Biology subjects

Escorcia, F. E.

Publications and source records attributed to Escorcia, F. E..

5 recordsLinked to original sources

bioRxiv2026

Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

Glypican-3 (GPC3) is an oncofetal protein widely being explored as a diagnostic and therapeutic target in hepatocellular carcinoma (HCC). Given that radiotherapy in the form of external beam and radioembolization are standard-of-care treatments for HCC, we aimed to determine whether there was any relationship between GPC3 and response to radiotherapy. Here, we demonstrate that GPC3 expression confers radioresistance in liver cancer through integrated in vitro, in vivo, and patient-level clinical analyses. Stable GPC3-knockout in liver cancer cell lines (HepG2, Hep3B, Huh7) and ectopic GPC3 expression in GPC3-negative liver cancer cells (SNU449), as well as in non-hepatic A431 cells, demonstrated that GPC3-mediated radioresistance is not restricted to hepatic lineage. Following irradiation, GPC3-deficient cells exhibited reduced proliferation, impaired clonogenic survival, persistent DNA damage, prolonged G2/M arrest, and increased apoptosis. Transcriptomic profiling demonstrated enrichment of cell-cycle and DNA damage response pathways in irradiated GPC3-deficient cells compared with GPC3-positive cells, and protein analyses confirmed sustained activation of the ATM/CHK2 axis. In vivo, GPC3 deletion markedly enhanced radiation-induced tumor growth delay in both HepG2 and A431 xenograft models. Consistent with these findings, high GPC3 expression was associated with inferior clinical outcomes in patients with HCC undergoing external-beam radiotherapy or radioembolization. Together, these findings identify GPC3 as a determinant of radioresistance in liver cancer and suggest its potential utility as a biomarker to guide radiotherapeutic strategies. Significance statementRadiotherapy is an important treatment option for HCC, but biomarkers that predict tumor response remain limited. GPC3 is highly expressed in most HCCs and is being investigated as an important biomarker for diagnosis and treatment of this disease, yet its relationship, if any, on radiosensitivity has not been previously reported. Here, we identify GPC3 as a modulator of radioresistance. GPC3 loss enhances radiosensitivity and is associated with persistent unresolved DNA damage, prolonged G2/M arrest, and sustained activation of the ATM/CHK2 pathway, resulting in delayed tumor growth after irradiation. In a clinical cohort of patients treated with radiotherapy, high GPC3 expression was associated with poorer overall survival. These findings suggest that GPC3 expressing tumors may necessitate either more dose-intense radiotherapy, radiobioligically ablative and/or combined with other modalities, or alternative therapeutic modalities to adequately treat HCC.

cancer biology↗

bioRxiv2025

Multimodal Approach for Identification and Validation of Hepatocellular Carcinoma Targets for Radiotheranostics

Identifying tumor selective targets is critical for the development of precision diagnostic and therapeutic agents in oncology. Despite advances in precision oncology elsewhere, there are no FDA-approved hepatocellular carcinoma (HCC)-selective treatments. HCC is the most common type of liver cancer and accounts for significant morbidity and mortality worldwide. Here, we sought to integrate bulk (371 cases) and single cell RNA sequencing (scRNAseq, n=2 datasets, 34 cases, 102,956 cells) of patient samples to enrich for molecules that are overexpressed in HCC, which could serve as HCC-selective targets. To guide definitions of tumor and normal cell clusters with higher fidelity, we also imported a normal liver scRNAseq dataset. Using this integrated approach, we identified several HCC-selective plasma membrane molecules. To validate these targets, we performed immunohistochemical staining of HCC and normal tissue microarrays and confirmed HCC-selective staining of identified targets. Next, we verified the presence of these targets in several commercially available HCC cell lines by flow cytometry and western blot. Finally, we designed, engineered, and tested novel antibody-based positron emission tomography (immunoPET) agents to these targets in various murine models of liver cancer. Our findings confirm that we can leverage this multimodal approach to identify and validate of HCC-selective targets, which can be used to develop tumor-selective diagnostic and therapeutic radiopharmaceuticals, or radiotheranostics, and other precision oncology agents. One Sentence SummaryA multimodal pipeline defines and validates tumor-selective surface targets for radiotheranostic use in hepatocellular carcinoma.

cancer biology↗

bioRxiv2025

Biodistribution-Driven Discovery Identifies a Glycosidase-Cleavable Linker to Reprogram Radiotheranostics

