In vitro microtumors provide a physiologically predictive tool for breast cancer therapeutic screening.
ABSTRACT: Many anti-cancer drugs fail in human trials despite showing efficacy in preclinical models. It is clear that the in vitro assays involving 2D monoculture do not reflect the complex extracellular matrix, chemical, and cellular microenvironment of the tumor tissue, and this may explain the failure of 2D models to predict clinical efficacy. We first optimized an in vitro microtumor model using a tumor-aligned ECM, a tumor-aligned medium, MCF-7 and MDA-MB-231 breast cancer spheroids, human umbilical vein endothelial cells, and human stromal cells to recapitulate the tissue architecture, chemical environment, and cellular organization of a growing and invading tumor. We assayed the microtumor for cell proliferation and invasion in a tumor-aligned extracellular matrix, exhibiting collagen deposition, acidity, glucose deprivation, and hypoxia. We found maximal proliferation and invasion when the multicellular spheroids were cultured in a tumor-aligned medium, having low pH and low glucose, with 10% fetal bovine serum under hypoxic conditions. In a 7-day assay, varying doses of fluorouracil or paclitaxel had differential effects on proliferation for MCF-7 and MDA-MB-231 tumor spheroids in microtumor compared to 2D and 3D monoculture. The microtumors exhibited a tumor morphology and drug response similar to published xenograft data, thus demonstrating a more physiologically predictive in vitro model.
Project description:The aim of our study was to evaluate the influence of low-intensity pulsed US on the delivery of doxorubicin (DOX) into MDA-MB-231 triple-negative breast cancer and A549 non-small cell lung cancer cell 2D and 3D cultures. US with pulse repetition frequency of 10 Hz and 1 MHz center frequency was generated with peak negative pressure of 0.5 MPa and 50% duty cycle. SonoVue microbubbles were used. Spheroids were formed using 3D Bioprinting method. DOX delivery in 2D and 3D cultures was assessed using fluorescence microscopy. US without the addition of microbubbles did not enhance the penetration of DOX into monolayer-cultured cells and tumor spheroids. In the presence of microbubbles US improved the delivery of DOX into the edge end middle zones of A549 and MDA-MB-231 spheroids. Application of low-intensity pulsed US in combination with microbubbles may be a promising approach to enhance the delivery of DOX into tumor spheroids.
Project description:Cepharanthine (CEP) is a bis-bynzelisoquinoline alkaloid from the same class as the anticancer agent tetrandrine (TET). However, the effects of CEP against breast cancer have not been extensively studied, despite its long therapeutic history with low toxicity against other types of cancer. 3D culture systems more accurately mimic the human body and address the limitations of determining drug effectiveness compared with 2D culture systems. In the present study, the antitumor activities of TET and CEP were compared in 3D culture systems in triple-negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor-positive breast cancer MCF-7 cell lines. Cell viability, apoptosis and cytotoxicity assays were performed to determine the total number of live or dead cells, the IC50 values, the number of apoptotic cells and spheroid roundness. Viability suppression of MDA-MB-231 cells was significantly greater with both TET and CEP compared with that of MCF-7 cells, and the roundness of MDA-MB-231 spheroids treated with CEP was decreased significantly compared with that of spheroid treated with TET. Cytoplasmic shrinkage in each cell line significantly increased with the treatment of TET compared with the control; however, this effect was stronger with CEP. The ratio of dead/live cells in each cell line treated with TET and CEP increased in a dose-dependent manner. Overall, the present study demonstrated that CEP had greater cell toxicity in 3D spheroids of breast cancer cells compared with TET, suggesting that CEP may have a stronger antitumor activity on TNBC spheroids compared with TET.
Project description:Traditionally, two-dimensional (2D) monolayer cell culture models have been used to study in vitro conditions for their ease of use, simplicity and low cost. However, recently, three-dimensional (3D) cell culture models have been heavily investigated as they provide better physiological relevance for studying various disease behaviors, cellular activity and pharmaceutical interactions. Typically, small-sized tumor spheroid models (100-500 ?m) are used to study various biological and physicochemical activities. Larger, millimetric spheroid models are becoming more desirable for simulating native tumor microenvironments (TMEs). Here, we assess the use of ultra-large spheroid models (~2000 ?m) generated from scaffolds made from a nozzle-free, ultra-high resolution printer; these models are explored for assessing chemotherapeutic responses with molecular doxorubicin (DOX) and two analogues of Doxil? (Dox-NP?, DoxovesTM) on MDA-MB-231 and MCF-7 breast cancer cell lines. To provide a comparative baseline, small spheroid models (~500 ?m) were developed using a self-aggregation method of MCF-7 breast cancer cell lines, and underwent similar drug treatments. Analysis of both large and small MCF-7 spheroids revealed that Dox-NP tends to have the highest level of inhibition, followed by molecular doxorubicin and then Doxoves. The experimental advantages and drawbacks of using these types of ultra-large spheroids for cancer research are discussed.
