skip to main content

Title: Fluid Shear Stress Induces Drug Resistance to Doxorubicin and Paclitaxel in the Breast Cancer Cell Line MCF7

Circulating tumor cells (CTCs) are known to have cancer stem cell (CSC) properties and survive physiological conditions of fluid shear stress (FSS). However, current chemotherapy screening techniques do not adequately recapitulate this FSS environment and are not predictive of a drug response. In this study, MCF7 and MDA‐MB‐231 cells under FSS are used as an in vitro model of CTCs. The effects of doxorubicin (DOX) and paclitaxel on sheared cells using WST8 assay and stemness (CD44+/CD24) and apoptosis (Annexin V+/7‐AAD+) using flow cytometry are tested. Quantitative polymerase chain reaction is used to test gene expression. It is shown that suspension‐cultured and FSS treated MCF7 cells increase in drug resistance, especially with DOX. There is a synergistic increase in the CD44+/CD24CSC‐like population and an increase in drug resistance‐related gene expression in MCF7 cells co‐treated with FSS and drugs. There is also a correlated increase in STAT3 and NANOG expression under FSS. To the best of the authors' knowledge, this is the first report to suggest that the increase in CSC‐like cells from FSS contributes to drug resistance via the STAT3/NANOG pathway. This increase in CTC drug resistance also highlights the importance of implementing FSS, which is unavailable in current drug screening techniques.

more » « less
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Advanced Therapeutics
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract There is a need for new in vitro systems that enable pharmaceutical companies to collect more physiologically-relevant information on drug response in a low-cost and high-throughput manner. For this purpose, three-dimensional (3D) spheroidal models have been established as more effective than two-dimensional models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in shape, size, and aggregate forming tendencies. In this study, we overcome these challenges by coupling tissue engineering toolsets with microfluidics technologies to create engineered cancer microspheres. Specifically, we employ biosynthetic hydrogels composed of conjugated poly(ethylene glycol) (PEG) and fibrinogen protein (PEG-Fb) to create engineered breast and colorectal cancer tissue microspheres for 3D culture, tumorigenic characterization, and examination of potential for high-throughput screening (HTS). MCF7 and MDA-MB-231 cell lines were used to create breast cancer microspheres and the HT29 cell line and cells from a stage II patient-derived xenograft (PDX) were encapsulated to produce colorectal cancer (CRC) microspheres. Using our previously developed microfluidic system, highly uniform cancer microspheres (intra-batch coefficient of variation (CV) ≤ 5%, inter-batch CV < 2%) with high cell densities (>20×106 cells/ml) were produced rapidly, which is critical for use in drug testing. Encapsulated cells maintained high viability and displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. For PDX CRC microspheres, the percentage of human (70%) and CRC (30%) cells was maintained over time and similar to the original PDX tumor, and the mechanical stiffness also exhibited a similar order of magnitude (103 Pa) to the original tumor. The cancer microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with staurosporine (1 μM) and doxorubicin (10 μM). Expected responses were quantified using CellTiter-Glo® 3D, demonstrating initial applicability to HTS drug discovery. PDX CRC microspheres were treated with Fluorouracil (5FU) (10 to 500 μM) and displayed a decreasing trend in metabolic activity with increasing drug concentration. Providing a more physiologically relevant tumor microenvironment in a high-throughput and low-cost manner, the PF hydrogel-based cancer microspheres could potentially improve the translational success of drug candidates by providing more accurate in vitro prediction of in vivo drug efficacy. Citation Format: Elizabeth A. Lipke, Wen J. Seeto, Yuan Tian, Mohammadjafar Hashemi, Iman Hassani, Benjamin Anbiah, Nicole L. Habbit, Michael W. Greene, Dmitriy Minond, Shantanu Pradhan. Production of cancer tissue-engineered microspheres for high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 175. 
    more » « less
  2. Abstract

    Temozolomide (TMZ) is the standard‐of‐care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug‐resistance mediated by a DNA repair protein, O6‐methylguanine‐DNA methyltransferase (MGMT), which eliminates the TMZ‐induced DNA lesions. Here, the development of an iron oxide nanoparticle (NP) system for targeted delivery of small interfering RNAs to suppress the TMZ‐resistance gene (MGMT) is reported. The NPs are able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially passaged patient‐derived xenografts. The treatment with sequential administration of this NP and TMZ result in increased apoptosis of GBM stem‐like cells, reduced tumor growth, and significantly prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.

    more » « less
  3. Inhibition of overexpressed enzymes is among the most promising approaches for targeted cancer treatment. However, many cancer-expressed enzymes are “nonlethal,” in that the inhibition of the enzymes’ activity is insufficient to kill cancer cells. Conventional antibody-based therapeutics can mediate efficient treatment by targeting extracellular nonlethal targets but can hardly target intracellular enzymes. Herein, we report a cancer targeting and treatment strategy to utilize intracellular nonlethal enzymes through a combination of selective cancer stem-like cell (CSC) labeling and Click chemistry-mediated drug delivery. A de novo designed compound, AAMCHO [N-(3,4,6-triacetyl- N-azidoacetylmannosamine)-cis-2-ethyl-3-formylacrylamideglycoside], selectively labeled cancer CSCs in vitro and in vivo through enzymatic oxidation by intracellular aldehyde dehydrogenase 1A1. Notably, azide labeling is more efficient in identifying tumorigenic cell populations than endogenous markers such as CD44. A dibenzocyclooctyne (DBCO)-toxin conjugate, DBCO-MMAE (Monomethylauristatin E), could next target the labeled CSCs in vivo via bioorthogonal Click reaction to achieve excellent anticancer efficacy against a series of tumor models, including orthotopic xenograft, drug-resistant tumor, and lung metastasis with low toxicity. A 5/7 complete remission was observed after single-cycle treatment of an advanced triple-negative breast cancer xenograft (~500 mm3).

    more » « less
  4. Abstract

    The controlled release of drugs using nanoparticle‐based delivery vehicles is a promising strategy to improve the safety and efficacy of chemotherapy. A simple, scalable, and reproducible strategy is developed to synthesize a drug delivery system (DDS) by loading 6‐maleimidocaproyl‐hydrazone doxorubicin (DOX‐EMCH) into the empty core of virus‐like particles (VLPs) derived from Physalis mottle virus (PhMV) via a combination of chemical conjugation to cysteine residues and π–π stacking interactions with the anchored doxorubicin molecule. The DOX‐EMCH prodrug features an acid‐sensitive hydrazine linker that triggers the release of doxorubicin in the slightly acidic extracellular tumor microenvironment or acidic endosomal or lysosomal compartments following cellular uptake. The VLP external surface is coated with polyethylene glycol (PEG) to prevent non‐specific uptake and improve biocompatibility. The DOX‐PhMV‐PEG particles are stable in vitro and show greater efficacy in vivo compared to free doxorubicin in a breast tumor mouse model (using MDA‐MB‐231 cells and nude mice): 92% of the tumor‐bearing mice treated with DOX‐PhMV‐PEG are completely cured compared to 27% of those treated with free doxorubicin under the same conditions, representing a 3.4‐fold improvement. These results lay a foundation for the further development of this biological drug delivery system for a new generation of chemotherapy products.

    more » « less
  5. Abstract

    Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.

    more » « less