Dataset Information

Chemoresistance is mediated by ovarian cancer leader cells in vitro.

ABSTRACT:

Background

Leader cells are a subset of cancer cells that coordinate the complex cell-cell and cell-matrix interactions required for ovarian cancer migration, invasion, tumour deposition and are negatively associated with progression-free survival and response to therapy. Emerging evidence suggests leader cells may be enriched in response to chemotherapy, underlying disease recurrence following treatment.

Methods

CRISPR was used to insert a bicistronic T2A-GFP cassette under the native KRT14 (leader cell) promoter. 2D and 3D drug screens were completed in the presence of chemotherapies used in ovarian cancer management. Leader cell; proliferative (Ki67); and apoptotic status (Cleaved Caspase 3) were defined by live cell imaging and flow cytometry. Quantitative real-time PCR defined "stemness" profiles. Proliferation was assessed on the xCELLigence real time cell analyser. Statistical Analysis was performed using unpaired non-parametric t-tests or one-way ANOVA and Tukey's multiple comparison post hoc.

Results

Leader cells represent a transcriptionally plastic subpopulation of ovarian cancer cells that arise independently of cell division or DNA replication, and exhibit a "stemness" profile that does not correlate with epithelial-to-mesenchymal transition. Chemotherapeutics increased apoptosis-resistant leader cells in vitro, who retained motility and expressed known chemo-resistance markers including ALDH1, Twist and CD44v6. Functional impairment of leader cells restored chemosensitivity, with leader cell-deficient lines failing to recover following chemotherapeutic intervention.

Conclusions

Our data demonstrate that ovarian cancer leader cells are resistant to a diverse array of chemotherapeutic agents, and are likely to play a critical role in the recurrence of chemo-resistant disease as drivers of poor treatment outcomes.

SUBMITTER: Karimnia N

PROVIDER: S-EPMC8408956 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundThe metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines.MethodsFT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence.ResultsSixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers.ConclusionsThe above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

Project description:Mechanical factors in the tumor microenvironment play an important role in response to a variety of cellular activities in cancer cells. Here, we utilized polyacrylamide hydrogels with varying physical parameters simulating tumor and metastatic target tissues to investigate the effect of substrate stiffness on the growth, phenotype, and chemotherapeutic response of ovarian cancer cells (OCCs). We found that increasing the substrate stiffness promoted the proliferation of SKOV-3 cells, an OCC cell line. This proliferation coincided with the nuclear translocation of the oncogene Yes-associated protein. Additionally, we found that substrate softening promoted elements of epithelial-mesenchymal transition (EMT), including mesenchymal cell shape changes, increase in vimentin expression, and decrease in E-cadherin and β-catenin expression. Growing evidence demonstrates that apart from contributing to cancer initiation and progression, EMT can promote chemotherapy resistance in ovarian cancer cells. Furthermore, we evaluated tumor response to standard chemotherapeutic drugs (cisplatin and paclitaxel) and found antiproliferation effects to be directly proportional to the stiffness of the substrate. Nanomechanical studies based on atomic force microscopy (AFM) have revealed that chemosensitivity and chemoresistance are related to cellular mechanical properties. The results of cellular elastic modulus measurements determined by AFM demonstrated that Young's modulus of SKOV-3 cells grown on soft substrates was less than that of cells grown on stiff substrates. Gene expression analysis of SKOV-3 cells showed that mRNA expression can be greatly affected by substrate stiffness. Finally, immunocytochemistry analyses revealed an increase in multidrug resistance proteins, namely, ATP binding cassette subfamily B member 1 and member 4 (ABCB1 and ABCB4), in the cells grown on the soft gel resulting in resistance to chemotherapeutic drugs. In conclusion, our study may help in identification of effective targets for cancer therapy and improve our understanding of the mechanisms of cancer progression and chemoresistance.