Targeting lung cancer stem cells using combination of Tel and Docetaxel liposomes in 3D cultures and tumor xenografts.
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ABSTRACT:
SUBMITTER: Arthur P
PROVIDER: S-EPMC7437978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Therapeutic efficacy of nanocarriers can be amplified by active targeting and overcoming the extracellular matrix associated barriers of tumors. The aim of the present study was to investigate the effect of oral antifibrotic agent (telmisartan) on tumor uptake and anticancer efficacy of EphA2 receptor targeted liposomes. Docetaxel loaded PEGylated liposomes (DPL) functionalized with nickel chelated phospholipid were prepared using a modified hydration method. DPL were incubated with various concentrations of histidine tagged EphA2 receptor specific peptide (YSA) to optimize particle size, zeta potential, and percentage YSA binding. Cellular uptake studies using various endocytosis blockers revealed that a caveolae dependent pathway was the major route for internalization of YSA anchored liposomes of docetaxel (YDPL) in A549 lung cancer cell line. Hydrodynamic diameter and zeta potential of optimized YDPL were 157.3 ± 11.8 nm and -3.64 mV, respectively. Orthotopic lung tumor xenograft (A549) bearing athymic nude mice treated with oral telmisartan (5 mg/kg) for 2 days showed significantly (p < 0.05) higher uptake of YDPL in tumor tissues compared to healthy tissue. Average lung tumor weight of the YDPL + telmisartan treated group was 4.8- and 3.8-fold lower than that of the DPL and YDPL treated groups (p < 0.05). Substantially lower expression (p < 0.05) of EphA2 receptor protein, proliferating cell nuclear antigen (PCNA), MMP-9, and collagen 1A level with increased E-cadherin and TIMP-1 levels in immunohistochemistry and Western blot analysis of lung tumor samples of the combination group confirmed antifibrotic effect with enhanced anticancer activity. Active targeting and ECM remodeling synergistically contributed to anticancer efficacy of YDPL in orthotopic lung cancer.
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Project description:Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.
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Project description:Allergic reactions and severe systemic toxicity are two major challenges for the clinical application of docetaxel (DTX) for treatment of non-small-cell lung cancer (NSCLC). We developed a novel lung-targeted DTX-loaded liposome (DTX-LP), an efficient drug delivery system, with a patented DBaumNC technology to overcome these deficiencies. In the present study, we describe the targeting activity, tumor inhibition rate (TIR), survival, pathology, tumor apoptosis and metabolism of DTX after intravenous injection of DTX-LP compared to the DTX injection (DTX-IN) formulation based on the VX2 orthotopic lung cancer rabbit model. Biodistribution studies revealed the highest accumulation in lung and tumor within 12 h after the injection of DTX-LP. The increased TIR indicates that the growth of tumor was slowed. Pathology tests demonstrated that DTX-LP can reduce metastasis and toxicity to non-targeted organs, leading to greatly extended survival time and improved survival of tumor-bearing rabbits. Flow cytometry and immunohistochemistry confirmed that DTX-LP is highly efficacious in tumor tissue, leading to a significant increase of tumor apoptosis and decrease of proliferation and angiogenesis. The results from this study demonstrate the increased intrapulmonary tumor targeting activity, enhanced antitumor effect and reduced toxicity of DTX-LP compared to DTX-IN and highlight its clinical prospects for NSCLC therapy.
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Project description:Docetaxel is a commonly used chemotherapeutic drug for patients with late stage prostate cancer. However, serious side effect and drug resistance limit its clinical success. Brefeldin A is a 16-membered macrolide antibiotic from mangrove-derived Fungus Aspergillus sp. (9Hu), which exhibited potent cytotoxicity against human cancer cells. In the present study, we determined the effect of brefeldin A on docetaxel-induced growth inhibition and apoptosis in human prostate cancer PC-3 cells. Brefeldin A in combination with docetaxel inhibited the growth of PC-3 cells in monolayer and in three dimensional cultures. The combination also potently stimulated apoptosis in PC-3 cells as determined by propidium iodide staining and morphological assessment. Mechanistic studies showed that growth inhibition and apoptosis in PC-3 cells treated with brefeldin A and docetaxel were associated with decrease in the level of Bcl-2. The present study indicates that combined brefeldin A with docetaxel may represent a novel approach for improving the efficacy of docetaxel, and Bcl-2 may serve as a target for brefeldin A to enhance the effects of docetaxel chemotherapy.
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Project description:In this work, we evaluated the ability of 3D co-cultures with mesenchymal stem cells (MSCs) to redifferentiate monolayer expanded articular chondrocytes (ACs) and produce cartilaginous extracellular matrix at varying stages of the dedifferentiation process and further examined the dependency of this effect on the culture medium composition. Primary bovine ACs were expanded in monolayers for up to nine population doublings to obtain seven cell stocks with gradually increasing levels of dedifferentiation. Culture expanded ACs were then seeded as monocultures and co-cultures with rabbit bone marrow-derived MSCs (30:70 ratio of ACs-to-MSCs) on porous scaffolds. Parallel cultures were established for each cell population in serum-containing growth medium and serum-free induction medium supplemented with dexamethasone and TGF-β3. After 3 weeks, all groups were analyzed for DNA content, glycosaminoglycan (GAG) and hydroxyproline (HYP) production, and chondrogenic gene expression. Significant enhancements in cellularity, GAG content and GAG/HYP ratio, and chondrogenic phenotype were observed in the induction medium compared to growth medium at all levels of AC expansion. Furthermore, primary co-cultures showed similarly enhanced chondrogenesis compared to monocultures in both culture media, whereas passaged ACs benefitted from co-culturing only in the induction medium. We conclude that co-cultures of ACs and MSCs can produce superior in vitro engineered cartilage in comparison to pure AC cultures, due to both heterotypic cellular interactions and decreased need for monolayer expansion of biopsied chondrocytes. While the initial level of AC dedifferentiation affected the quality of the engineered constructs, co-culture benefits were realized at all stages of AC expansion when suitable chondroinductive culture medium was used.
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Project description:The integration of microfluidics and cell biology has reached a significant milestone with the development of "organ-on-chips", smart technological platforms that, once applied to the study of human diseases, such as cancer, might ultimately contribute to design personalised treatments and hence improve health outcomes. This paper reports that the combination of microfluidics and dielectrophoresis (DEP) allows to culture different pancreatic ductal adenocarcinoma (PDAC) human cell lines into a cyclic olefin polymer (COP) chamber (HepaChip®), enriched by the extracellular matrix (ECM) protein collagen. We show that PDAC cells cultured into the HepaChip® (1) are vital and grow, provided they properly attach to collagen; (2) show morphological appearance and growth characteristics closer to those of cells grown as spheroids than as classical 2 dimensional (2D) in vitro cultures. Finally, preliminary experiments show that PDAC cells respond to high doses of Cisplatin perfused through the chip. Overall, the present microfluidic platform could be exploited in the future for a personalised approach to PDAC.
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Project description:The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young's modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-? and TNF-? with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-? and TNF-?. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.
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