Ling Yu

Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in hematologic malignancies but has limited efficacy in solid tumors due to tumor microenvironment (TME) barriers that impede CAR T cell recognition, infiltration, and sustained function. Traditional 2D assays inadequately recapitulate these constraints, necessitating improved in vitro models. This study validated a 3D tumor spheroid platform using an agarose microwell system to generate uniform B7-H3-positive spheroids from multiple solid tumor cell lines, enabling the evaluation of CAR T cell activity. TME-relevant immune modulation under 3D conditions was analyzed by flow cytometry for B7-H3, MHC I/II, and antigen processing machinery (APM), followed by co-culture with B7-H3 CAR T cells to assess cytotoxicity, spheroid integrity, tumor viability, and CAR T cell activation, exhaustion, and cytokine production. Two human cancer-cell-line-derived spheroids, DU 145 (prostate cancer) and SUM159 (breast cancer), retained B7-H3 expression, while MC38 (mouse colon cancer)-derived spheroids served as a B7-H3 negative control. Under 3D culture conditions, DU 145 and SUM159 spheroids acquire TME-like immune evasion characteristics and specifically downregulated MHC-I and APM (TAP1, TAP2, LMP7) with concurrent upregulation of MHC-II and calreticulin. Co-culture showed effective spheroid infiltration, cytotoxicity, and structural disruption, with infiltrating CAR T cells displaying higher CD4⁺ fraction, activation, exhaustion, effector/terminal differentiation, and IFN-γ/TNF-α production. This 3D platform recapitulates critical TME constraints and provides a cost-effective, feasible preclinical tool to assess CAR T therapies beyond conventional 2D assays.

Recapitulating the dynamic microenvironment is essential for advanced 3D cell culture and reliable drug testing, yet conventional static platforms lack physiologically relevant fluid dynamics. Here, a pump‐free vertical dual‐circulation centrifugal microfluidic platform is presented, enabling unidirectional medium flow and efficient nutrient exchange for hydrogel‐based 3D cultures. By combining centrifugal and gravitational forces, this integrated system achieves stable long‐term circulation and establishes tunable concentration gradients, supporting sustained tumor clusters growth under dynamic conditions. Compared with static systems, the platform significantly enhances tumor spheroid expansion, promotes complex morphological remodeling, and facilitates fusion events. Functional drug perfusion studies demonstrate improved doxorubicin distribution and reduced local drug accumulation. This new type of vertical centrifugal microfluidic platform provides a versatile solution for dynamic tissue culture and drug screening, advancing the capabilities of in vitro disease modeling and personalized medicine.

Thread-based analytical devices are low-cost, portable, and easy to use, making them ideal for detecting various biomolecules like glucose and DNA with minimal sample requirements, while also offering environmental benefits through their biodegradability. This study explores the potential of zwitterionic poly(sulfobetaine methacrylate) brushes modified cotton thread (PSBMA@threads) as an innovative substitute for DNA solid-phase extraction. The PSBMA polymer brushes were synthesized on cotton threads via surface-initiated atom transfer radical polymerization (SI-ATRP). The usability of the PSBMA@threads for DNA extraction from cell lysates containing cell debris, proteins, and detergents was evaluated. Characterization using SEM, FTIR, and EDS confirmed the successful functionalization with PSBMA polymer brushes. The antifouling properties of PSBMA@threads, including resistance to non-specific protein adsorption and underwater oil repellency, were assessed. The results demonstrated selective DNA capture from protein and lipid-rich lysates. Optimized extraction parameters improved DNA yield, enabling efficient extraction from tumor cells, which successfully underwent PCR amplification. Comparative experiments with commercial silica membrane-based columns revealed that PSBMA@threads exhibited comparable DNA extraction capability. The PSBMA@threads maintained extraction capability after six months of ambient storage, highlighting its stability and cost-effectiveness for nucleic acid isolation in analytical applications.

Immunohistochemistry (IHC) is a well-established method in tissue pathology and finds critical applications in both clinical and fundamental medical science. Generally, labeled antibodies are a requisite for IHC to visualize the spatial distribution of the biomarker (antigen) of interest in the tissue slices. In this work, a label-free IHC imaging strategy is reported for the first time by using an oblique-incidence reflectivity difference (OIRD) to obviate the need for labeled antibodies. The position-resolved relative abundance of biomarkers was successfully mapped by the differential OIRD image upon the binding of native antibody on the tissue slices. The dynamic binding kinetics of antibody on the tissue are also monitored. The efficacy of such a label-free IHC strategy is further verified with fluorescent IHC images. This work explores the application of the OIRD imaging technique in tissue slices and offers a label-free IHC imaging strategy for pathological analysis, disease diagnosis, and drug screening.

For traditional laboratory microscopy observation, the multi-dimensional, real-time, in situ observation of three-dimensional (3D) tumor spheroids has always been the pain point in cell spheroid observation. In this study, we designed a side-view observation petri dish/device that reflects light, enabling in situ observation of the 3D morphology of cell spheroids using conventional inverted laboratory microscopes. We used a 3D-printed handle and frame to support a first-surface mirror, positioning the device within a cell culture petri dish to image cell spheroid samples. The imaging conditions, such as the distance between the mirror and the 3D spheroids, the light source, and the impact of the culture medium, were systematically studied to validate the in situ side-view observation. The results proved that placing the surface mirror adjacent to the spheroids enables non-destructive in situ real-time tracking of tumor spheroid formation, migration, and fusion dynamics. The correlation between spheroid thickness and dark core appearance under light microscopy and the therapeutic effects of chemotherapy doxorubicin and natural killer cells on spheroids’ 3D structure was investigated.

For traditional laboratory microscopy observation, the multi-dimensional, real-time, in situ observation of three-dimensional (3D) tumor spheroids has always been the pain point in cell spheroid observation. In this study, we designed a side-view observation petri dish/device that reflects light, enabling in situ observation of the 3D morphology of cell spheroids using conventional inverted laboratory microscopes. We used a 3D-printed handle and frame to support a first surface mirror, positioning the device within a cell culture petri dish to image cell spheroid samples. The imaging conditions, such as the distance between the mirror and the 3D spheroids, the light source, and the impact of the culture medium, were systematically studied to validate the in-situ side-view observation. The results proved that placing the surface mirror adjacent to the spheroids enables non-destructive in situ real-time tracking of tumor spheroid formation, migration, and fusion dynamics. The correlation between spheroid thickness and dark core appearance under light microscopy and the therapeutic effects of chemotherapy doxorubicin and Natural Killer cells on spheroids’s 3D structure was investigated.