Biology subjects

Kan, E. L.

Publications and source records attributed to Kan, E. L..

3 recordsLinked to original sources

bioRxiv2025

Simultaneous Label-free Imaging of Nucleolar Dynamics and Subcellular Metabolic Shifts Across Tissue Contexts

The nucleolus is essential for ribosome biogenesis and for regulating cellular responses to growth and stress, and its relationship to cellular metabolic activity and functional state highlights its potential as a biomarker of cellular health. However, challenges in contrast multiplexing and high-resolution isotropic three-dimensional (3D) imaging hinder the non-invasive, simultaneous assessment of nucleolar activity and subcellular metabolic maps across different tissue contexts, especially in complex 3D environments. To fully harness the nucleoluss potential as a biomarker and diagnostic target, we present a multimodal imaging platform that combines third harmonic generation (THG) imaging with metabolic autofluorescence of NAD(P)H and FAD to study structural and metabolic nucleolar dynamics. Enabled by a high-power multimode fiber source and an axial deblurring network, we achieved [~] 400 nm isotropic resolution in deep 3D imaging and confirmed the high accuracy of our method for label-free nucleolus identification using co-registered immunostaining and electron microscopy. To establish the biological relevance of our approach, we demonstrate that nucleolar stress leads to an unexpected depletion of NADH across cellular compartments. Furthermore, in the human endometrium--where nucleolar dynamics are central to the tissues response to progesterone--our label-free imaging strategy delineated endometrial structures in freshly excised tissues and revealed that progesterone treatment induces distinct changes in nucleolar translocation and metabolic adaptation in organoids derived from diseased patients compared to controls. This capacity to non-invasively visualize and quantify features of the nucleolus and its local metabolic microenvironment at single-cell resolution in human tissues--and dynamically track these changes over time in patient-derived organoids--provides a powerful tool for uncovering the roles of the nucleolus in development, disease progression, and therapeutic response. Together, these findings establish our platform as a significant advance for both fundamental research and organelle-based tissue diagnostics.

cell biology↗

bioRxiv2025

A Vascularized Liver Microphysiological System Captures Key Features of Hepatic Insulin Resistance and Monocyte Infiltration

Three-dimensional in vitro liver models are a promising means to recapitulate key aspects of human liver disease pathologies, thereby aiding therapeutic development. Spheroidal aggregates of hepatocytes, sometimes including non-parenchymal cells, are an established approach for modeling facets of metabolism and drug responses, yet these models often lack dynamic interactions with vascular and immune cells that also contribute to disease development and progression. To address this, we developed a microphysiological system (MPS) that integrates multicellular human hepatic spheroids with self-organized microvascular networks. We show extensive interaction between primary human spheroids and functional vasculature while maintaining key hepatic functions. We demonstrate the utility of this MPS by modeling an insulin resistance state through chronic exposure to disease-mimetic media conditions. This disease model displays altered hepatocyte metabolism, dysregulated vascular features, and increased inflammation state. Enabled by the functional vasculature, we further extend this disease model to capture changes in immune cell recruitment. When culturing CD14+ monocytes in our liver MPS, a subset of monocytes extravasate, localize to hepatic spheroids, and begin differentiating into CD163+ macrophages. These cells infiltrate with greater frequency in insulin resistant samples, consistent with known clinical findings. All together, this vascularized MPS model captures disease-relevant liver biology including inflammatory features.

bioengineering↗

bioRxiv2023

Patient-Specific Vascularized Tumor Model: Blocking TAM Recruitment with Multispecific Antibodies Targeting CCR2 and CSF-1R

Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-{beta}, referred to as CSF1R/CCR2/TGF-{beta} Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-{beta} Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).

bioengineering↗