Collective Cancer Invasion Research Articles

Three-dimensional computer vision for exploring heterogeneity in collective Cancer Invasion

Collective cancer invasion exhibits a hierarchical structure characterized by leader-follower organization. Dynamic gene expression analysis of invading cells using nanobiosensors within 3D microenvironments provides a valuable means to explore the regulation of leader cells during collective cancer invasion. Nonetheless, the analysis of time-lapse, multimodal images that capture the intricacies of complex invading structures and gene expression profiles in 3D tumor spheroids poses a significant technological challenge. Here, we present a computer vision-based workflow that streamlines the identification of protrusions and detached clusters from 3D tumor spheroids. This methodology not only discerns invading multicellular structures and quantifies their physical properties, but also captures gene expression patterns associated with these invasive mechanisms using an intracellular nanobiosensor. Consequently, it empowers a systematic exploration of the genotypic and phenotypic heterogeneities inherent in cancer invasion. To illustrate the effectiveness of this approach, we applied it to the analysis of a long noncoding RNA, MALAT1, in tumor spheroids derived from patients with muscle-invasive bladder cancer. Our investigation delved into the heterogeneity of cancer invasion and its relationship to MALAT1 expression. Overall, this workflow represents a valuable tool for gaining insights into the complexities of cancer invasion.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Long noncoding RNA MALAT1 is dynamically regulated in leader cells during collective cancer invasion

Cancer cells collectively invade using a leader-follower organization, but the regulation of leader cells during this dynamic process is poorly understood. Using a dual double-stranded locked nucleic acid (LNA) nanobiosensor that tracks long noncoding RNA (lncRNA) dynamics in live single cells, we monitored the spatiotemporal distribution of lncRNA during collective cancer invasion. We show that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is dynamically regulated in the invading fronts of cancer cells and patient-derived spheroids. MALAT1 transcripts exhibit distinct abundance, diffusivity, and distribution between leader and follower cells. MALAT1 expression increases when a cancer cell becomes a leader and decreases when the collective migration process stops. Transient knockdown of MALAT1 prevents the formation of leader cells and abolishes the invasion of cancer cells. Taken together, our single-cell analysis suggests that MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

Dynamic Regulation of MALAT1 in Leader Cell Formation and Collective Cancer Invasion

Dynamic Regulation of MALAT1 in Leader Cell Formation and Collective Cancer Invasion

Stiffness-dependent active wetting enables optimal collective cell durotaxis

The directed migration of cellular clusters enables morphogenesis, wound healing and collective cancer invasion. Gradients of substrate stiffness direct the migration of cellular clusters in a process called collective durotaxis, but the underlying mechanisms remain unclear. Here we unveil a connection between collective durotaxis and the wetting properties of cellular clusters. We show that clusters of cancer cells dewet soft substrates and wet stiff ones. At intermediate stiffness—at the crossover from low to high wettability—clusters on uniform-stiffness substrates become maximally motile, and clusters on stiffness gradients exhibit optimal durotaxis. Durotactic velocity increases with cluster size, stiffness gradient and actomyosin activity. We demonstrate this behaviour on substrates coated with the cell–cell adhesion protein E-cadherin and then establish its generality on substrates coated with extracellular matrix. We develop an active wetting model that explains collective durotaxis in terms of a balance between in-plane active traction and tissue contractility and out-of-plane surface tension. Finally, we show that the distribution of cluster displacements has a heavy tail, with infrequent but large cellular hops that contribute to durotactic migration. Our study demonstrates a physical mechanism of collective durotaxis, through both cell–cell and cell–substrate adhesion ligands, based on the wetting properties of active droplets. Substrate stiffness influences cellular cluster migration through collective durotaxis. Now, the underlying mechanism of this process is explained by considering the wetting dynamics of the clusters.

Intratumoral Heterogeneity Promotes Collective Cancer Invasion through NOTCH1 Variation.

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

Decoding leader cells in collective cancer invasion.

