Invadopodia enable cooperative invasion and metastasis of breast cancer cells

Metastasis, the spread of cancer to distant sites, is responsible for the majority of cancer deaths. In breast cancer, recent research indicates that metastases often originate from polyclonal seedings, involving collective dissemination of cell clusters rather than individual cells. This highlights the potential for cooperativity between different cancer cell clones in facilitating metastasis. The metastatic cascade begins when cancer cells gain invasive properties, including motility and extracellular matrix (ECM) degradation. These are often associated with epithelial-mesenchymal transition (EMT), a process where epithelial cells lose cell-cell contacts, strengthen cell-matrix adhesions, and become motile. Cancer cells may also develop invadopodia, membrane protrusions rich in matrix metalloproteinases (MMPs), enabling ECM degradation and increasing metastatic potential. The EMT program is complex and can lead to clones with varying degrees of invasiveness. Previous 3D in vitro studies have shown that invasive collective strands contain cells with different invasive traits, with leader cells exhibiting increased contractility, cell-ECM adhesion, and ECM remodeling compared to follower cells. Leader cells enable the invasion of follower cells, a phenomenon known as cooperative invasion. Previous research suggests a potential link between invadopodia and leader cell function during collective invasion, but the precise role of invadopodia and the spatial dynamics preceding cooperative invasion remain unclear. Furthermore, the role of cooperation in actual metastasis in vivo has not been fully explored. This study aims to elucidate how cancer clones with differing invasive abilities cooperate during invasion and metastasis, using isogenic breast cancer cell lines in 3D in vitro and in vivo models.

The literature extensively documents the critical role of metastasis in cancer mortality (Hanahan & Weinberg, 2000; Vanharanta & Massagué, 2013). Studies reveal that breast cancer metastases frequently arise from polyclonal seedings (Cheung et al., 2016; Aceto et al., 2014), underscoring the significance of cooperative cell behavior. The connection between invasion and EMT is well-established (Yeung & Yang, 2017), with invadopodia playing a key role in ECM degradation (Ignatelli et al., 2012; Eckert et al., 2011, 2017; Karamanou et al., 2020). Invadopodia, described in detail by Murphy and Courtneidge (2011) and Eddy et al. (2017), are crucial for cancer cell invasion and metastasis (Gligorijevic et al., 2014; Gligorijevic et al., 2012). The non-binary nature of EMT and the existence of diverse EMT pathways (Ye & Weinberg, 2015) contribute to the phenotypic heterogeneity observed in cancer cell populations. Studies using 3D in vitro models of breast cancer have revealed the role of leader cells in collective invasion (Cheung et al., 2013; Zhang et al., 2019; Wolf et al., 2007; Carey et al., 2013; Westcott et al., 2015; Chapman et al., 2014), with leader cells driving the invasion of follower cells (Bayarmagnai et al., 2019). However, the detailed mechanisms and the in vivo relevance of this cooperative invasion remain areas of active research.

This study utilized the isogenic murine breast cancer cell lines 4T1 (highly invasive and metastatic) and 67NR (non-invasive), syngeneic to Balb/C mice. The invasive capacity of these cell lines was assessed using 3D spheroid invasion assays in collagen I, a method described in detail by Perrin et al. (2021). MMP-dependent invasion was confirmed using a pan-MMP inhibitor (GM6001). The role of invadopodia was investigated by analyzing the expression of key invadopodia components (cortactin and Tks5) via Western blotting, and by assessing invadopodia function using a gelatin degradation assay (Sharma et al., 2013; Pourfarhangi et al., 2018). Cell migration was analyzed using a scratch assay, tracking individual cells using the TrackMate plugin in Fiji (Tinevez et al., 2017). To study cooperative invasion, 4T1 and 67NR cells expressing fluorescent proteins (mScarlet and GFP, respectively) were mixed at a 1:50 ratio and embedded in collagen I. Longitudinal imaging was performed to analyze cell sorting and invasion dynamics (Perrin et al., 2021). Cell sorting was quantified using a Distance Index (DI), measuring the relative distance of each cell to the spheroid center. The role of cell contractility and MMPs in cell sorting was investigated using inhibitors (Y-27632 and GM6001). Time-lapse imaging was used to track individual cells and analyze their motility, using mean square displacement (MSD) analysis to assess persistence (Wu & Berland, 2008). The role of E-cadherin in cell sorting was examined using a blocking antibody and E-cadherin knockdown cell lines. A 2D cell-ECM adhesive competition assay was developed to compare the adhesive properties of 4T1 and 67NR cells to ECM. Cell-matrix contact angles were measured to assess adhesion strength. To investigate the role of invadopodia in cooperative invasion, invadopodia were stably eliminated in 4T1 cells using Tks5 knockdown (Gligorijevic et al., 2014), generating Tks5-KD and control Tks5-CTL cell lines. The effect of invadopodia elimination on cooperative invasion was evaluated using mixed spheroid invasion assays. In vivo metastasis was assessed by orthotopically implanting single or mixed cell lines in mice, and quantifying lung metastasis using a clonogenic assay (https://doi.org/10.5281/zenodo.6639302). Western blotting and immunofluorescence were used to validate knockdown efficiencies and analyze tumor sections.

