Biology subjects

Choyke, P. L.

Publications and source records attributed to Choyke, P. L..

8 recordsLinked to original sources

bioRxiv2026

Complementary cytotoxicity of GD2-targeted photoimmunotherapy and 5-aminolevulinic acid photodynamic therapy in neuroblastoma and osteosarcoma

Phototherapy, a light-activated anticancer treatment, enables localized tumor-cell killing with distinct mechanisms of action. Photoimmunotherapy (PIT) produces immunogenic tumor cell death upon near-infrared light activation of a photoabsorber through antigen-specific targeting. Photodynamic therapy (PDT) produces reactive oxygen species through red-light activation of intracellular protoporphyrin IX generated from 5-aminolevulinic acid uptake and metabolism. PIT may have limited activity in antigen-low cells, whereas PDT has less precise tumor selectivity. We combined these modalities to define their interaction, broaden cytotoxicity, and determine whether dual treatment could reduce light-dose requirements. We conjugated dinutuximab, which targets the GD2 antigen, to IRDye 700DX and characterized plasma-membrane localization by confocal and widefield microscopy. PIT and PDT monotherapies were evaluated across agent and light doses in neuroblastoma (NB) and osteosarcoma (OS) cell lines. Combination matrices were tested using interaction, highest-single-agent, and Bliss analyses. Both monotherapies demonstrated significant light-dose-dependent effects in NB and OS. PIT produced no measurable cytotoxicity in antigen-blunted control cells, whereas PDT remained effective, confirming antigen-dependence of PIT and antigen-independence of PDT. The combination interaction was significant in SK-N-BE(2) but not LM7. At selected combinations, however, dual treatment produced greater killing than the more effective matched monotherapy in both SK-N-BE(2) and LM7 (Padj<0.022). Notably, lowest combination of PIT 10 J/cm2 plus PDT 10 J/cm2 achieved 90.3% killing in SK-N-BE(2), exceeding higher light-dose PIT or PDT monotherapy, suggesting a light-dose sparing effect. These findings establish potent and complementary PIT-PDT activity, supporting dual phototherapy to broaden cytotoxicity and reduce light-dose requirements in GD2-expressing tumor phototherapy.

cancer biology↗

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

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↗

bioRxiv2024

In vivo tracking of ex vivo generated 89Zr-oxine labeled plasma cells by PET in a non-human primate model

B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody secreting cells. Zirconium-89-oxine labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 hours of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.

bioengineering↗

bioRxiv2023

Metabolic and imaging phenotypes associated with RB1 loss in castrate resistant prostate cancer

Advanced prostate cancer is treated with androgen receptor (AR) signaling inhibitors, which are initially effective, but most patients eventually develop resistance and progress to castrate-resistant prostate cancer (CRPC). Loss of RB1 in CRPC tumors is correlated with rapid progression and poor patient survival and, in combination with TP53 loss, predisposes patients to the development of transitional neuroendocrine prostate cancer (NEPC). Although progressive CRPC is clinically associated with higher 18FDG-PET SUVmax values, it is unknown whether inactivation of RB1 and/or TP53 is a driver of increased glucose import. Using a cohort of patient-derived xenograft (PDX)-derived CRPC organoids, we found that NEPC could not be conclusively distinguished from adenocarcinoma by 18FDG uptake alone, and PSMA protein levels did not correlate with cancer phenotype or 18FDG uptake. Castration-resistant models showed higher 18FDG uptake, but lower pyruvate-to-lactate conversion compared to their castration-sensitive counterparts. In parallel studies using castration-sensitive prostate cancer models, RB1/TP53 knockdown did not affect 18FDG uptake, but increased basal respiration and glycolytic activity, with combined depletion leading to glucose diversion into glycogenesis. These metabolic changes were reflected in increased lactate dehydrogenase flux detected by 13C-hyperpolarized magnetic resonance spectroscopy upon RB1 loss, but not in 18FDG uptake. The metabolic heterogeneity revealed here suggests that a multimodal molecular imaging approach can improve tumor characterization, potentially leading to a better prognosis in cancer treatment.

cancer biology↗

bioRxiv2022

MRI Guided Fresh Tissue Procurement in Radical Prostatectomy Specimens: An Evolutionary Paradigm

PurposeThe aim of this work is to assess the feasibility of novel targeted methods for fresh tissue procurement that facilitate accurate identification of lesion location and tissue sample extraction in an excised prostate gland, while minimizing the tissue deformation or damage. Methods and ResultsProposed fresh tissue procurement methods are based on utilizing a custom 3D printed patient-specific prostate mold. In the semi-freehand method, the access cut is guided by a set of markers on the bottom of the mold that define the boundary of a lesion in two orthogonal directions. The procurement site is identified by palpation. In the guided cut with-locator card method the procurement sites are identified by a cutout in the locator cards inserted into the access cuts. The two approaches were utilized to procure >50 samples. In the biopsy needle method guiding channels were designed into the prostate mold to procure fresh tissue. This method was used 4 times. Finally, a mixed-reality biopsy needle method was developed for phantom testing. ConclusionsPreliminary results of fresh tissue procurement utilizing novel methods showed improve precision of obtaining cancerous fresh tissue for molecular and genomic cancer research compared to current methods which rely on surgical estimation of lesion location.

bioengineering↗

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↗

bioRxiv2018

PET by MRI: Glucose Imaging by 13C-MRS without Dynamic Nuclear Polarization by Noise Suppression through Tensor Decomposition Rank Reduction

Metabolic reprogramming is one of the defining features of cancer and abnormal metabolism is associated with many other pathologies. Molecular imaging techniques capable of detecting such changes have become essential for cancer diagnosis, treatment planning, and surveillance. In particular, 18F-FDG (fluorodeoxyglucose) PET has emerged as an essential imaging modality for cancer because of its unique ability to detect a disturbed molecular pathway through measurements of glucose uptake. However, FDG-PET has limitations that restrict its usefulness in certain situations and the information gained is limited to glucose uptake only. 13C magnetic resonance spectroscopy theoretically has certain advantages over FDG-PET, but its inherent low sensitivity has restricted its use mostly to single voxel measurements. We show here a new method of imaging glucose metabolism in vivo that relies on a simple, but robust and efficient, post-processing procedure by the higher dimensional analog of singular value decomposition, tensor decomposition. Using this procedure, we achieve an order of magnitude increase in signal to noise in both dDNP and non-hyperpolarized non-localized experiments without sacrificing accuracy. In CSI experiments an approximately 30-fold increase was observed, enough that the glucose to lactate conversion indicative of the Warburg effect can be imaged without hyper-polarization with a time resolution of 12 s and an overall spatial resolution that compares favorably to 18F-FDG PET.

cancer biology↗