Exploring doxorubicin transport in 2D and 3D models of MDA-MB-231 sublines: impact of hypoxia and cellular heterogeneity on doxorubicin accumulation in cells.

Triple-negative breast cancer (TNBC) treatment is challenging due to its aggressive nature and heterogeneity of this type of cancer, characterized by various subtypes and intratumoral diversity. Doxorubicin (DOX) plays a crucial role in TNBC chemotherapy reducing the tumor size and improving patient survival. However, decreased drug uptake and increased resistance in specific cell subpopulations reduce the effectiveness of the treatment. This study explored the differences in DOX transport in MDA-MB-231 phenotypic sublines in cell monolayer (2D model) and cell spheroids (3D cultures). Cell spheroids were formed using magnetic 3D Bioprinting method. DOX transport into cells and spheroids was evaluated using fluorescence microscopy after different incubation durations with DOX in normoxia and hypoxia. In hypoxia, DOX transport into cells was 2.5 to 5-fold lower than in normoxia. The subline F5 monolayer-cultured cells exhibited the highest DOX uptake, while subline H2 cells showed the lowest uptake in normoxia and hypoxia. In 3D cultures, DOX transport was up to 2-fold lower in spheroids formed from subline H2 cells. Spheroids from subline D8 and MDA-MB-231 parent cells had the highest DOX uptake. A correlation was observed between the characteristics of the cells and their resistance to anticancer drugs. The results indicate that different cancer cell subpopulations in tumours due to differences in drug uptake could significantly impact treatment efficacy.

Interaction of phenotypic sublines isolated from triple-negative breast cancer cell line MDA-MB-231 modulates their sensitivity to paclitaxel and doxorubicin in 2D and 3D assays.

Breast cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. These result in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to analyze cell phenotypic sublines and the influence of their interaction on drug resistance, spheroid formation, and migration. Seven sublines were derived from the MDA-MB-231 breast cancer cell line using a multiple-cell suspension dilution. The growth rate, CD133 receptor expression, migration ability, and chemosensitivity of these sublines to anticancer drugs doxorubicin (DOX) and paclitaxel (PTX) were determined. Three sublines (F5, D8, H2) have been chosen to study their interaction in 2D and 3D assays. In the 2D model, the resistance of all sublines composition to DOX decreased, but in the 3D model, the resistance of all sublines except H2, increased to both PTX and DOX. In the 3D model, the combined sublines F5 and D8 had higher resistance to DOX and statistically significantly lower resistance for PTX compared to the control. The interaction between cancer stem-like cells (F5) and increased migration cells (D8) increased resistance to PTX in cell monolayer and increased resistance against both DOX and PTX in the spheroids. The interaction of DOX-resistant (H2) cells with other cell subpopulations (D8, F5, HF) decreased the resistance to DOX in cell monolayer and both DOX and PTX in spheroids.

Nongenotoxic ABCB1 activator tetraphenylphosphonium can contribute to doxorubicin resistance in MX-1 breast cancer cell line.

Hyperactivation of ABC transporter ABCB1 and induction of epithelial-mesenchymal transition (EMT) are the most common mechanism of acquired cancer chemoresistance. This study describes possible mechanisms, that might contribute to upregulation of ABCB1 and synergistically boost the acquisition of doxorubicin (DOX) resistance in breast cancer MX-1 cell line. DOX resistance in MX-1 cell line was induced by a stepwise increase of drug concentration or by pretreatment of cells with an ABCB1 transporter activator tetraphenylphosphonium (TPP+) followed by DOX exposure. Transcriptome analysis of derived cells was performed by human gene expression microarrays and by quantitative PCR. Genetic and epigenetic mechanisms of ABCB1 regulation were evaluated by pyrosequencing and gene copy number variation analysis. Gradual activation of canonical EMT transcription factors with later activation of ABCB1 at the transcript level was observed in DOX-only treated cells, while TPP+ exposure induced considerable activation of ABCB1 at both, mRNA and protein level. The changes in ABCB1 mRNA and protein level were related to the promoter DNA hypomethylation and the increase in gene copy number. ABCB1-active cells were highly resistant to DOX and showed morphological and molecular features of EMT. The study suggests that nongenotoxic ABCB1 inducer can possibly accelerate development of DOX resistance.

3D Tumor Spheroid Models for In Vitro Therapeutic Screening of Nanoparticles.

The anticancer activity of compounds and nanoparticles is most often determined in the cell monolayer. However, three-dimensional (3D) systems, such as tumor spheroids, are more representing the natural tumor microenvironment. They have been shown to have higher invasiveness and resistance to cytotoxic agents and radiotherapy compared to cells growing in 2D monolayer. Furthermore, to improve the prediction of clinical efficacy of drugs, in the past decades, even more sophisticated systems, such as multicellular 3D cultures, closely representing natural tumor microenvironment have been developed. Those cultures are formed from either cell lines or patient-derived tumor cells. Such models are very attractive and could improve the selection of tested materials for clinical trials avoiding unnecessary expensive tests in vivo. The microenvironment in tumor spheroids is different, and those differences or the interaction between several cell populations may contribute to different tumor response to the treatment. Also, different types of nanoparticles may have different behavior in 3D models, depending on their nature, physicochemical properties, the presence of targeting ligands on the surface, etc. Therefore, it is very important to understand in which cases which type of tumor spheroid is more suitable for testing specific types of nanoparticles, which conditions should be used, and which analytical method should be applied.

Evaluation of low-intensity pulsed ultrasound on doxorubicin delivery in 2D and 3D cancer cell cultures.

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.

Heterogeneity of breast cancer: The importance of interaction between different tumor cell populations.

INTRODUCTION: Breast cancer is the most common cancer and the second leading cause of cancer-related death in women worldwide. Despite the early detection of breast cancer and increasing knowledge of its biology and chemo-resistance, metastatic breast cancer is largely incurable disease. We provide a review of the intertumor and intratumor heterogeneity, explain the differences between triple-negative breast cancer subtypes. Also, we describe the interaction of breast tumor cells with their microenvironment cells and explain how this interaction contributes to the tumor progression, metastasis formation and resistance to the treatment. DISCUSSION: One of the main causes that complicate the treatment is tumor heterogeneity. It is observed among patients (intertumor heterogeneity) and in each individual tumor (intratumor heterogeneity). In the case of intratumor heterogeneity, the tumor consists of different phenotypical cell populations. During breast cancer subtype identification, a small piece of solid tumor tissue does not necessarily represent all the tumor composition. Breast tumor cell phenotypical differences may appear due to cell localization in different tumor sites, unique response to the treatment, cell interaction with tumor microenvironment or tumor cell interaction with each other. This heterogeneity may lead to breast cancer aggressiveness and challenging treatment. CONCLUSION: Understanding the molecular and cellular mechanisms of tumor heterogeneity that are relevant to the development of treatment resistance is a major area of research. Identification of differences between populations and their response to anticancer drugs would help to predict the potential resistance to chemotherapy and thus would help to select the most suitable anticancer treatment.