The study found that 4T1 cells, but not 67NR cells, exhibited robust MMP-dependent invasion in 3D collagen I. 4T1 cells showed significantly higher persistence in cell migration than 67NR cells. When mixed in spheroids, 4T1 and 67NR cells sorted, with 4T1 cells accumulating at the spheroid-matrix interface. This sorting was driven by the higher persistence of 4T1 cells and their preferential adhesion to the ECM, and was independent of E-cadherin-mediated cell-cell adhesions. Inhibiting MMPs or cell contractility blocked cell sorting. Time-lapse imaging revealed that 4T1 cells actively moved from the spheroid core to the edge, whereas 67NR cells remained within their initial compartments. 4T1 cells led 67NR cells in cooperative invasion, with 67NR cells following 4T1 cells into the collagen matrix. This cooperative invasion was MMP-dependent and maintained the spatial organization observed in the spheroids, with 4T1 cells at the spheroid-matrix interface. Eliminating invadopodia in 4T1 cells (via Tks5 knockdown) abolished their individual invasive capacity but did not affect the ability of non-invasive Tks5-KD cells to undergo cooperative invasion when mixed with Tks5-CTL cells. However, mixed spheroids of Tks5-KD and 67NR cells did not exhibit cooperative invasion, suggesting that invadopodia in leader cells are essential for this process. In vivo studies demonstrated that 67NR cells and Tks5-KD cells failed to metastasize alone. However, mixtures of Tks5-CTL and Tks5-KD cells exhibited cooperative metastasis, with both cell types present in lung metastases, indicating that leader cells with functional invadopodia can enable the metastasis of follower cells lacking invadopodia.

This study demonstrates that invasive breast cancer cells can sort and lead non-invasive cells in cooperative invasion and metastasis. The observed cell sorting is driven by differential persistence in cell migration and differential sensitivity to ECM, a mechanism different from the classical differential adhesion hypothesis. The crucial role of invadopodia in leader cells for cooperative invasion and metastasis is highlighted by the findings that invadopodia-deficient cells fail to initiate invasion and, importantly, that invadopodia-positive cells enable the metastasis of invadopodia-negative cells. This suggests that targeting invadopodia could be a promising therapeutic strategy to inhibit metastasis. The observation that cooperative metastasis is dependent on strong E-cadherin interactions highlights a potential therapeutic target and an area for future research.

This research reveals the critical role of invadopodia in leader cells for cooperative invasion and metastasis in breast cancer. The findings demonstrate a novel mechanism for collective metastasis driven by differential cell motility and ECM interaction. Targeting invadopodia may offer a powerful approach for inhibiting both individual and collective cancer cell invasion and metastasis. Future studies should focus on the interplay between cell cycle, energy metabolism, and invadopodia function in determining leader and follower cell identities.

The study primarily focused on two isogenic murine breast cancer cell lines and one human cell line, limiting the generalizability of the findings to other cancer types or cellular contexts. The in vivo models used were preclinical, and further investigation is needed to confirm these findings in human patients. The precise molecular mechanisms underlying the differential persistence and ECM sensitivity observed between the cell lines remain to be fully elucidated.