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New Born Calf Serum Can Induce Spheroid Formation in Breast Cancer KAIMRC1 Cell Line.

ABSTRACT: Three-dimensional (3D) cell culture systems have become very popular in the field of drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. We have developed a novel and easy method to produce spheroids from the newly isolated KAIMRC1 cell line in vitro. It can be used as a 3D model to study proliferation, differentiation, cell death, and drug response of cancer cells. Our procedure requires growth media supplemented with 10% new born calf serum (NBCS) and regular cell culture plates to generate KAIMRC1 spheroids without the need for any specialized 3D cell culture system. This procedure generates multiple spheroids within a 12-24-h culture. KAIMRC1 spheroids are compact, homogeneous in size and morphology with a mean size of 55.8 µm (±3.5). High content imaging (HCI) of KAIMRC1 spheroids treated with a panel of 240 compounds resulted in the identification of several highly specific compounds towards spheroids. Immunophenotyping of KAIMRC1 spheroids revealed phosphorylation of FAK, cJUN, and E-cadherin, which suggests the involvement of JNK/JUN pathway in the KAIMRC1 spheroids formation. Gene expression analysis showed upregulation of cell junction genes, GJB3, DSC1, CLDN5, CLDN8, and PLAU. Furthermore, co-culture of KAIMRC1 cells with primary cancer-associated-fibroblasts (CAFs) showcased the potential of these cells in drug discovery application.

SUBMITTER: Ali R

PROVIDER: S-EPMC8740237 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Breast cancer is the second leading cause of cancer-related mortality worldwide as most patients often suffer cancer relapse. The reason is often attributed to the presence of cancer stem cells (CSCs). Recent studies revealed that dysregulation of microRNA (miRNA) are closely linked to breast cancer recurrence and metastasis. However, no specific study has comprehensively characterised the CSC characteristic and miRNA transcriptome in spheroid-enriched breast cells. This study described the generation of spheroid MCF-7 cell in serum-free condition and the comprehensive characterisation for their CSC properties. Subsequently, miRNA expression differences between the spheroid-enriched CSC cells and their parental cells were evaluated using next generation sequencing (NGS). Our results showed that the MCF-7 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, spheroid-enriched CSCs possessed greater cell proliferation, migration, invasion, and wound healing ability. A total of 134 significantly (p<0.05) differentially expressed miRNAs were identified between spheroids and parental cells using miRNA-NGS. MiRNA-NGS analysis revealed 25 up-regulated and 109 down-regulated miRNAs which includes some miRNAs previously reported in the regulation of breast CSCs. A number of miRNAs (miR-4492, miR-4532, miR-381, miR-4508, miR-4448, miR-1296, and miR-365a) which have not been previously reported in breast cancer were found to show potential association with breast cancer chemoresistance and self-renewal capability. The gene ontology (GO) analysis showed that the predicted genes were enriched in the regulation of metabolic processes, gene expression, DNA binding, and hormone receptor binding. The corresponding pathway analyses inferred from the GO results were closely related to the function of signalling pathway, self-renewability, chemoresistance, tumorigenesis, cytoskeletal proteins, and metastasis in breast cancer. Based on these results, we proposed that certain miRNAs identified in this study could be used as new potential biomarkers for breast cancer stem cell diagnosis and targeted therapy.

Project description:Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGFβ1. Knockdown of BSP or its inactivation by specific antibodies were found to reduce the metastatic potential of MDA-MB-231 triple-negative breast cancer cells in xenografts. While the role of BSP in bone metastasis was studied using such in vivo models, valid in vitro test systems to investigate BSP biology have been lacking since this protein is expressed at very low levels in classical 2D cell cultures and the frequently used breast cancer cell line MDA-MB-231 is difficult to grow in 3D. Here, we have developed a long-term 3D spheroid culture model using MDA-MB-231 cells in a sandwich approach using cell embedding between a non-adherent surface and basement membrane extracts. This allowed consistent growth of spheroids for more than 21 days. Also, co-culturing of MDA-MB-231 with CCD-1137Sk fibroblasts yielded stably growing spheroids, suggesting the importance of extracellular matrix (ECM) in this process. In addition, we have set up a novel and simple open source analysis tool to characterize protein expression in 2D cultures and spheroids by immunofluorescence. Using this approach in combination with Western blot analysis, the expression profile of BSP was analyzed. BSP was enriched at the rims of spheroids, both in mono- and co-cultures and its abundance in general correlated with that of TGFβ1 under different conditions, including spheroid maturation, cytostatic treatment, and fibroblast co-culture. Conversely, correlation of IGF-1 and BSP was limited to mono-culture time course profiles. In conclusion, we present novel tools to study the regulation of gene expression in combination with cell proliferation and apoptosis in a long-term 3D model of breast cancer and find dynamic abundance profiles of the metastasis-relevant protein BSP and its regulators.

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New Born Calf Serum Can Induce Spheroid Formation in Breast Cancer KAIMRC1 Cell Line.

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