Multicompartmentalized microvascularized tumor-on-a-chip to study tumor-stroma interactions and drug resistance in ovarian cancer.

Introduction: The majority of ovarian cancer (OC) patients receiving standard of care chemotherapy develop chemoresistance within 5 years. The tumor microenvironment (TME) is a dynamic and influential player in disease progression and therapeutic response. However, there is a lack of models that allow us to elucidate the compartmentalized nature of TME in a controllable, yet physiologically relevant manner and its critical role in modulating drug resistance. Methods: We developed a 3D microvascularized multiniche tumor-on-a-chip formed by five chambers (central cancer chamber, flanked by two lateral stromal chambers and two external circulation chambers) to recapitulate OC-TME compartmentalization and study its influence on drug resistance. Stromal chambers included endothelial cells alone or cocultured with normal fibroblasts or cancer-associated fibroblasts (CAF). Results: The tumor-on-a-chip recapitulated spatial TME compartmentalization including vessel-like structure, stromal-mediated extracellular matrix (ECM) remodeling, generation of oxygen gradients, and delayed drug diffusion/penetration from the circulation chamber towards the cancer chamber. The cancer chamber mimicked metastasis-like migration and increased drug resistance to carboplatin/paclitaxel treatment in the presence of CAF when compared to normal fibroblasts. CAF-mediated drug resistance was rescued by ECM targeted therapy. Critically, these results demonstrate that cellular crosstalk recreation and spatial organization through compartmentalization are essential to determining the effect of the compartmentalized OC-TME on drug resistance. Conclusions: Our results present a functionally characterized microvascularized multiniche tumor-on-a-chip able to recapitulate TME compartmentalization influencing drug resistance. This technology holds the potential to guide the design of more effective and targeted therapeutic strategies to overcome chemoresistance in OC.

Reprogramming of normal fibroblasts into ovarian cancer-associated fibroblasts via non-vesicular paracrine signaling induces an activated fibroblast phenotype.

Cancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present that conditioned media from ovarian cancer lines leads to an increase in the activated state of fibroblasts demonstrated by functional assays and up-regulation of known CAF-related genes and ECM pathways. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression. Critically, the conditioned CAFs resemble a transcriptional signature with involvement of ECM remodeling. The present study provides mechanistic and functional insights about the activation and reprogramming of CAFs in the ovarian tumor microenvironment mediated by non-vesicular paracrine signaling. Moreover, it provides a translational based approach to reprogram normal fibroblasts from both uterine and ovarian origin into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF/ECM-mediated chemoresistance in OC are further warranted.

Normal Uterine Fibroblast Are Reprogramed into Ovarian Cancer-Associated Fibroblasts by Ovarian Tumor-derived Conditioned Media.

Cancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present a translatable-based approach for the reprogramming of normal uterine fibroblasts into ovarian CAFs using ovarian tumor-derived conditioned media to establish two well-characterized ovarian conditioned CAF lines. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression and resembled a CAF-like subtype associated with worse prognosis. The present study provides a reproducible, cost-effective, and clinically relevant protocol to reprogram normal fibroblasts into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF-mediated chemoresistance in OC are further warranted.

Reduction of Metastasis via Epigenetic Modulation in a Murine Model of Metastatic Triple Negative Breast Cancer (TNBC).

This study investigates the effects of a dual selective Class I histone deacetylase (HDAC)/lysine-specific histone demethylase 1A (LSD1) inhibitor known as 4SC-202 (Domatinostat) on tumor growth and metastasis in a highly metastatic murine model of Triple Negative Breast Cancer (TNBC). 4SC-202 is cytotoxic and cytostatic to the TNBC murine cell line 4T1 and the human TNBC cell line MDA-MB-231; the drug does not kill the normal breast epithelial cell line MCF10A. Furthermore, 4SC-202 reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model, which closely mimics human TNBC in terms of sites of metastasis, reveal reduced tumor burden and lung metastasis. The mechanism of action of 4SC-202 may involve effects on cancer stem cells (CSC) which can self-renew and form metastatic lesions. Approximately 5% of the total 4T1 cell population grown in three-dimensional scaffolds had a distinct CD44high/CD24low CSC profile which decreased after treatment. Bulk transcriptome (RNA) sequencing analyses of 4T1 tumors reveal changes in metastasis-related pathways in 4SC-202-treated tumors, including changes to expression levels of genes implicated in cell migration and cell motility. In summary, 4SC-202 treatment of tumors from a highly metastatic murine model of TNBC reduces metastasis and warrants further preclinical studies.

Single chain variable fragment fused to maltose binding protein: a modular nanocarrier platform for the targeted delivery of antitumorals.

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular ß-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element.

