Advanced Healthcare Materials

Immunocompetent PDMS‐Free Organ‐on‐Chip Model of Cervical Cancer Integrating Patient‐Specific Cervical Fibroblasts and Neutrophils

AbstractDespite preventive measures and available treatments, cervical cancer still ranks as the fourth most prevalent cancer among women worldwide and remains the leading cause of cancer death in women in many developing countries. To gain further insights into pathogenesis and to develop novel (immuno)therapies, more sophisticated human models recreating patient heterogeneities and including aspects of the tumor microenvironment are urgently required. A novel polydimethylsiloxane‐free microfluidic platform, designed specifically for the generation and ccultivation of cervical cancerous tissue, is introduced. The microscale open‐top tissue chambers of the cervical cancer‐on‐chip (CCoC) enable facile generation and long‐term cultivation of SiHa spheroids in co‐culture with donor‐derived cervical fibroblasts. The resulting 3D tissue emulates physiological architecture and allows dissection of distinct effects of the stromal tissue on cancer viability and growth. Treatment with cisplatin at clinically‐relevant routes of administration and dosing highlights the platform's applicability for drug testing. Moreover, the model is amenable for integration and recruitment of donor‐derived neutrophils from the microvasculature‐like channel into the tissue, all while retaining their ability to produce neutrophil extracellular traps. In the future, the immunocompetent CCoC featuring donor‐specific primary cells and tumor spheroids has the potential to contribute to the development of new (immuno)therapeutic options.

A Novel 3D High‐Throughput Phenotypic Drug Screening Pipeline to Identify Drugs with Repurposing Potential for the Treatment of Ovarian Cancer

AbstractOvarian cancer (OC) poses a significant clinical challenge due to its high recurrence rates and resistance to standard therapies, particularly in advanced stages where recurrence is common, and treatment is predominantly palliative. Personalized treatments, while effective in other cancers, remain underutilized in OC due to a lack of reliable biomarkers predicting clinical outcomes. Accordingly, precision medicine approaches are limited, with PARP inhibitors showing efficacy only in specific genetic contexts. Drug repurposing offers a promising, rapidly translatable strategy by leveraging existing pharmacological data to identify new treatments for OC. Patient‐derived polyclonal spheroids, isolated from ascites fluid closely mimic the clinical behavior of OC, providing a valuable model for drug testing. Using these spheroids, a high‐throughput drug screening pipeline capable of evaluating both cytotoxicity and anti‐migratory properties of a diverse drug library, including FDA‐approved, investigational, and newly approved compounds is developed. The findings highlight the importance of 3D culture systems, revealing a poor correlation between drug efficacy in traditional 2D models and more clinically relevant 3D spheroids. This approach has expedited the identification of promising candidates, such as rapamycin, which demonstrated limited activity as a monotherapy but synergized effectively with standard treatments like cisplatin and paclitaxel in vitro. In combination with platinum‐based therapy, Rapamycin led to significant in vitro cytotoxicity and a marked reduction in tumor burden in a syngeneic in vivo model. This proof‐of‐concept study underscores the potential of drug repurposing to rapidly advance new treatments into clinical trials for OC, offering renewed hope for patients with advanced disease.

TLR Agonist Nano Immune Therapy Clears Peritoneal and Systemic Ovarian Cancer

AbstractIntraperitoneal (IP) administration of immunogenic mesoporous silica nanoparticles (iMSN) in a mouse model of metastatic ovarian cancer promotes the development of tumor‐specific CD8+ T cells and protective immunity. IP delivery of iMSN functionalized with the Toll‐like receptor (TLR) agonists polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPLA) stimulated rapid uptake by all peritoneal myeloid subsets. Myeloid cells quickly transported iMSN to milky spots and fat‐associated lymphoid clusters (FALCs) present in tumor‐burdened adipose tissues, leading to a reduction in suppressive T cells and an increase in activated memory T cells. Two doses of iMSN cleared or reduced ovarian and colorectal cancer and protected against future tumor engraftment. In contrast, subcutaneous (SC) and intravenous (IV) delivery of iMSN were without therapeutic effect in mice with peritoneal metastases, supporting the need for activation of regional immune cells. Remarkably, intraperitoneal delivery of iMSN cleared subcutaneously implanted ovarian cancer, supporting homing of antigen specific T cells to extraperitoneal tumor sites.