Collective cancer invasion with leader-follower organization is increasingly recognized as a predominant mechanism in the metastatic cascade. Leader cells support cancer invasion by creating invasion tracks, sensing environmental cues and coordinating with follower cells biochemically and biomechanically. With the latest developments in experimental and computational models and analysis techniques, the range of specific traits and features of leader cells reported in the literature is rapidly expanding. Yet, despite their importance, there is no consensus on how leader cells arise or their essential characteristics. In this Perspective, we propose a framework for defining the essential aspects of leader cells and provide a unifying perspective on the varying cellular and molecular programmes that are adopted by each leader cell subtype to accomplish their functions. This Perspective can lead to more effective strategies to interdict a major contributor to metastatic capability.

Engineering a 3D collective cancer invasion model with control over collagen fiber alignment

Prior to cancer cell invasion, the structure of the extracellular matrix (ECM) surrounding the tumor is remodeled, such that circumferentially oriented matrix fibers become radially aligned. This predisposed radially aligned matrix structure serves as a critical regulator of cancer invasion. However, a biomimetic 3D model recapitulating a tumor's behavioral response to these ECM structures is not yet available. In this study, we have developed a phase-specific, force-guided method to establish a 3D dual topographical tumor model in which each tumor spheroid/organoid is surrounded by radially aligned collagen I fibers on one side and circumferentially oriented fibers on the opposite side. A coaxial rotating cylinder system was employed to construct the dual fiber topography and to pre-seed tumor spheroids/organoids within a single device. This system enables the application of different force mechanisms in the nucleation and elongation phases of collagen fiber polymerization to guide fiber alignment. In the nucleation phase, fiber alignment is enhanced by a horizontal laminar Couette flow driven by the inner cylinder rotation. In the elongation phase, fiber growth is guided by a vertical gravitational force to form a large aligned collagen matrix gel (35 × 25 × 0.5 mm) embedded with >1000 tumor spheroids. The fibers above each tumor spheroid are radially aligned along the direction of gravitational force in contrast to the circumferentially oriented fibers beneath each tumor spheroid/organoid, where the presence of the tumor interferes with the gravity-induced fiber alignment. After tumor invasion, there are more disseminated multicellular clusters on the radially aligned side, compared to the side of the tumor spheroid/organoid facing circumferentially oriented fibers. These results indicate that our 3D dual topographical model recapitulates the preference of tumors to invade and disseminate along radially aligned fibers. We anticipate that this 3D dual topographical model will have broad utility to those studying collective tumor invasion and that it has the potential to identify cancer invasion-targeted therapeutic agents.

Morphology of Active Deformable 3D Droplets

We numerically investigate the morphology and disclination line dynamics of active nematic droplets in three dimensions. Although our model only incorporates the simplest possible form of achiral active stress, active nematic droplets display an unprecedented range of complex morphologies. For extensile activity finger-like protrusions grow at points where disclination lines intersect the droplet surface. For contractile activity, however, the activity field drives cup-shaped droplet invagination, run-and-tumble motion or the formation of surface wrinkles. This diversity of behaviour is explained in terms of an interplay between active anchoring, active flows and the dynamics of the motile dislocation lines. We discuss our findings in the light of biological processes such as morphogenesis, collective cancer invasion and the shape control of biomembranes, suggesting that some biological systems may share the same underlying mechanisms as active nematic droplets.

Collective Polarization of Cancer Cells at the Monolayer Boundary.

