement of any nucleic acid amplification. Particularly, the proposed method has significant applications in early cancer diagnosis as it can accurately identify breast cancer cell-derived exosomes in clinical serum samples and can differentiate pancreatic cancer patients and healthy individuals.

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells.

The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high-grade prostate cancers. We describe a non-invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD-CTCs) from blood samples followed by their identification by immunostaining for pan-cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl-acyl coenzyme-A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case-control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. The test accurately detects PrAD-CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection.

Epithelial cell adhesion molecule 1 (EpCAM) expression in human tumours: a comparison with pan-cytokeratin in 11,053 tumours

Epithelial cell adhesion molecule 1 (EpCAM) is an epithelial membrane glycoprotein, often used as a surrogate pan-epithelial marker. To compare the expression patterns of EpCAM and pankeratin antibody (CKpan), the gold standard of epithelial cell detection, tissue microarrays containing 11,053 samples from 101 different tumor types were analysed by immunohistochemistry. EpCAM staining was detectable in 99 (82.5%) of 120 tumour categories including 85 (70.8%) entities with at least one strongly positive case.

A clinically feasible circulating tumor cell sorting system for monitoring the progression of advanced hepatocellular carcinoma

BackgroundHematogenous metastasis is essential for the progression of advanced hepatocellular carcinoma (HCC) and can occur even after patients receive multidisciplinary therapies, including immunotherapy and hepatectomy; circulating tumor cells (CTCs) are one of the dominant components of the metastatic cascade. However, the CTC capture efficiency for HCC is low due to the low sensitivity of the detection method. In this study, epithelial cell adhesion molecule (EpCAM)/vimentin/Glypican-3 (GPC3) antibody-modified lipid magnetic spheres (LMS) were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype, respectively, in order to capture more CTCs with a more comprehensive phenotype for monitoring tumor metastasis.ResultsThe novel CTC detection system of Ep-LMS/Vi-LMS/GPC3-LMS was characterized by low toxicity, strong specificity (96.94%), high sensitivity (98.12%) and high capture efficiency (98.64%) in vitro. A sudden increase in CTC counts accompanied by the occurrence of lung metastasis was found in vivo, which was further validated by a clinical study. During follow-up, the rapid increase in CTCs predicted tumor progression in HCC patients. Additionally, genetic testing results showed common genetic alterations in primary tumors, CTCs and metastatic tissues. The proportion of patients predicted to benefit from immunotherapy with the CTC detection method was higher than that for the tissue detection method (76.47% vs. 41.18%, P = 0.037), guiding the application of clinical individualized therapy.ConclusionsThe Ep-LMS/Vi-LMS/GPC3-LMS sequential CTC capture system is convenient and feasible for the clinical prediction of HCC progression. CTCs captured by this system could be used as a suitable alternative to HCC tissue detection in guiding immunotherapy, supporting the clinical application of CTC liquid biopsy.

Antitumor activities of a defucosylated anti‑EpCAM monoclonal antibody in colorectal carcinoma xenograft models.

Epithelial cell adhesion molecule (EpCAM) is a typeI transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (KD)=2.2x10‑8 M] using flow cytometry. EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderate binding‑affinity (KD=1.5x10‑8M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.

Abstract A011: Detection of disseminated tumor cells in advanced clear cell renal cell carcinoma through research autopsy

Abstract Background: The vast majority of cancer deaths can be attributed to tumor metastases. Disseminated tumor cells (DTCs) are thought to act as a reservoir of tumor clones which can lie dormant before causing overt metastases. Their presence has been shown to be a poor prognostic indicator in several tumor types. Despite their importance, the timing and source of DTCs is unclear across most solid cancers and even less is known about them in late-stage disease. The Posthumous Evaluation of the Advanced Cancer Environment (PEACE) post-mortem study enables extensive sampling of malignant lesions but also normal tissues, thereby allowing study of DTCs in patients with advanced cancer. Methods: We performed research autopsy sampling of macroscopically normal bone, lung and liver in 3 patients with advanced clear cell renal cell carcinoma (RCC). In addition, we performed bone marrow aspiration at autopsy from vertebral bodies and the ilium using an anterior approach. Mechanical disaggregation followed by collagenase digestion was used to generate single cell suspensions. Detection of tumor cells was carried out using antibodies against known tumor or epithelial markers carbonic anhydrase IX (CAIX). Epithelial cell adhesion molecule (EpCAM) was also used for bone marrow samples as native bone marrow cells do not express this marker. These sub-populations were then isolated using fluorescence activated cell sorting and DNA was extracted and amplified from single cells. Results: We performed and optimised collagenase-based digestion on all solid tissues collected. The percentage of viable cells generated varied significantly between subjects and tissues with bone marrow samples more viable than lung and liver. Rare and distinct populations of CAIX+ and EpCAM+ cells were detected in normal tissues between 0.5-2%. DNA amplification was successful on single cells isolated from normal lung and liver but not from bone tissue. Pilot shallow coverage whole genome sequencing revealed genomically aberrant cells ranging from single chromosome arm losses to widespread copy number aberrations. Conclusion: We demonstrate feasibility of sequencing single cells from autopsy study subjects. Rare populations of single cells with markers of clear cell RCC were detected and isolated both in normal tissues of patients with advanced disease. Genomic analyses of these cells will lead to insights into their relationship to the primary tumor and overt metastatic lesions. Citation Format: Haixi Yan, Cristina Cotobal Martin, Christie English, Annelien Verfaillie, Jonas Demeulemeester, Andrew Rowan, Annika Fendler, Anne-Laure Cattin, Sucheta Mahapatra, Lewis Au, Scott Shepherd, Ben Shum, Charlotte Spencer, Zayd Tippu, Ula Mahadeva, Anna Green, Eleanor Carlyle, Cristina Naceur-Lombardelli, PEACE consortium, Mariam Jamal-Hanjani, Charles Swanton, Samra Turajlic, Peter Van Loo. Detection of disseminated tumor cells in advanced clear cell renal cell carcinoma through research autopsy [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A011.

