Biology subjects

Antmen, E.

Publications and source records attributed to Antmen, E..

6 recordsLinked to original sources

bioRxiv2026

Microfluidic T-Chip enables one-step clinical-scale T-cell purification from blood products for CAR T-cell manufacturing

Treatment with chimeric antigen receptor (CAR) T cells has emerged as a promising immune therapy for relapsed and refractory hematologic malignancies. The CAR T cells are manufactured in a series of steps that involve isolating T cells from the patients leukapheresis product, genetically modifying them to express the CAR against the target antigen, and reinfusing them into the patient. Efficient T-cell enrichment from leukapheresis products is critical to the success of these therapies. Current methods for T-cell sorting on a clinical scale involve several washing steps to remove red blood cells and platelets, followed by T-cell selection and activation. These multi-step processes result in cell loss during processing and involve several handling steps. Here, we utilize fluidically assembled micromagnetic lenses to develop a high-throughput, continuous-flow microfluidic T-cell sorter, designated as the T-Chip, for sorting magnetic bead-labeled CD3+ T cells in a single step. Our approach allows direct sorting of T cells in expansion media from leukopaks without any washing steps, effectively removing 99.999% of RBCs and platelets from the leukapheresis product. A single 1-inch x 3-inch T-Chip can process leukapheresis product at a throughput of 60 mL/hr and 2.56 {+/-} 0.12 billion cells/hr. Using this optimized workflow, we demonstrate clinical-scale enrichment of highly pure CD3+ T cells (97.7 {+/-} 1.3%) with high viability (97.0 {+/-} 1.1%) and recovery (87.3 {+/-} 14.8%) in a functionally closed manner. Downstream processing of T cells isolated using the T-Chip yielded potent anti-mesothelin CAR T cells with demonstrated anti-tumor efficacy. Overall, by exploiting precisely engineered magnetic forces and laminar flow, the microfluidic T-Chip overcomes bottlenecks caused by low throughput and enables single-step large-scale T-cell purification for the rapid development of CAR T cells.

bioengineering↗

bioRxiv2025

The E3 ligase HECTD4 regulates COX-2 dependent tumor progression and metastasis

E3 ubiquitin ligases mediating turnover of proteins engaged in cancer progression point to key regulatory nodes. To uncover modifiers of metastatic competency, we conducted an in vivo genome-wide CRISPR-inactivation screen using cultured breast circulating tumor cells, following intravascular seeding and lung colonization. We identified HECTD4, a previously uncharacterized gene encoding a conserved potential HECT domain-containing ubiquitin transferase, as a potent tumor and metastasis suppressor. We show that purified HECTD4 mediates ubiquitin conjugation in vitro, and proteomic studies combined with ubiquitin remnant profiling identify a major degradation target as the prostaglandin synthetic enzyme cyclooxygenase-2 (COX-2; PTGS2). In addition to COX-2 itself, HECTD4 targets its regulatory kinase MKK7. In breast cancer models, HECTD4 expression is induced as cells lose adherence to the matrix, and its depletion massively increases COX-2 expression, enhancing anchorage-independent proliferation and tumorigenesis. Genetic or pharmacologic suppression of COX-2 reverses the pro-tumorigenic and pro-metastatic phenotype of HECTD4-depleted cells. Thus, HECTD4 encodes an E3 ubiquitin ligase that downregulates COX-2 suppressing anchorage-independence in epithelial cancer cells. Significance StatementA genome-wide CRISPR-inactivation screen identified the previously uncharacterized E3 ubiquitin ligase HECTD4, as a tumor and metastasis suppressor, with COX-2 as its major degradation target. The pro-tumorigenic and pro-metastatic effect of HECTD4 suppression depends on COX-2 stabilization, which is critical for anchorage-independent growth, providing a basis for investigating COX-2 inhibition to prevent metastatic recurrence.

cancer biology↗

bioRxiv2024

Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

bioengineering↗

bioRxiv2024

CDKN1B (p27kip1) enhances drug tolerant persister CTCs by restricting polyploidy following mitotic inhibitors.