Cell polarization, a process depending on both intracellular and intercellular interactions, is crucial for collective cell migration that commonly emerges in embryonic development, tissue morphogenesis, wound healing and cancer metastasis. Although invasive cancer cells display weak cell–cell interactions, they can invade host tissues through a collective mode. Yet, how cancer cells without stable cell–cell junctions polarize collectively to migrate and invade is not fully understood. Here, using a wound-healing assay, we elucidate the polarization of carcinoma cells at the population level. We show that with loose intercellular connections, the highly polarized leader cells can induce the polarization of following cancer cells and subsequent transmission of polarity information by membrane protrusions, leading to gradient polarization at the monolayer boundary. Unlike the polarization of epithelial monolayer where Rac1/Cdc42 pathway functions primarily, our data show that collective polarization of carcinoma cells is predominantly controlled by Golgi apparatus, a disruption of which results in the destruction of collective polarization over a large scale. We reveal that the Golgi apparatus can sustain membrane protrusion formation, polarized secretion, intracellular trafficking, and F-actin polarization, which contribute to collective cancer cell polarization and its transmission between cells. These findings could advance our understanding of collective cancer invasion in tumors.

Diversity in cancer invasion phenotypes indicates specific stroma regulated programs.

Tumor dissemination into the surrounding stroma is the initial step in a metastatic cascade. Invasion into stroma is a non-autonomous process for cancer, and its progression depends upon the stage of cancer, as well as the cells residing in the stroma. However, a systems framework to understand how stromal fibroblasts resist, collude, or aid cancer invasion has been lacking, limiting our understanding of the role of stromal biology in cancer metastasis. We and others have shown that gene perturbation in stromal fibroblasts can modulate cancer invasion into the stroma, highlighting the active role stroma plays in regulating its own invasion. However, cancer invasion into stroma is a complex higher-order process and consists of various sub-phenotypes that together can result in an invasion. Stromal invasion exhibits a diversity of modalities in vivo. It is not well understood if these diverse modalities are correlated, or they emanate from distinct mechanisms and if stromal biology could regulate these characteristics. These characteristics include the extent of invasion, formation, and persistence of invasive forks by cancer as opposed to a collective frontal invasion, the persistence of invading velocity by leader cells at the tip of invasive forks, etc. We posit that quantifying distinct aspects of collective invasion can provide useful suggestions about the plausible mechanisms regulating these processes, including whether the process is regulated by mechanics or by intercellular communication between stromal cells and cancer. Here, we have identified the sub-characteristics of invasion, which might be indicative of broader mechanisms regulating these processes, developed methods to quantify these metrics, and demonstrated that perturbation of stromal genes can modulate distinct aspects of collective invasion. Our results highlight that the genetic state of stromal fibroblasts can regulate complex phenomena involved in cancer dissemination and suggest that collective cancer invasion into stroma is an outcome of the complex interplay between cancer and stromal fibroblasts.

Epigenetically heterogeneous tumor cells direct collective invasion through filopodia-driven fibronectin micropatterning.

Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely underexplored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and three-dimensional (3D) collective invasion. We further identified JAG1 signaling as a previously unknown upstream activator of MYO10 expression in leader cells. Using live-cell imaging, we found that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 up-regulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning.

Prognostic significance of an invasive leader cell-derived mutation cluster on chromosome 16q.

Intratumoral heterogeneity is defined by subpopulations with varying genotypes and phenotypes. Specialized, highly invasive leader cells and less invasive follower cells are phenotypically distinct subpopulations that cooperate during collective cancer invasion. Because leader cells are a rare subpopulation that would be missed by bulk sequencing, a novel image-guided genomics platform was used to precisely select this subpopulation. This study identified a novel leader cell mutation signature and tested its ability to predict prognosis in non-small cell lung cancer (NSCLC) patient cohorts. Spatiotemporal genomic and cellular analysis was used to isolate and perform RNA sequencing on leader and follower populations from the H1299 NSCLC cell line, and it revealed a leader-specific mutation cluster on chromosome 16q. Genomic data from patients with lung squamous cell carcinoma (LUSC; n=475) and lung adenocarcinoma (LUAD; n=501) from The Cancer Genome Atlas were stratified by 16q mutation cluster (16qMC) status (16qMC+ vs 16qMC-) and compared for overall survival (OS), progression-free survival (PFS), and gene set enrichment analysis (GSEA). Poorer OS, poorer PFS, or both were found across all stages and among early-stage patients with 16qMC+ tumors within the LUSC and LUAD cohorts. GSEA revealed 16qMC+ tumors to be enriched for the expression of metastasis- and survival-associated gene sets. This represents the first leader cell mutation signature identified in patients and has the potential to better stratify high-risk NSCLC and ultimately improve patient outcomes.