Project description:The 3-dimensional (3D) microenvironment of breast carcinomas is characterized by profoundly altered pH homeostasis, reflecting increased metabolic acid production and a confined extracellular space characterized by poor diffusion, yet the relative contributions of specific pH-regulatory transporters to 3D growth are poorly understood. The aim of this work was to determine how 3D spheroid growth of breast cancer cells impacts the expression and spatial organization of major acid extruding proteins, and how these proteins in turn are required for spheroid growth.MCF-7 (Luminal-A) and MDA-MB-231 (Triple-negative) human breast cancer cells were grown as ~700-950 ?m diameter spheroids, which were subjected to Western blotting for relevant transporters (2- and 3D growth), quantitative immunohistochemical analysis, and spheroid growth assays. Individual transporter contributions were assessed (i) pharmacologically, (ii) by stable shRNA- and transient siRNA-mediated knockdown, and (iii) by CRISPR/Cas9 knockout.In MCF-7 spheroids, expression of the lactate-H(+) cotransporter MCT1 (SLC16A1) increased from the spheroid periphery to its core, the Na(+),HCO3 (-) cotransporter NBCn1 (SLC4A7) was most highly expressed at the periphery, and the Na(+)/H(+) exchanger NHE1 (SLC9A1) and MCT4 (SLC16A3) were evenly distributed. A similar pattern was seen in MDA-MB-231 spheroids, except that these cells do not express MCT1. The relative total expression of NBCn1 and NHE1 was decreased in 3D compared to 2D, while that of MCT1 and MCT4 was unaltered. Inhibition of MCT1 (AR-C155858) attenuated MCF-7 spheroid growth and this was exacerbated by addition of S0859, an inhibitor of Na(+),HCO3 (-) cotransporters and MCTs. The pharmacological data was recapitulated by stable knockdown of MCT1 or NBCn1, whereas knockdown of MCT4 had no effect. CRISPR/Cas9 knockout of NHE1, but neither partial NHE1 knockdown nor the NHE1 inhibitor cariporide, inhibited MCF-7 spheroid growth. In contrast, growth of MDA-MB-231 spheroids was inhibited by stable or transient NHE1 knockdown and by NHE1 knockout, but not by knockdown of NBCn1 or MCT4.This work demonstrates the distinct expression and localization patterns of four major acid-extruding transporters in 3D spheroids of human breast cancer cells and reveals that 3D growth is dependent on these transporters in a cell type-dependent manner, with potentially important implications for breast cancer therapy.
Project description:Pseudopterosin, produced by the sea whip of the genus Antillogorgia, possesses a variety of promising biological activities, including potent anti-inflammatory effects. However, few studies examined pseudopterosin in the treatment of cancer cells and, to our knowledge, the ability to inhibit triple-negative breast cancer (TNBC) proliferation or invasion has not been explored. Thus, we evaluated the as-yet unknown mechanism of action of pseudopterosin: Pseudopterosin was able to inhibit proliferation of TNBC. Interestingly, analyzing breast cancer cell proliferation after knocking down glucocorticoid receptor ? (GR?) revealed that the antiproliferative effects of pseudopterosin were significantly inhibited when GR? expression was reduced. Furthermore, pseudopterosin inhibited the invasion of MDA-MB-231 3D tumor spheroids embedded in an extracellular-like matrix. Remarkably, the knockdown of GR? in 3D tumor spheroids revealed increased ability of cells to invade the surrounding matrix. In a coculture, encompassing peripheral blood mononuclear cells (PBMC) and MDA-MB-231 cells, and the production of interleukin 6 (IL-6) and interleukin 8 (IL-8) significantly increased compared to a monoculture. Notably, pseudopterosin indicated to block cytokine elevation, representing key players in tumor progression in the coculture. Thus, our results reveal pseudopterosin treatment as a potential novel approach in TNBC therapy.
Project description:BACKGROUND: Aggressive metastatic breast cancer cells seemingly evade surgical resection and current therapies, leading to colonization in distant organs and tissues and poor patient prognosis. Therefore, high-throughput in vitro tools allowing rapid, accurate, and novel anti-metastatic drug screening are grossly overdue. Conversely, aligned nanofiber constitutes a prominent component of the late-stage breast tumor margin extracellular matrix. This parallel suggests that the use of a synthetic ECM in the form of a nanoscale model could provide a convenient means of testing the migration potentials of cancer cells to achieve a long-term goal of providing clinicians an in vitro platform technology to test the efficacy of novel experimental anti-metastatic compounds. METHODS: Electrospinning produces highly aligned, cell-adhesive nanofiber matrices by applying a strong electric field to a polymer-containing solution. The resulting fibrous microstructure and morphology closely resembles in vivo tumor microenvironments suggesting their use in analysis of migratory potentials of metastatic cancer cells. Additionally, a novel interface with a gel-based delivery system creates CXCL12 chemotactic gradients to enhance CXCR4-expressing cell migration. RESULTS: Cellular dispersions of MCF-10A normal mammary epithelial cells or human breast cancer cells (MCF-7 and MDA-MB-231) seeded on randomly-oriented nanofiber exhibited no significant differences in total or net distance traveled as a result of the underlying topography. Cells traveled ~2-5 fold greater distances on aligned fiber. Highly-sensitive MDA-MB-231 cells displayed an 82% increase in net distance traversed in the presence of a CXCL12 gradient. In contrast, MCF-7 cells exhibited only 31% increase and MCF-10A cells showed no statistical difference versus control or vehicle conditions. MCF-10A cells displayed little sensitivity to CXCL12 gradients, while MCF-7 cells displayed early sensitivity when CXCL12 concentrations were higher. MDA-MB-231 cells displayed low relative expression levels of CXCR4, but high sensitivity resulting in 55-fold increase at late time points due to CXCL12 gradient dissipation. CONCLUSIONS: This model could create clinical impact as an in vitro diagnostic tool for rapid assessment of tumor needle biopsies to confirm metastatic tumors, their invasiveness, and allow high-throughput drug screening providing rapid development of personalized therapies.
