Resistance Of Spheroids Research Articles

The response and resistance to drugs in ovarian cancer cell lines in 2D monolayers and 3D spheroids

Ovarian cancer is the most common type of gynecologic cancer. One of the leading causes of high mortality is chemoresistance, developed primarily or during treatment. Different mechanisms of drug resistance appear at the cellular and cancer tissue organization levels. We examined the differences in response to the cytotoxic drugs CIS, MTX, DOX, VIN, PAC, and TOP using 2D (two-dimensional) and 3D (three-dimensional) culture methods. We tested the drug-sensitive ovarian cancer cell line W1 and established resistant cell lines to appropriate cytotoxic drugs. The following qualitative and quantitative methods were used to assess: 1) morphology - inverted microscope and hematoxylin & eosin staining; 2) viability - MTT assay; 3) gene expression – a quantitative polymerase chain reaction; 4) identification of proteins – immunohistochemistry, and immunofluorescence. Our results indicate that the drug-sensitive and drug-resistant cells cultured in 3D conditions exhibit stronger resistance than the cells cultured in 2D conditions. A traditional 2D model shows that drug resistance of cancer cells is caused mainly by changes in the expression of genes encoding ATP-binding cassette transporter proteins, components of the extracellular matrix, “new” established genes related to drug resistance in ovarian cancer cell lines, and universal marker of cancer stem cells. Whereas in a 3D model, the drug resistance in spheroids can be related to other mechanisms such as the structure of the spheroid (dense or loose), the cell type (necrotic, quiescent, proliferating cells), drug concentrations or drug diffusion into the dense cellular/ECM structure.

Drug resistance evaluation in novel 3D in vitro model

Ovarian cancer rates the highest mortality among all gynecological malignancies. The main reason for high mortality is the development of drug resistance. It can be related to changes in the expression of many drug resistance genes as well as expression of extracellular matrix proteins and cell density in the tumor. We developed a simple two-dimensional and three-dimensional model of drug sensitive A2780 and resistant to cisplatin and paclitaxel variants of ovarian cancer cell line. Using MTT assay, we compared drug resistance in two-dimensional and three-dimensional cell culture conditions. Real-time polymerase chain reaction analysis was used to compare the expression of drug resistance genes. The expression of proteins in spheroids was determined by immunohistochemistry. We observed a moderate increase in cisplatin resistance and a significant increase in paclitaxel resistance between two-dimensional and three-dimensional cell culture conditions. Our findings show that changes in the expression of drug resistance genes may play a crucial role in the drug resistance of cancer cells in traditional cell culture. On the other hand, the drug resistance in spheroids may result from different mechanisms such as cell density in the spheroid, extracellular matrix proteins expression and drug capacity to diffuse into the spheroid.

Effect of Radiation on self-renewality of prostate cancer stem cells.

Introduction: CSCs have been identified in prostate cancer (PCa), one of the most diagnosed malignancies in men over the world, for which radiation resistance is a major problem in the treatment of advanced stages. Cancer stem cells (CSCs) have the ability to self-renew and differentiate to give rise to heterogeneous phenotype of the tumor cells. It is believed that CSCs are involved in metastasis, recurrence and therapy resistance in various cancers. Recently CSCs have been identified in prostate cancer. In vitro one of the main properties used to enriched cancer stem cells is their capacity to form spheres in non-adherent culture conditions. Materials and Methods: Human prostate carcinoma cell line (DU145) were plated in serum-free suspension culture system allowed for tumorsphere forming that enriched cancer stem cells. To identify prostate CSCs in adherent cells and nonadherent sphere cells of prostate cancer cell lines, the expression of the CD133 and CD44 cell-surface markers were assessed by flowcytometry. To evaluate of different doses of radiation (electron 6 MeV) on the self-renewal and proliferation of the Du145, sphere-formation assay (SFA) and plating efficiency (colony formation assay (CFA)) were assessed. The results were comparing with the parental cells. SFA is based on the self-renewing properties unique to CSCs. CFA is used as a measure of the proliferative potential of cells. Results: The results show that both SF (CFA) and SF (SFA) declined with the increase of radiation dose in cells treated as monolayer or sphere culture. However, the extent of reduction in cells treated as monolayer culture was significantly higher compared to cells treated as spheroids, suggesting higher resistance of spheroids to radiation. According to these results, tumorspheres from PCa showed more radioresistant than bulk of tumor. Conclusion: Due to the relative resistance of CSCs to radiotherapy, determination and characterization of radioresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