The mitotic inhibitor docetaxel (DTX) is often used to treat endocrine-refractory metastatic breast cancer, but initial responses are mitigated as patients eventually have disease progression. Using a cohort of ex vivo cultures of circulating tumor cells (CTCs) from patients with heavily pretreated breast cancer (n=18), we find two distinct patterns of DTX susceptibility, independent of clinical treatment history. In CTCs cultured from some patients, treatment with a single dose of DTX results in complete cell killing, associated with accumulation of non-viable polyploid ([≥]8N) cells arising from endomitosis. In others, a transient viable drug-tolerant persister (DTP) population emerges, ultimately enabling renewed proliferation of CTCs with preserved parental cell ploidy and DTX sensitivity. In these CTC cultures, efficient cell cycle exit generates a [≤]4N drug-tolerant state dependent on CDKN1B (p27Kip1). Exposure to DTX triggers stabilization of CDKN1B through AKT-mediated phosphorylation at serine 10. Suppression of CDKN1B reduces the number of persister CTCs, increases [≥]8N mitotic cells and abrogates regrowth after DTX exposure. Thus, CDKN1B-mediated suppression of endomitosis contributes to a reversible persister state following mitotic inhibitors in patient-derived treatment refractory breast cancer cells. Summary in bulletsO_LITransient DTX tolerant persister cells emerge in some patient-derived cultured CTCs. C_LIO_LIDTX-tolerant persisters restrict endoreduplication and polyploidy through CDKN1 (p27kip1). C_LIO_LIDTX exposure induces CDKN1B stabilization through AKT mediated phosphorylation at serine 10. C_LIO_LISuppression of polyploidy underlies a drug tolerant persister state specific to mitotic inhibitors. C_LI

cancer biology↗

bioRxiv2019

Loss Of Nuclear Deformability Of Breast Cancer Cells By The Disruption Of Actin Filaments

It is well known that chemical and biomechanical interactions between the nucleus and cytoskeleton are involved in and critical for movement, migration and nuclear positioning of cancer cells. Through nucleo-cytoskeletal coupling, proteins of the LINC complex and the nuclear envelope are capable of transducing cytoplasmic mechanical input across the nuclear membrane; however, their functional importance in the behavior of cancer cells and their nuclei has never been directly tested. In this study, our assumption was that the differences in the malignancies of breast cancer cells are the result of the differences in their nuclear deformation and its expression can be amplified on micropatterned surfaces. Based on this, our hypothesis was that the level of completeness of polymerization of actin filaments can affect nuclear deformability, and as a result, the metastatic capability of the cancer cells. In order to prove this we disrupted the polymerization of the actin filaments by using two drugs, Cytochalasin D and Jasplakinolide, which caused impaired propagation of intracellular forces, prevented nuclear deformation and increased in the expression levels of Lamin A/C and Nesprin-2 in malignant breast cancer cells. Our findings suggest that activity of these two proteins is critical for nucleo-cytoskeletal force transmission. More importantly, actin filament disruption can prevent the distortions in nuclear morphology and as a result avoid the development of cancer metastasis.

bioengineering↗

bioRxiv2019

A 2-Compartment Bone Tumor Model for Testing the Efficacy of Cancer Drugs

We produced a three dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor tissue microenvironment, migration of the tumor cells and the efficacy of an anticancer drug, Doxorubicin, for personalized medicine applications. The model consisted of two compartments: (a) a healthy bone tissue mimic, poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate ({beta}-TCP) sponge that was seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, a lyophilized collagen sponge that was seeded with human osteosarcoma cells (Saos-2). The tumor component was introduced to a central cavity created in the healthy bone mimic and together they constituted the total 3D model (3D-BTM). The scaffolds were characterized by determining their mechanical properties, studying their topography and stability with compression tests, microCT, SEM, confocal microscopy and gravimetry. Porosities of the sponges were determined from {micro}CT data as 96.7% and 86% for PLGA/TCP and collagen sponges, respectively. The average diameters of the pores were measured by using ImageJ (NIH, USA) as 199{+/-}52 {micro}m for PLGA/TCP and 50-150 m for collagen scaffolds. Young modulus of the PLGA/TCP and collagen sponges were determined as 4.76MPa and 140kPa, respectively. Cells seeded on the two sponges were studied independently and together on the BTM. Cell proliferation, morphology, calcium phosphate forming capacity and ALP production were studied on both healthy bone and tumor mimics. All types of cells showed cellular extensions and spread on and in the scaffolds indicating good cell-material interactions. Angiogenic developments in BTM were studied along with migration of cells between the components with immunocytochemistry, SEM, microCT, qRT-PCR and agarose gel electrophoresis. Confocal microscopy showed that a direct contact was established between the cells present in different parts of the BTM; and the HUVEC cells within the healthy bone mimic were observed to migrate into the tumor mimic. This was confirmed by the increase in the levels of angiogenic factors VEGF, bFGF, and IL-8 in the tumor component. The IC50 of Doxorubicin on Saos-2 cells was determined as 0.1876 {micro}g.mL-1. Doxorubicin was administered to the BTM at 2.7 {micro}g.mL-1 concentration and after allowing one day for interaction, the cell number was determined with Alamar Blue cell viability test as 7-fold lesser compared to 24 h earlier. Apoptosis of the osteosarcoma cells was measured by caspase-3 enzyme activity assay. These results demonstrate the suitability of the 3D BTM model for use in the investigation of activities and migrations of cells in a tumor tissue. These will be very useful in studying metastatic capabilities of cells in addition to personalized drug treatments.

bioengineering↗