Paracrine Ovarian Cancer Cell‐Derived CSF1 Signaling Regulates Macrophage Migration Dynamics in a 3D Microfluidic Model that Recapitulates In Vivo Infiltration Patterns in Patient‐Derived Xenografts

Abstract A high density of macrophages in the ovarian cancer microenvironment is associated with disease progression and poor outcomes. Understanding cancer‐macrophage interaction mechanisms that establish this pro‐tumorigenic microenvironment is critical for developing macrophage‐targeted therapies. Here, 3D microfluidic assays and patient‐derived xenografts are utilized to define the role of cancer‐derived colony stimulating factor 1 (CSF1) on macrophage infiltration dynamics toward ovarian cancer cells. It is demonstrated that multiple ovarian cancer models promote the infiltration of macrophages into a 3D extracellular matrix in vitro in a cell density‐dependent manner. Macrophages exhibit directional migration and increased migration speed under both direct interactions with cancer cells embedded within the matrix and paracrine crosstalk with cancer cells seeded in an independent microchannel. It is also found that platinum‐based chemotherapy increases macrophage recruitment and the levels of cancer cell‐derived CSF1. Targeting CSF1 signaling under baseline or chemotherapy‐treatment conditions reduces the number of infiltrated macrophages. It is further shown that results obtained with the 3D microfluidic model reflect the recruitment profiles of macrophages in patient‐derived xenografts in vivo. These findings highlight the role of CSF1 signaling in establishing macrophage‐rich ovarian cancer microenvironments, as well as the utility of microfluidic models in recapitulating 3D tumor ecosystems and dissecting cancer‐macrophage signaling.

Angiogenesis‐Enabled Human Ovarian Tumor Microenvironment‐Chip Evaluates Pathophysiology of Platelets in Microcirculation

AbstractThe tumor microenvironment (TME) promotes angiogenesis for its growth through the recruitment of multiple cells and signaling mechanisms. For example, TME actively recruits and activates platelets from the microcirculation to facilitate metastasis, but platelets may simultaneously also support tumor angiogenesis. Here, to model this complex pathophysiology within the TME that involves a signaling triad of cancer cells, sprouting endothelial cells, and platelets, an angiogenesis‐enabled tumor microenvironment chip (aTME‐Chip) is presented. This platform recapitulates the convergence of physiology of angiogenesis and platelet function within the ovarian TME and describes the contribution of platelets in promoting angiogenesis within an ovarian TME. By including three distinct human ovarian cancer cell‐types, the aTME‐Chip quantitatively reveals the following outcomes—first, introduction of platelets significantly increases angiogenesis; second, the temporal dynamics of angiogenic signaling is dependent on cancer cell type; and finally, tumor‐educated platelets either activated exogenously by cancer cells or derived clinically from a cancer patient accelerate tumor angiogenesis. Further, analysis of effluents available from aTME‐Chip validate functional outcomes by revealing changes in cytokine expression and several angiogenic and metastatic signaling pathways due to platelets. Collectively, this tumor microphysiological system may be deployed to derive antiangiogenic targets combined with antiplatelet treatments to arrest cancer metastasis.

Sonosensitizer‐Functionalized Graphene Nanoribbons for Adhesion Blocking and Sonodynamic Ablation of Ovarian Cancer Spheroids

AbstractAdvanced stage ovarian cancer is challenging to treat due to widespread seeding of tumor spheroids throughout the mesothelial lining of the peritoneal cavity. In this work, a therapeutic strategy using graphene nanoribbons (GNR) functionalized with 4‐arm polyethylene glycol (PEG) and chlorin e6 (Ce6), a sonosensitizer, to target metastatic ovarian cancer spheroids is reported. GNR‐PEG‐Ce6 adsorbs onto the spheroids and disrupts their adhesion to extracellular matrix proteins or LP‐9 mesothelial cells. Furthermore, for spheroids that do adhere, GNR‐PEG‐Ce6 delays spheroid disaggregation and spreading as well as mesothelial clearance, key metastatic processes following adhesion. Owing to the sonodynamic effects of Ce6 and its localized delivery via the biomaterial, GNR‐PEG‐Ce6 can kill ovarian cancer spheroids adhered to LP‐9 cell monolayers when combined with mild ultrasound irradiation. The interaction with GNR‐PEG‐Ce6 also loosens cell–cell adhesions within the spheroids, rendering them more susceptible to treatment with the chemotherapeutic agents cisplatin and paclitaxel, which typically have difficulty in penetrating ovarian cancer spheroids. Thus, this material can facilitate effective chemotherapeutic and sonodynamic combination therapies. Finally, the adhesion inhibiting and sonodynamic effects of GNR‐PEG‐Ce6 are also validated with tumor spheroids derived from the ascites fluid of ovarian cancer patients, providing evidence of the translational potential of this biomaterial approach.