Role of extracellular signal-regulated kinase (ERK)1/2 in multicellular resistance to docetaxel in MCF-7 cells

Cancer cells frequently fail to respond to chemotherapy due to acquisition of chemoresistance. Tumour cells are prone to die by necrosis when they are metabolically stressed by hypoxic and glucose depletion (OGD) due to insufficient vascularization, a common feature of solid tumours. Tumour necrosis indicates poor prognosis and emergence of drug resistance in cancer patients; however, its molecular mechanism remains unclear. In this study, we used multicellular tumour spheroids (MTS) as an in vitro tumour model to investigate the molecular mechanisms underlying necrosis-linked drug resistance. MCF-7 cells formed tight and spherical shape of spheroids and started to form the necrotic core at 8 days of culture. We found that docetaxel (DOC)-induced apoptosis was gradually reduced during MCF-7 spheroid culture compared to that in monolayers and that more prominent resistance to DOC was observed when spheroids containing the necrotic core were treated. ERK1/2 and Akt appeared to be activated in MCF-7 spheroids with necrotic core, but not in 2D culture cells and in spheroids without necrotic core. DOC resistance in spheroids was reversed by inhibition of ERK1/2, but not of Akt, suggesting an important role for ERK1/2 in the DOC resistance in MCF-7 spheroids. These results provide new insight into the possible relation between necrosis-linked ERK1/2 activation and acquisition of multicellular resistance.

Bcl-2 Family Proteins Contribute to Apoptotic Resistance in Lung Cancer Multicellular Spheroids

Combinatorial therapies using the proteasome inhibitor, bortezomib, have been found to induce synergistic apoptosis in cancer cells grown as monolayers; however, three-dimensional spheroid culture may be a better model for the multicellular resistance found in solid tumors, such as lung cancer. We tested the combinatorial apoptotic strategy of using bortezomib together with TNF-related apoptosis-inducing ligand (TRAIL), both in monolayers and in spheroids of A549 lung cancer cells. Indeed, bortezomib plus TRAIL induced synergistic apoptosis in A549 cells grown as monolayers, but had little effect on A549 cells grown as three-dimensional multicellular spheroids. The acquired resistance of spheroids was not due to a limitation of diffusion, to survival pathways, such as NF-kappaB or PI3K/Akt/mTOR, or to the up-regulation of FLIP(S) (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein, short). We then investigated a role for the Bcl-2 family of anti- and proapoptotic proteins. When cells formed spheroids, antiapoptotic Bcl-2 increased, whereas antiapoptotic Mcl-1 decreased. ABT-737, a small molecule that inhibits Bcl-2, but not Mcl-1, abolished the multicellular resistance of A549 spheroids to bortezomib plus TRAIL. In another lung cancer cell line, H1299, acquisition of multicellular resistance in spheroids was also accompanied by an increase in Bcl-2 and decrease in Mcl-1. In H1299 spheroids compared with those of A549, however, Mcl-1 remained higher, and Mcl-1 knockdown was more effective than ABT-737 in removing multicellular resistance. Our study suggests that the balance of Bcl-2 family proteins contributes to the acquired multicellular resistance of spheroids, and suggests a possible target for improving the response of lung cancer to bortezomib therapies.

Drug and radiation resistance in spheroids: cell contact and kinetics.

Cells from multicellular spheroids are often more resistant than monolayers to drugs and radiation. While explanations for resistance can be based on differences in cell cycle distribution, inability of the drug to penetrate the spheroid, or the presence of hypoxic cells, these mechanisms do not adequately explain resistance to all agents. Small spheroids (containing about 25-50 cells) exposed to ionizing radiation, hyperthermia, photodynamic therapy, or topoisomerase II inhibitors, are more resistant to killing than monolayers; the close three-dimensional contact in spheroids has been implicated in this resistance. Proposed mechanisms for the 'contact effect' include gap junctional 'reciprocity', cell shape mediated changes in (repair-related) gene expression, and alterations in chromatin packaging which influence DNA repair. The consequences of the contact effect are especially important for multifraction exposures. Another form of resistance can be demonstrated during repetitive treatments; 'regrowth resistance' reflects the capacity of spheroid cells to proliferate more efficiently to compensate for cell killing.