While radiopharmaceutical therapy (RPT) has become part of the standard-of-care for patients with advanced prostate cancers and neuroendocrine tumors (NETs), cures are elusive and normal tissue toxicity remain a challenge. Chemical groups susceptible to cleavage by enzymes present in tumors, tumor microenvironment or in normal tissues, have the potential to improve the therapeutic index for RPT. Using DOTA-TATE as an example and drawing from strategies used to develop antibody-drug conjugates, we designed, and synthesized, a chemically diverse series of linkers between the chelator (DOTA) and the targeting vector (TATE). Of the 10 agents we tested, two with cleavable linker domains reduced kidney retention compared to DOTA-TATE: the previously reported DOTA-MVK({varepsilon})-TATE, and a novel agent bearing cleavable beta-galactose ({beta}-Gal) unit, DOTA-{beta}-Gal-TATE. In murine models of NETs, positron emission tomography (PET) was used to image yttrium-86 (86Y)-labeled variants and show that, while the 86Y-DOTA-MVK({varepsilon})-TATE exhibits similar tumor uptake to the parent non-cleavable 86Y-DOTA-TATE, 86Y-DOTA-{beta}-Gal-TATE shows enhanced tumor uptake, resulting in up to 10-fold improvement in the tumor-to-kidney ratios compared to 86Y-DOTA-TATE. In vitro and in vivo studies confirm high efficiency, enzyme-specific cleavage of 86Y-DOTA-MVK({varepsilon})-TATE and 86Y-DOTA-{beta}-Gal-TATE, supporting a key role for cleavable linker chemistry in the observed outcomes. RPT studies using actinium-225 (225Ac)-labeled variants confirm that all agents are therapeutically effective and well tolerated. While both cleavable variants exhibit superior local control, overall survival, and more favorable toxicity profile when compared with 225Ac-DOTA-TATE, 225Ac-DOTA-{beta}-Gal-TATE demonstrated lower nephrotoxicity. Our findings suggest a potentially generalizable strategy for improving the pharmacokinetics of radiopharmaceutical therapy agents. One Sentence SummaryA {beta}-galactose-cleavable linker reduces kidney toxicity, enhances tumor targeting and therapeutic efficacy in radiopharmaceutical therapy.

cancer biology↗

bioRxiv2022

Upregulation of Somatostatin Receptor Type 2 in a Receptor-Deficient In Vivo Pancreatic Neuroendocrine Tumor Model Improves Tumor Response to Targeted 177Lu-DOTATATE

PurposeThe goal of this study was to test whether histone deacetylase inhibitors (HDACis) restore somatostatin receptor type 2 (SSTR2) expression in models of high-grade pancreatic neuroendocrine tumors (PNETs), thereby facilitating effective treatment with 177Lu-DOTATATE therapy. MethodsTo assess tumor grade correlation with SSTR2 expression, we assessed human SSTR2 promoter methylation and expression levels in 96 NIH patient samples and merged the GSE149395 and GSE117852 datasets. We used three NET cell lines (QGP-1, BON-1, GOT-1) characterized by variable SSTR2 expression profiles for functional in vitro studies using HDACis. Finally, the QGP-1 xenograft mouse model, with low basal SSTR2 expression, was used to analyze the therapeutic efficacy of combined HDACi and 177Lu-DOTATATE therapies. ResultsHuman PNET SSTR2 promoter methylation showed a significant positive correlation with higher tumor grades (P = 0.000014). We also found a significant negative correlation (P < 0.0001) between SSTR2 promoter methylation and SSTR2 expression in three NET cell lines. In vitro, SSTR2 expression increased significantly in BON-1 and QGP-1 cells at 48 and 72 hours in a dose-dependent fashion using two different HDACis, valproic acid and CI-994. In vivo studies demonstrated a significant increase in 177Lu-DOTATATE tumor uptake in QGP-1-engrafted mice after 10 days of CI-994 pretreatment (P = 0.0175). Treatment with 177Lu-DOTATATE reduced tumor size in mice pretreated with CI-994 compared to 177Lu-DOTATATE alone (at 15 days, P = 0.0028). ConclusionHDACis increase SSTR2 surface expression in models of high-grade, SSTR2-deficient PNETs. This approach has the potential to improve tumor response to targeted therapy with 177 Lu-DOTATATE in patients with receptor-negative, metastatic PNETs. Translational Relevance StatementPancreatic neuroendocrine tumors (PNETs) express high levels of somatostatin receptor type 2 (SSTR2), a unique target for both tumor imaging and therapy. Unfortunately, high-grade PNETs lose SSTR2 surface expression and thus become ineligible for SSTR2-targeted 177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT). Restoring SSTR2 expression through the reversal of inhibitory epigenetic gene silencing mechanisms has the potential for improving tumor responsiveness to PRRT. We demonstrate that histone deacetylase inhibitors (HDACis) upregulate SSTR2 surface expression in three NET cell lines in vitro. In an in vivo PNET xenograft model with low basal SSTR2 expression, our studies validate a significantly higher tumor uptake of SSTR2-targeted 177Lu-DOTATATE in animals pretreated with HDACis compared to controls. Furthermore, we show that this higher tumor uptake results in significant anti-tumor response when compared to standard PRRT alone. Our preclinical results thus provide a rationale for utilizing HDACi pretreatment to improve targeted radionuclide therapy in patients with SSTR2-negative, metastatic PNETs.

molecular biology↗

bioRxiv2020

ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3-(A431) and GPC3+ cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

cancer biology↗