A Photoacoustic Contrast Nanoagent with a Distinct Spectral Signature for Ovarian Cancer Management

AbstractPhotoacoustic imaging (PAI) has tremendous potential for improving ovarian cancer detection. However, the lack of effective exogenous contrast agents that can improve PAI diagnosis accuracy significantly limits this application. This study presents a novel contrast nanoagent with a specific spectral signature that can be easily distinguished from endogenous chromophores in cancer tissue, allowing for high‐contrast tumor visualization. Constructed as a 40 nm biocompatible polymeric nanoparticle loaded with two naphthalocyanine dyes, this agent is capable of efficient ovarian tumor accumulation after intravenous injection. The developed nanoagent displays a spectral signature with two well‐separated photoacoustic peaks of comparable PA intensities in the near‐infrared (NIR) region at 770 and 860 nm, which remain unaffected in cancer tissue following systemic delivery. In vivo experiments in mice with subcutaneous and intraperitoneal ovarian cancer xenografts validate that this specific spectral signature allows for accurate spectral unmixing of the nanoagent signal from endogenous contrast in cancer tissue, allowing for sensitive noninvasive cancer diagnosis. In addition, this nanoagent can selectively eradicate ovarian cancer tissue with a single dose of photothermal therapy by elevating the intratumoral temperature to ≈49 °C upon exposure to NIR light within the 700–900 nm range.

Modification‐Driven Nanocarriers: Ovarian Cancer Cell Membrane– Camouflaged Indoximod/Doxorubicin Co‐Delivery Systems for Synergistic Immunochemotherapy

ABSTRACT Among the three primary gynecological malignancies, ovarian cancer has the highest mortality rate, and its onset is often insidious. Despite standard treatments, relapse and drug resistance remain major challenges. Doxorubicin (DOX) is known to induce immunogenic cell death (ICD); however, some patients still experience tumor resistance and recurrence owing to tumor‐driven immunosuppression. Indoleamine 2,3‐dioxygenase (IDO), which is highly expressed in tumor tissues, impairs T‐cell function and differentiation, thereby promoting immunosuppression. Consequently, combining the IDO inhibitor indoximod (IND) with DOX may reverse immunosuppression and enhance both T‐cell–mediated and ICD‐driven anticancer effects. However, both drugs are limited by high systemic toxicity and poor tumor targeting, necessitating the use of nanocarriers to improve delivery efficiency and minimize toxicity. This study aims to develop novel cell membrane–camouflaged liposomes capable of co‐delivering IND and DOX (DOX/IND@cmLPs) for ovarian cancer therapy and to evaluate its anticancer effects in vitro and in vivo. The particle size of DOX/IND@cmLPs is measured as 111.7 ± 2.7 nm using a Malvern Zetasizer Pro, with a zeta potential of −22.4 ± 4.00 mV. Entrapment efficiency (EE) is assessed using ultra‐high performance liquid chromatography and ultraviolet spectrophotometry, yielding EE values of 85.1% ± 3.4% for DOX and 23.9% ± 1.3% for IND. At both pH 7.4 and pH 5.5, DOX release from DOX/IND@cmLPs is rapid during the first 24 hours, followed by a slower, more sustained release. Coomassie Brilliant Blue staining and Western Blot analysis confirmed successful encapsulation of the cell membrane in the liposomes. The potent antitumor effect of DOX/IND@cmLPs is demonstrated via CellTiter‐Glo assays in vitro. Flow cytometry and immunofluorescence staining revealed an increased ratio of CD8 + T cells to Treg cells in tumor tissues, suggesting that DOX/IND@cmLPs may partially reverse local tumor‐induced immunosuppression. Reduced Ki‐67 expression and increased TdT‐mediated dUTP nick‐end labeling positive cell ratios in tumor sections indicated that DOX/IND@cmLPs treatment suppressed tumor proliferation and promoted apoptosis. Immunohistochemistry showed alterations in mammalian target of rapamycin (mTOR)‐related pathway proteins in tumors. Furthermore, DOX/IND@cmLPs could induce an abscopal effect and provide long‐lasting tumor suppression in a subcutaneous mouse model. In this study, a formulation of DOX/IND‐loaded liposomes camouflaged with ovarian cancer cell membranes is successfully developed, and their stable physicochemical properties are confirmed. As an effective nanodrug delivery system, DOX/IND@cmLPs exhibited enhanced tumor‐targeting and immune‐mediated anticancer activity both in vitro and in vivo, indicating their potential as a platform for future combined chemotherapy and immunotherapy.