Diagnostic Value of Epithelial Cell Adhesion Molecule Flow Cytometry for Diagnosis of Metastatic Carcinoma in Serous Effusions

Introduction: Malignant serous effusions are common in metastatic carcinomas. Although cytomorphology is recognized as the gold standard diagnostic method, it exhibits moderate sensitivity. This study aimed to assess the diagnostic value of immunophenotyping with a single epithelial marker, known as the epithelial cell adhesion molecule (EpCAM, CD326), in discriminating malignant metastatic carcinomas of serous fluids. Methods: This prospective study was conducted on suspicious or confirmed cases of malignant tumors from September 16, 2019, to June 21, 2020. Serous fluid samples were assessed via cytomorphology using the Wright-Papanicolaou method and the anti-EpCAM mouse monoclonal antibody (clone VU-1D9) flow cytometry. The EpCAM(+)/CD45(−) immunophenotype was defined as the metastatic involvement of carcinoma in the serous cavity. Results: A total of 118 samples (90 females and 28 males; mean age, 54.04 ± 16.14 years), collected from peritoneal and pleural fluids, were examined in this study. Five samples (4.24%) were positive in both EpCAM flow cytometry and cytology, while 102 samples (86.44%) were negative for both EpCAM flow cytometry and cytology, yielding an overall agreement of 92%, 84%, and 90.7% for the peritoneal, pleural, and total samples, respectively. Based on the Bayesian latent class model, the EpCAM flow cytometry showed sensitivity and specificity of 58.5% (95% CI: 0.3, 99.7) and 96.2% (95% CI: 47.8, 100), respectively. The corresponding values were 68.7% (95% CI: 0.3, 99.9) and 96.1% (95% CI: 47, 100) for cytology, respectively. Conclusion: The EpCAM flow cytometry and cytology showed comparable performance in detecting metastatic effusions. The EpCAM flow cytometry might have a diagnostic value in decreasing the false-negative rate of cytomorphology, while maintaining excellent specificity.

ImmunoInertial microfluidics: A novel strategy for isolation of small EV subpopulations

Cancer-specific small extracellular vesicles (sEVs), known as exosomes, have shown a great promise to serve as novel biomarkers for cancer diagnosis and prognosis in liquid biopsies. However, the high heterogeneity of sEVs posed great technical challenges to acquiring their molecular information. A simple and reproducible method for isolating subpopulations of sEVs can significantly enhance the detection and stratification of these circulating biomarkers and their function. This study used the synergic effects of the immunoaffinity-based approach and inertial microfluidics (ImmunoInertial microfluidics) to isolate specific subpopulations of sEVs. At first, the cancer cell-derived sEVs were captured on microbeads of varying sizes which were functionalized with specific capture antibodies such as epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and the programmed death-ligand 1 (PD-L1), facilitating the selective capture of sEVs. The sEVs-bearing microbeads were subsequently introduced to a series of inertial microfluidic channels, called iZExoSub (inertial zigzag microfluidics for exosome subpopulation separation), for size-based bead separation. Results revealed more than 90% efficiency in sEVs subpopulation separation, further proved via flow cytometry analysis data. Our approach is capable of selective isolation and quantitative detection of important biomarkers from sEVs subpopulations with high sensitivity and low cost and has the capacity to process samples of varying volumes, ranging from µL up to mL continuously. This system can outperform FACS machines in terms of sample throughput by orders of magnitudes. In addition, this study emphasized the necessity of using a consistent sEV marker (as capture and detector) across different samples for accurate assessment of subpopulations.

Using magnetic mesoporous silica nanoparticles armed with EpCAM aptamer as an efficient platform for specific delivery of 5-fluorouracil to colorectal cancer cells.

Background: Theranostic nanoparticles with both imaging and therapeutic capacities are highly promising in successful diagnosis and treatment of advanced cancers. Methods: Here, we developed magnetic mesoporous silica nanoparticles (MSNs) loaded with 5-fluorouracil (5-FU) and surface-decorated with polyethylene glycol (PEG), and epithelial cell adhesion molecule (EpCAM) aptamer (Apt) for controlled release of 5-FU and targeted treatment of colorectal cancer (CRC) both in vitro and in vivo. In this system, Au NPs are conjugated onto the exterior surface of MSNs as a gatekeeper for intelligent release of the anti-cancer drug at acidic conditions. Results: Nanocarriers were prepared with a final size diameter of 78nm, the surface area and pore size of SPION-MSNs were calculated as 636m2g-1, and 3nm based on the BET analysis. The release of 5-FU from nanocarriers was pH-dependent, with an initial rapid release (within 6h) followed by a sustained release for 96h at pH 5.4. Tracking the cellular uptake by flow cytometry technique illustrated more efficient and higher uptake of targeted nanocarriers in HT-29 cells compared with non-targeted formula. In vitro results demonstrated that nanocarriers inhibited the growth of cancer cells via apoptosis induction. Furthermore, the targeted NPs could significantly reduce tumor growth in immunocompromised C57BL/6 mice bearing HT-29 tumors, similar to those injected with free 5-FU, while inducing less side effects. Conclusion: These findings suggest that application of Apt-PEG-Au-NPs@5-FU represents a promising theranostic platform for EpCAM-positive CRC cells, although further experiments are required before it can be practiced in the clinic.