Chemoresistance is mediated by ovarian cancer leader cells in vitro

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.

Notch controls the cell cycle to define leader versus follower identities during collective cell migration.

Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, as cancer. How cell cycle progression affects migration, and vice-versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish Trunk Neural Crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. We propose that migratory behaviours are defined through the interaction of Notch signalling and cell cycle progression.

Collective invasion induced by an autocrine purinergic loop through connexin-43 hemichannels

Progression of epithelial cancers predominantly proceeds by collective invasion of cell groups with coordinated cell–cell junctions and multicellular cytoskeletal activity. Collectively invading breast cancer cells express the gap junction protein connexin-43 (Cx43), yet whether Cx43 regulates collective invasion remains unclear. We here show that Cx43 mediates gap-junctional coupling between collectively invading breast cancer cells and, via hemichannels, adenosine nucleotide/nucleoside release into the extracellular space. Using molecular interference and rescue strategies, we identify that Cx43 hemichannel function, but not intercellular communication, induces leader cell activity and collective migration through the engagement of the adenosine receptor 1 (ADORA1) and AKT signaling. Accordingly, pharmacological inhibition of ADORA1 or AKT signaling caused leader cell collapse and halted collective invasion. ADORA1 inhibition further reduced local invasion of orthotopic mammary tumors in vivo, and joint up-regulation of Cx43 and ADORA1 in breast cancer patients correlated with decreased relapse-free survival. This identifies autocrine purinergic signaling, through Cx43 hemichannels, as a critical pathway in leader cell function and collective invasion.

Leader cell activity and collective invasion by an autocrine nucleotide loop through connexin-43 hemichannels and ADORA1

Progression of epithelial cancers predominantly proceeds by collective invasion of cell groups with coordinated cell-cell junctions and multicellular cytoskeletal activity. Collectively invading breast cancer cells co-express adherens junctions and connexin-43 (Cx43) gap junctions in vitro and in patient samples, yet whether gap junctions contribute to collective invasion remains unclear. We here show that Cx43 is required for chemical coupling between collectively invading breast cancer cells and, by its hemichannel function, adenosine nucleotide release into the extracellular space. Using molecular interference and rescue strategies in vitro and in orthotopic mammary tumors in vivo, Cx43-dependent adenosine nucleotide release was identified as essential mediator engaging the nucleoside receptor ADORA1, to induce leader cell activity and collective migration. In clinical samples joint-upregulation of Cx43 and ADORA1 predicts decreased relapse-free survival. This identifies autocrine nucleotide signaling, through a Cx43/ADORA1 axis, as critical pathway in leader cell function and collective cancer cell invasion.Graphical abstract

Energetic regulation of coordinated leader–follower dynamics during collective invasion of breast cancer cells

The ability of primary tumor cells to invade into adjacent tissues, followed by the formation of local or distant metastasis, is a lethal hallmark of cancer. Recently, locomoting clusters of tumor cells have been identified in numerous cancers and associated with increased invasiveness and metastatic potential. However, how the collective behaviors of cancer cells are coordinated and their contribution to cancer invasion remain unclear. Here we show that collective invasion of breast cancer cells is regulated by the energetic statuses of leader and follower cells. Using a combination of in vitro spheroid and ex vivo organoid invasion models, we found that cancer cells dynamically rearrange leader and follower positions during collective invasion. Cancer cells invade cooperatively in denser collagen matrices by accelerating leader–follower switching thus decreasing leader cell lifetime. Leader cells exhibit higher glucose uptake than follower cells. Moreover, their energy levels, as revealed by the intracellular ATP/ADP ratio, must exceed a threshold to invade. Forward invasion of the leader cell gradually depletes its available energy, eventually leading to leader–follower transition. Our computational model based on intracellular energy homeostasis successfully recapitulated the dependence of leader cell lifetime on collagen density. Experiments further supported model predictions that decreasing the cellular energy level by glucose starvation decreases leader cell lifetime whereas increasing the cellular energy level by AMP-activated kinase (AMPK) activation does the opposite. These findings highlight coordinated invasion and its metabolic regulation as potential therapeutic targets of cancer.