Tactics of cancer invasion: solitary and collective invasion.

Much attention has been paid on the mechanism of cancer invasion from the viewpoint of the behaviour of individual cancer cells. On the other hand, histopathological analyses of specimens from cancer patients and of cancer invasion model animals have revealed that cancer cells often exhibit collective invasion, characterized by sustained cell-to-cell adhesion and polarized invasion as cell clusters. Interestingly, it has recently become evident that during collective invasion of cancer cells, the cells localized at invasion front (leader cells) and the cells following them (follower cells) exhibit distinct cellular characteristics, and that there exist the cells expressing representative proteins related to both epithelial and mesenchymal properties simultaneously, designated as hybrid epithelial-to-mesenchymal transition (EMT)-induced cells, in cancer tissue. Furthermore, the findings that cells adopted in hybrid EMT state form clusters and show collective invasion in vitro emphasize an importance of hybrid EMT-induced cells in collective cancer invasion. In this article, we overview recent findings of the mechanism underlying collective invasion of cancer cells and discuss the possibility of controlling cancer invasion and metastasis by targeting this process.

Genetic heterogeneity within collective invasion packs drives leader and follower cell phenotypes.

Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.

Abstract 4590: Dissecting the biology of leader and follower cells in collective cancer invasion

Abstract Previous work has demonstrated heterogeneity within collectively invading packs of lung cancer cells, including leader and follower cells that cooperate to facilitate invasion into the microenvironment. In order to better characterize the genetic differences between these two distinct cell types, we performed RNA-seq on purified populations of leader and follower cells from the H1299 non-small cell lung cancer cell line. Through this analysis, we identified that leaders and followers each contain distinct mutational and gene expression profiles, despite originating from the same parental cell line. Importantly, we identified 17 point mutations found uniquely in leaders and 18 point mutations found uniquely in followers, thus representing the first known compilation of leader- and follower-enriched genetic variants. Notable leader-enriched mutated genes included NAE1, NUP93 and ACTR3, while notable follower-enriched mutated genes included NADK, NDUFS1 and LEO1, suggesting possible roles for these genes in the biological function of leader and follower cells. After validating these mutations in the genomic DNA of leaders and followers, we sought to determine whether these mutational signatures are correlated with leader and follower gene expression markers within individual cells in a heterogeneous tumor cell population. To this end, we performed single-cell RNA-seq on H1299 parental, leader and follower cells isolated directly from collectively invading packs in a 3-D matrix. Hierarchical clustering and tSNE analysis based upon most variably expressed genes revealed four distinct cell clusters; two expressing higher levels of leader cell genes including MYO10 and JAG1 (clusters 1 and 4), and two expressing higher levels of follower genes including IL13RA2 and HTATIP2 (clusters 2 and 3). Interestingly, clusters 1 and 4 are composed exclusively of cells with leader mutational profiles, while nearly all cells in clusters 2 and 3 contain follower mutational profiles, suggesting that these mutations could be driving the differential gene expression, and ultimately the unique biological behaviors, of leaders and followers. In addition, clusters 1 and 2 display higher levels of proliferative markers compared with clusters 3 and 4, an indication that there are proliferative and non-proliferative subpopulations within the leader and follower populations. Taken together, these data give us new insight into the multiple levels of heterogeneity that exist within an invasive tumor cell population, suggest novel drivers of leader and follower cell biology in collective invasion, and open the door to new potential strategies for targeting and inhibiting metastasis in human lung cancer. Citation Format: Brian A. Pedro, Jessica Konen, Emily Summerbell, Janna K. Mouw, Manali Rupji, Bhakti Dwivedi, Jeanne Kowalski, Paula M. Vertino, Adam I. Marcus. Dissecting the biology of leader and follower cells in collective cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4590.