The Balance Between Shear Flow and Extracellular Matrix in Ovarian Cancer‐on‐Chip

Abstract Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor‐on‐chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors’ previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient‐derived cells and sera.

Human Omental Mature Adipocytes used as Paclitaxel Reservoir for Cell‐Based Therapy in Ovarian Cancer

AbstractPrimary human omental adipocytes and ovarian cancer(OC) cells establish a bidirectional communication in which tumor driven lipolysis is induced in adipocytes and the resulting fatty acids are delivered to cancer cells within the tumor microenvironment. Despite meaningful improvement in the treatment of OC, its efficacy is still limited by hydrophobicity and untargeted effects related to chemotherapeutics. Herein, omental adipocytes are firstly used as a reservoir for paclitaxel, named Living Paclitaxel Bullets (LPB) and secondly benefit from the established dialogue between adipocytes and cancer cells to engineer a drug delivery process that target specifically cancer cells. These results show that mature omental adipocytes can successfully uptake paclitaxel and deliver it to OC cells in a transwell coculture based in vitro model. In addition, the efficacy of this proof‐of‐concept has been demonstrated in vivo and induces a significant inhibition of tumor growth on a xenograft tumor model. The use of mature adipocytes can be suitable for clinical prospection in a cell‐based therapy system, due to their mature and differentiated state, to avoid risks related to uncontrolled cell de novo proliferation capacity after the delivery of the antineoplastic drug as observed with other cell types when employed as drug carriers.

Engineering Improved CAR T Cell Products with A Multi‐Cytokine Particle Platform for Hematologic and Solid Tumors

AbstractDespite the remarkable clinical efficacy of chimeric antigen receptor (CAR) T cells in hematological malignancies, only a subset of patients achieves a durable complete response (dCR). DCR has been correlated with CAR T cell products enriched with T cells memory phenotypes. Therefore, reagents that consistently promote memory phenotypes during the manufacturing of CAR T cells have the potential to significantly improve clinical outcomes. A novel modular multi‐cytokine particle (MCP) platform is developed that combines the signals necessary for activation, costimulation, and cytokine support into a single “all‐in‐one” stimulation reagent for CAR T cell manufacturing. This platform allows for the assembly and screening of compositionally diverse MCP libraries to identify formulations tailored to promote specific phenotypes with a high degree of flexibility. The approach is leveraged to identify unique MCP formulations that manufacture CAR T cell products from diffuse large B cell patients   with increased proportions of memory‐like phenotypes MCP‐manufactured CAR T cells demonstrate superior anti‐tumor efficacy in mouse models of lymphoma and ovarian cancer through enhanced persistence. These findings serve as a proof‐of‐principle of the powerful utility of the MCP platform to identify “all‐in‐one” stimulation reagents that can improve the effectiveness of cell therapy products through optimal manufacturing.

Isolation Defines Identity: Functional Consequences of Extracellular Vesicle Purification Strategies

ABSTRACT The biological activity of extracellular vesicles (EVs) is largely defined by their molecular cargo, yet the impact of isolation workflows on EV proteomes and function remains incompletely understood. Here, we compared four isolation strategies for EVs derived from malignant ascites and ES‐2 ovarian cancer cell culture supernatants, assessing yield, particle size, protein cargo, and EV‐associated enzymatic activity. Proteomic analyses of particle‐normalized preparations were performed according to MISEV2023 guidelines, and vesicle‐associated protease activity was profiled using a FRET‐based assay with inhibitor panels. Principal component and overlap analyses identified a common EV proteome signature for ascites and ES‐2 EVs, which was complemented by workflow‐dependent detection of additional proteins. Ultracentrifugation/density gradient (UC‐DG) and tangential flow filtration/size exclusion chromatography (TFF‐SEC) achieved the highest enrichment of canonical EV markers, whereas TFF/ultrafiltration (TFF‐UF) was enriched in lipoproteins and secreted proteins. Functionally, UC‐DG and TFF‐SEC samples exhibited strong ADAM10‐associated activity, while TFF‐UF retained residual non‐metalloprotease activity. These results reveal to what extent EV purification methods impact both, EV composition and function. This methodological awareness is critical for advancing EV‐based biomarker discovery, diagnostics, and therapeutic platforms.

