Construction and Preclinical Evaluation of 211At Labeled Anti-mesothelin Antibodies as Potential Targeted Alpha Therapy Drugs.

Targeted alpha therapy (TAT) is a promising tumor therapy that can specifically transport α particle to the vicinity of tumor cells while the normal cells are only slightly irradiated. Mesothelin is a highly promising molecular signature for many types of solid tumors including malignant mesothelioma, pancreatic cancer, ovarian cancer and lung adenocarcinoma etc., while the expression in normal human tissues are limited, thus making mesothelin a promising antigen for TAT. Previously we developed a theoretical model that could predict and optimize in vitro screening of potential TAT drugs. The aim of the study is construction and preclinical evaluation of 211At labeled anti-mesothelin antibodies as potential TAT drugs. Mesothelin expression of two tumor cell lines were confirmed by flow cytometry, and their radiosensitivities were also evaluated. We used two kinds of anti-mesothelin antibodies, ET210-6 and ET210-28, to construct TAT drugs. Then, radiochemical purity, stability in vitro, affinity of the conjugates and mesothelin expression level were assessed. The specific killing of mesothelin-positive cancer cells treated by 211At-ET210-28 and 211At-ET210-6 were studied via Cell Counting Kit-8 assay and colony formation assay. 211At-ET210-28 and 211At-ET210-6 revealed excellent affinity and stability in both phosphate buffer saline and fetal bovine serum environment. Radiolabeled antibody conjugates bound specifically to mesothelin-positive cells in vitro. Both 211At-ET210-28 and 211At-ET210-6 could specifically kill mesothelin-positive cells with negligible damages to mesothelin-negative cells. Our findings provide initial proof-of-concept for the potential use of 211At labeled ET210-28/ET210-6 anti-mesothelin antibody in specific killings of mesothelin-positive tumor cells.

Association of extracellular matrix microarchitecture and three-dimensional collective invasion of cancer cells.

As much as 90% of cancer associated mortality follows metastasis of a primary tumor. Circulating tumor cells (CTCs) and CTC clusters are important for metastasis. Compared to CTCs, CTC clusters formed by collective invasion exhibit a 23-50 fold increase in metastatic potential, but the factors that influence collective invasion are largely unknown. Using well defined three-dimensional matrices and different extracellular matrix (ECM) concentrations, we found that cancer cells were more prone to collective invasion at low ECM concentration. Moreover, despite variation of biological factors, changes in ECM microarchitecture, especially the pore size of the matrix, was correlated with the probability of collective invasion, which indicates that the physical microarchitecture of ECM plays an important role in collective invasion of cancer cells.

Mixed secondary chromatin structure revealed by modeling radiation-induced DNA fragment length distribution.

Spatial chromatin structure plays fundamental roles in many vital biological processes including DNA replication, transcription, damage and repair. However, the current understanding of the secondary structure of chromatin formed by local nucleosome-nucleosome interactions remains controversial, especially for the existence and conformation of 30 nm structure. Since chromatin structure influences the fragment length distribution (FLD) of ionizing radiation-induced DNA strand breaks, a 3D chromatin model fitting FLD patterns can help to distinguish different models of chromatin structure. Here, we developed a novel "30-C" model combining 30 nm chromatin structure models with Hi-C data, which measured the spatial contact frequency between different loci in the genome. We first reconstructed the 3D coordinates of the 25 kb bins from Hi-C heatmaps. Within the 25 kb bins, lower level chromatin structures supported by recent studies were filled. Simulated FLD patterns based on the 30-C model were compared to published FLD patterns induced by heavy ion radiation to validate the models. Importantly, the 30-C model predicted that the most probable chromatin fiber structure for human interphase fibroblasts in vivo was 45% zig-zag 30 nm fibers and 55% 10 nm fibers.

Cancer stem-like cells with hybrid epithelial/mesenchymal phenotype leading the collective invasion.

Collective invasion of cancer cells is the key process of circulating tumor cell (CTC) cluster formation, and greatly contributes to metastasis. Cancer stem-like cells (CSC) have a distinct advantage of motility for metastatic dissemination. To verify the role of CSC in the collective invasion, we performed 3D assays to investigate the collective invasion from cancer cell spheroids. The results demonstrated that CSC can significantly promote both collective and single-cell invasion. Further study showed that CSC prefer to move outside and lead the collective invasion. More interestingly, approximately 60% of the leader CSC in collective invasion co-expressed both epithelial and mesenchymal genes, while only 4% co-expressed in single invasive CSC, indicating that CSC with hybrid epithelial/mesenchymal phenotype play a key role in cancer cell collective invasion.

A Theoretical Model for Predicting and Optimizing In Vitro Screening of Potential Targeted Alpha-Particle Therapy Drugs.

One highly promising approach to cancer treatment, especially for tumors that have undergone micrometastasis, is targeted alpha-particle therapy (TAT). However, the development of a TAT drug has been impeded due to numerous unsuccessful attempts to establish effective in vitro screening methods. The goal of this study was to construct a model to predict and optimize in vitro screening of potential TAT drugs. Based on mean field hypothesis, microdosimetry and the classic linear-quadratic equation, a novel model was built, which can predict our own in vitro experiments and replicate published data from others. Interestingly, this model can also be used to quickly optimize several key parameters in in vitro screening of potential TAT drugs, instructing the optimal combinations of the expression level of antigen, the binding affinity of antibody and drug antibody ratio, as well as others. In addition, to conveniently evaluate the therapeutic benefit of different drugs, a simple but universal parameter, the death ratio, is proposed. To our knowledge, this is the first model that can predict and guide the optimization of in vitro potential targeted alpha-particle therapy drug screening, which may then accelerate the development of potential targeted alpha-particle therapy drugs dramatically.