Probing three-dimensional collective cancer invasion with DIGME

BackgroundMulticellular pattern formation plays an important role in developmental biology, cancer metastasis and wound healing. While many physical factors have been shown to regulate these multicellular processes, the role of ECM micro-to-meso scale geometry has been poorly understood in 3D collective cancer invasion.ResultsWe have developed a mechanical-based strategy, Diskoid In Geometrically Micropatterned ECM (DIGME). DIGME allows easy engineering of the shape of 3D tissue organoid, the mesoscale ECM heterogeneity, and the fiber alignment of collagen-based ECM all at the same time. We have employed DIGME to study the 3D invasion of MDA-MB-231 diskoids in engineered collagen matrix. We find that the collective cancer invasion is closely regulated by the micro-to-meso scale geometry of the ECM.ConclusionsWe conclude that DIGME provides a simple yet powerful tool to probe 3D dynamics of tissue organoids in physically patterned microenvironments.

Probing cooperative force generation in collective cancer invasion

Collective cellular dynamics in the three-dimensional extracellular matrix (ECM) plays a crucial role in many physiological processes such as cancer invasion. Both chemical and mechanical signaling support cell–cell communications on a variety of length scales, leading to collective migratory behaviors. Here we conduct experiments using 3D in vitro tumor models and develop a phenomenological model in order to probe the cooperativity of force generation in the collective invasion of breast cancer cells. In our model, cell–cell communication is characterized by a single parameter that quantifies the correlation length of cellular migration cycles. We devise a stochastic reconstruction method to generate realizations of cell colonies with specific contraction phase correlation functions and correlation length a. We find that as a increases, the characteristic size of regions containing cells with similar contraction phases grows. For small a values, the large fluctuations in individual cell contraction phases smooth out the temporal fluctuations in the time-dependent deformation field in the ECM. For large a values, the periodicity of an individual cell contraction cycle is clearly manifested in the temporal variation of the overall deformation field in the ECM. Through quantitative comparisons of the simulated and experimentally measured deformation fields, we find that the correlation length for collective force generation in the breast cancer diskoid in geometrically micropatterned ECM (DIGME) system is , which is roughly twice the linear size of a single cell. One possible mechanism for this intermediate cell correlation length is the fiber-mediated stress propagation in the 3D ECM network in the DIGME system.

Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer invasion

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape.

Characterisation of Prognosis and Invasion of Cutaneous Squamous Cell Carcinoma by Podoplanin and E-Cadherin Expression

Background: Around 5% of all cutaneous squamous cell carcinoma (cSCC) metastasise. Metastases usually locate in regional skin and lymph nodes, suggesting collective cancer invasion. The cellular level of tumour invasion and prognostic parameters remain to be characterised. Methods: We performed immunohistochemical analyses of E-cadherin (marker for collective cancer invasion) and podoplanin (marker for epithelial-mesenchymal transition [EMT], single-cell invasion) expression in 102 samples of metastatic and non-metastatic cSCC and 18 corresponding skin and lymph node metastases to characterise the invasion of cSCC. Immunohistochemical results were retrospectively correlated with clinical data. Results: E-cadherin was highly expressed in metastatic and non-metastatic cSCC and skin metastases. This suggests collective cancer invasion. However, E-cadherin was downregulated in poorly differentiated cSCC and lymph node metastases, suggesting partial EMT. Podoplanin was significantly upregulated in metastatic (p = 0.002) and poorly differentiated (p = 0.003) cSCC. Overexpression of podoplanin represented a statistically independent prognostic factor for disease-free survival (p = 0.014). Conclusion: Collective cancer invasion is likely in cSCC. In lymph node metastases and poorly differentiated cSCC, partial EMT is possible. Podoplanin is an independent prognostic parameter for metastasis.