In Situ Formed Nanocomposite Hydrogel Improve Local Delivery of Antiangiogenic Agents and Immune Checkpoint Inhibitor for Cervical Carcinoma Therapy

Abstract Immune checkpoint blockade targeting the programmed cell death protein‐1 (PD‐1)/ligand (PD‐L1) axis has emerged as a promising therapeutic strategy for cervical carcinoma. However, its clinical application remains limited by the immunosuppressive tumor microenvironment (TME) and poor targeting efficiency, particularly in solid tumors. To address these challenges, a nanocomposite hydrogel system (Apa/BPNPs@Gel) is developed by encapsulating PD‐L1 inhibitor BMS202 nanoparticles coated with polyvinyl alcohol (BPNPs) into a polyvinyl alcohol/alginate hybrid hydrogel. This in situ formed hydrogel exhibits favorable biocompatibility and reactive oxygen species‐dependent sequential drug release. Initially, the antiangiogenic agent apatinib (Apa) is released to alleviate tumor hypoxia through vascular normalization and enhance PD‐L1 suppression, priming the TME for subsequent anti‐PD(L)1 therapy. The hydrogel framework extends the residence time of BMS202 (a skeleton component), improving therapeutic efficacy. Notably, in preclinical cervical carcinoma models, Apa/BPNPs@Gel mediated combination therapy significantly inhibited tumor growth and prolonged survival by activating tumor‐suppressed CD8+ T cells. Hence, this locally administrable hydrogel offers a versatile platform to modulate the immunosuppressive TME and enhance immunotherapeutic outcomes.

Phototheranostic LPP‐QDs‐IR‐820 Nanocomposites for Specific NIR‐II Imaging of Lymphatic and Photothermal Therapy of Cervical Tumors

AbstractPrecise theranostics of tumors is intricately linked to the early detection and monitoring of lymph nodes (LN) and metastases, making the targeted localization of LNs essential for tumor identification. However, designing LN‐targeting probes remains a significant challenge due to issues such as lymphatic uptake, biocompatibility, and fluorescence stability. To address these challenges, near‐infrared II (NIR‐II) fluorescence probes are developed through meticulous analysis of LN physiological structure and passive targeting strategy for LN detection and tumor therapy. An LPP‐QDs‐IR‐820 nanocomposite (NCs) is engineered, comprising the IR‐820 molecules and ultrabright PbS@CdS quantum dots (QDs), which are encapsulated within a liposome‐SH‐mPEG2000 polymer matrix. These NCs demonstrates remarkable lymphatic enrichment, facilitating real‐time tracking of LN via electrostatic repulsion and extracellular matrix effects. Importantly, the NCs exhibit negligible in vivo toxicity and high biocompatibility. The intense NIR‐II fluorescence emissions of IR‐820 and PbS@CdS QDs confer upon the NCs a high NIR‐II fluorescence quantum yield (6%). The cervical tumors and their deep microvessels are clearly observed via NIR‐II fluorescence imaging. Moreover, the photothermal properties of IR‐820 enable the NCs to achieve a photothermal conversion efficiency of 36.56%, leading to effective photothermal therapy in cervical tumor mice.

3D Microtumors Representing Ovarian Cancer Minimal Residual Disease Respond to the Fatty Acid Oxidation Inhibitor Perhexiline

AbstractThe poor survival of ovarian cancer patients is linked to their high likelihood of relapse. In spite of full apparent macroscopic clearance, tumor recurrences arise from cells that are resistant to primary chemotherapy in the form of minimal residual disease (MRD). MRD exhibits distinct molecular drivers from bulk cancer and therefore necessitates alternative therapeutic strategies. However, there is a lack of 3D models that faithfully recapitulate MRD ex vivo for therapy development. This study constructs microfluidics‐based 3D microtumors to generate a clinically‐relevant model for ovarian cancer MRD. The microtumors recapitulate the non‐genetic heterogeneity of ovarian cancer, capturing the “Oxford Classic” five molecular signatures. Gene expression in the 3D microtumors aligns closely with MRD from ovarian cancer patients and features the upregulation of fatty acid metabolism genes. Finally, the MRD 3D microtumors respond to the approved fatty acid oxidation inhibitor, perhexiline, demonstrating their utility in drug discovery. This system might be used as a drug‐testing platform for the discovery of novel MRD‐specific therapies in ovarian cancer.