Project description:Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer.
Project description:<h4>Background</h4>In this study we evaluated the interactions of human adipose tissue-derived stem cells (ADSCs) and different human breast cancer cell lines (BRCAs) with regard to the safety of cell-assisted lipotransfers for breast reconstruction and a thereby unintended co-localization of ADSCs and BRCAs.<h4>Methods</h4>ADSCs were co-cultured with five different human BRCAs (MCF-7, MDA-MB-231, SK-BR-3, ZR-75-30, and EVSA-T) and primary BRCAs from one patient in a transwell system, and cell-cell-interactions were analyzed by assessing doubling time, migration and invasion, angiogenesis, quantitative real-time polymerase chain reaction (PCR) of more than 300 tumor-associated genes, and multiplex protein assays of 20 chemokines and growth factors and eight matrix metalloproteinases (MMPs). Results of co-culture were compared to those of the respective monoculture.<h4>Results</h4>Quantitative real-time PCR revealed remarkable changes in the expression of multiple tumor-associated genes in co-culture compared to monocultures of both ADSCs and BRCAs. Concomitantly, the concentration of several tumor-associated proteins, such as cytokines and MMPs, were strongly increased in co-culture. Furthermore, exclusively in co-culture with ADSCs, the different BRCAs were exposed to several important tumor-modulating proteins, such as CCL2, HGF, or interleukins. Co-culture did not significantly affect cellular proliferation of either ADSCs or BRCAs (p?>?0.05). The migration of MCF-7 and MDA-MB-231 BRCAs was significantly increased in co-culture with ADSCs by a mean of 11% and 23%, respectively (p?=?0.04 and 0.012), as well as that of ADSCs in co-culture with MDA-MB-231, ZR-75-30, and EVSA-T (+11-15%, p?=?0.035-0.045). Co-culture with MDA-MB-231, SK-BR-3, and EVSA-T BRCAs significantly increased the invasive behavior of ADSCs by a mean of 24-41% (p?=?0.014-0.039). There were no significant differences in the in vitro invasive properties of BRCAs in co-culture compared to monoculture. An in vitro angiogenesis assay revealed an increased tube formation of conditioned media from co-cultured BRCAs and ADSCs compared to the respective monocultures.<h4>Conclusion</h4>This study further elucidates the possible interactions of primary human ADSCs with human BRCAs, pointing towards a potential increased oncological risk which should not be neglected when considering a clinical use of cell-assisted lipoaspirates in breast reconstruction.
Project description:Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.
Project description:Many dietary flavonoids possess anti-cancer activities. Here, the effect of apple peel flavonoid fraction 4 (AF4) on the growth of triple-negative (MDA-MB-231, MDA-MB-468), estrogen receptor-positive (MCF-7), and HER2-positive (SKBR3) breast cancer cells was determined and compared with the effect of AF4 on normal mammary epithelial cells and dermal fibroblasts. AF4 inhibited breast cancer cell growth in monolayer cultures, as well as the growth of MCF-7 spheroids, without substantially affecting the viability of non-malignant cells. A sub-cytotoxic concentration of AF4 suppressed the proliferation of MDA-MB-231 cells by inhibiting passage through the G0/G1 phase of the cell cycle. AF4-treated MDA-MB-231 cells also exhibited reduced in vitro migration and invasion, and decreased Akt (protein kinase B) signaling. Higher concentrations of AF4 were selectively cytotoxic for MDA-MB-231 cells. AF4 cytotoxicity was associated with the intracellular accumulation of reactive oxygen species. Importantly, intratumoral administration of AF4 suppressed the growth of MDA-MB-231 xenografts in non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice. The selective cytotoxicity of AF4 for breast cancer cells, combined with the capacity of sub-cytotoxic AF4 to inhibit breast cancer cell proliferation, migration, and invasion suggests that flavonoid-rich AF4 (and its constituents) has potential as a natural therapeutic agent for breast cancer treatment.