Poly(ethylene-imine)-Functionalized Magnetite Nanoparticles Derivatized with Folic Acid: Heating and Targeting Properties.

Magnetite nanoparticles (MNPs) coated by branched poly (ethylene-imine) (PEI) were synthesized in a one-pot. Three molecular weights of PEI were tested, namely, 1.8 kDa (sample MNP-1), 10 kDa (sample MNP-2), and 25 kDa (sample MNP-3). The MNP-1 particles were further functionalized with folic acid (FA) (sample MNP-4). The four types of particles were found to behave magnetically as superparamagnetic, with MNP-1 showing the highest magnetization saturation. The particles were evaluated as possible hyperthermia agents by subjecting them to magnetic fields of 12 kA/m strength and frequencies ranging between 115 and 175 kHz. MNP-1 released the maximum heating power, reaching 330 W/g at the highest frequency, in the high side of reported values for spherical MNPs. In vitro cell viability assays of MNP-1 and MNP-4 against three cell lines expressing different levels of FA receptors (FR), namely, HEK (low expression), and HeLa (high expression), and HepG2 (high expression), demonstrated that they are not cytotoxic. When the cells were incubated in the presence of a 175 kHz magnetic field, a significant reduction in cell viability and clone formation was obtained for the high expressing FR cells incubated with MNP-4, suggesting that MNP-4 particles are good candidates for magnetic field hyperthermia and active targeting.

Polyethylenimine-Bisphosphonate-Cyclodextrin Ternary Conjugates: Supramolecular Systems for the Delivery of Antineoplastic Drugs.

Bisphosphonates (BPs) are bone-binding molecules that provide targeting capabilities to bone cancer cells when conjugated with drug-carrying polymers. This work reports the design, synthesis, and biological evaluation of polyethyleneimine-BP-cyclodextrin (PEI-BP-CD) ternary conjugates with supramolecular capabilities for the loading of antineoplastic drugs. A straightforward, modular, and versatile strategy based on the click aza-Michael addition reaction of vinyl sulfones (VSs) allows the grafting of BPs targeting ligands and ßCD carrier appendages to the PEI polymeric scaffold. The in vitro evaluation (cytotoxicity, cellular uptake, internalization routes, and subcellular distribution) for the ternary conjugates and their doxorubicin inclusion complexes in different bone-related cancer cell lines (MC3T3-E1 osteoblasts, MG-63 sarcoma cells, and MDA-MB-231 breast cancer cells) confirmed specificity, mitochondrial targeting, and overall capability to mediate a targeted drug transport to those cells. The in vivo evaluation using xenografts of MG-63 and MDA-MB-231 cells on mice also confirmed the targeting of the conjugates.

Human Plasma-Derived 3D Cultures Model Breast Cancer Treatment Responses and Predict Clinically Effective Drug Treatment Concentrations.

Lack of efficacy and a low overall success rate of phase I-II clinical trials are the most common failures when it comes to advancing cancer treatment. Current drug sensitivity screenings present several challenges including differences in cell growth rates, the inconsistent use of drug metrics, and the lack of translatability. Here, we present a patient-derived 3D culture model to overcome these limitations in breast cancer (BCa). The human plasma-derived 3D culture model (HuP3D) utilizes patient plasma as the matrix, where BCa cell lines and primary BCa biopsies were grown and screened for drug treatments. Several drug metrics were evaluated from relative cell count and growth rate curves. Correlations between HuP3D metrics, established preclinical models, and clinical effective concentrations in patients were determined. HuP3D efficiently supported the growth and expansion of BCa cell lines and primary breast cancer tumors as both organoids and single cells. Significant and strong correlations between clinical effective concentrations in patients were found for eight out of ten metrics for HuP3D, while a very poor positive correlation and a moderate correlation was found for 2D models and other 3D models, respectively. HuP3D is a feasible and efficacious platform for supporting the growth and expansion of BCa, allowing high-throughput drug screening and predicting clinically effective therapies better than current preclinical models.

Acid anhydride coated carbon nanodots: activated platforms for engineering clicked (bio)nanoconstructs.

Activated carbon nanodots functionalized with acid anhydride groups (AA-CNDs) are prepared by one-pot water-free green thermolysis of citric acid. As a proof of concept of their capabilities as appealing and versatile platforms for accessing engineering nanoconstructs, the as-prepared AA-CNDs have been reacted to yield clickable CNDs. Their click bioconjugation with relevant recognizable complementary clickable sugars has led to multivalent CND-based glyconanoparticles that are non-toxic and biorecognizable. The accessibility and intrinsic reactivity of AA-CNDs expand the current toolbox of covalent surface grafting methodologies and provide a wide range of potential applications for engineering (bio)nanoconstructs.