Radiation-Induced DNA Unwinding Is Influenced by Cell Shape and Trypsin

Incubation of cells in high salt/alkali typically leads to denaturation and unwinding of DNA, yet DNA from Chinese hamster V79 cells grown for 1 day as spheroids stops unwinding after only 5-10 min. We previously postulated that this was a result of "constraints" to DNA unwinding present in cells in spheroids but not in monolayers, and that these constraints could be responsible for the increased resistance of spheroids of V79 cells to killing by ionizing radiation (i.e., the contact effect). However, studies reported here indicate that this limited DNA unwinding is correlated with a round cell shape and lack of cell surface fibronectin. In round cells which continue to synthesize fibronectin, demonstration of constraints requires prior exposure to trypsin in order to digest cell surface fibronectin. However, trypsin did not influence cell killing by ionizing radiation. Therefore, the increase in radiation resistance of V79 spheroids and the change in their DNA unwinding kinetics both appear contingent upon a change in cell shape; differences in DNA denaturation rates which are detected in spheroids using the unwinding assay are apparently not directly responsible for the contact effect.

Resistance of V79 multicell spheroids to mitoxantrone: drug uptake and cytotoxicity.

We have previously shown that V79 multicell spheroids are resistant to the anthracenedione mitoxantrone (1). In this paper we demonstrate that this resistance is not solely due to restricted drug penetration into the spheroid, but also to an altered intrinsic resistance of the cells when grown as a three-dimensional structure. We have studied the uptake and toxicity of mitoxantrone in V79-OCF4 monolayers, 100 micron spheroids, 650 micron spheroids, and outer and inner cells of 650 micron spheroids. The LD90 for cells exposed as monolayers to mitoxantrone for two hours was 0.016 microgram/ml, 0.055 microgram/ml for 100 micron spheroids, 1.5 micrograms/ml for outer spheroid cells and 6.2 micrograms/ml for inner spheroid cells. Uptake of [14C]mitoxantrone was linear for all populations with no plateau up to the highest doses used. The uptake of drug required to kill 90% of the cells in a population (UP90) of monolayers was 3.7 ng/10(6) cells, 10.7 ng/10(6) cells for 100 micron spheroids, 169 ng/10(6) cells for outer spheroid cells, and 146 ng/10(6) cells for inner spheroid cells. The relative resistance of spheroids compared to monolayers, based on drug concentration in the medium, was 3.4 for small spheroids, 92 for outer cells, and 390 for inner cells. When cell survival was normalized to drug uptake, the relative resistance of spheroids to monolayers was 2.9 for small spheroids, 46 for outer cells and 40 for inner cells of large spheroids. The data demonstrate that the resistance of multicell spheroids to mitoxantrone is not solely due to restricted drug penetration into the cell aggregate but is also due to a spheroid-induced altered intrinsic resistance of the V79 cells.

In vitro chemosensitivity testing using the multicellular tumor spheroid model.

Using growth delay and clonogenic cell survival as end points, we have shown that the 3-dimensional structure of human lung tumour spheroids confers a degree of resistance to the anthracyclines doxorubicin and 4'-deoxydoxorubicin, relative to cells grown as monolayer. 4'-deoxydoxorubicin induces a longer growth delay and greater clonogenic cell kill than doxorubicin in spheroids, although it is no more cytotoxic in monolayer. Using fluorescent microscopy we have demonstrated, qualitatively, that lipophilic anthracycline analogues partition into the spheroid more rapidly and to a greater degree than doxorubicin. It is apparent that penetration is an important aspect of anthracycline drug resistance in spheroids, and the spheroid model may represent a better in vitro system for testing lipophilic analogues of cytotoxic drugs.

Radiation-Induced DNA Damage in V79 Spheroids and Monolayers

Chinese hamster V79 cells can be grown as monolayers or in suspension as multicell spheroids. When irradiated, cells from spheroids can accumulate more sublethal damage than cells from monolayers as evidenced by a wider shoulder on the survival curve. We have attempted to determine whether this increased resistance of spheroids to damage by ionizing radiation is reflected by DNA damage. The production and repair of single-strand breaks was quantified using alkaline sucrose gradient sedimentation and hydroxylapatite chromatography. Both methods indicated that equal amounts of radiation damage per rad were produced in cells grown as single cells or as spheroids, and that postirradiation rejoining of DNA single-strand breaks also occurred with similar efficiency. Thus, these results suggest that the enhanced survival of cells irradiated as spheroids is dependent on processes other than (or in addition to) initial radiation damage to DNA.