Collective Cell Invasion Research Articles

Assembly of glioblastoma tumoroids and cerebral organoids: a 3D in vitro model for tumor cell invasion.

Glioblastoma (GBM) has a fatal prognosis because of its aggressive and invasive characteristics. Understanding the mechanism of invasion necessitates an elucidation of the relationship between tumor cells and the tumor microenvironment. However, there has been a scarcity of suitable models to investigate this. In this study, we established a glioblastoma-cerebral organoid assembloid (GCOA) model by co-culturing patient-derived GBM tumoroids and human cerebral organoids. Tumor cells from the tumoroids infiltrated the cerebral organoids, mimicking the invasive nature of the parental tumors. Using time-lapse imaging, various invasion patterns of cancer cells within cerebral organoids resembling a normal tissue milieu were monitored. Both single- and collective-cell invasion was captured in real-time. We also confirmed the formation of an intercellular tumor network and tumor-normal-cell interactions. Furthermore, the transcriptomic characterization of GCOAs revealed distinct features of invasive tumor cells. Overall, this study established the GCOA as a three-dimensional (3D) in vitro assembloid model to investigate invasion mechanisms and interactions between tumor cells and their microenvironment.

Back Cover Image

ON THE BACK COVER: DA‐Raf inhibits ERK activation, resulting in the suppression of collective cell migration and invasion of KRAS‐mutant cancer cells. In human pancreatic carcinoma MIA PaCa‐2 cells, phospho‐ERK1/2 (magenta) is suppressed by the expression of EGFP‐DA‐Raf (green). EGFP‐DA‐Raf is concentrated on the plasma membrane.Credit: Aoi Matsuda and Takeshi Endo (Chiba University, Japan) image

The highly metastatic 4T1 breast carcinoma model possesses features of a hybrid epithelial/mesenchymal phenotype.

Epithelial-mesenchymal transitions (EMTs) are thought to promote metastasis via downregulation of E-cadherin (also known as Cdh1) and upregulation of mesenchymal markers such as N-cadherin (Cdh2) and vimentin (Vim). Contrary to this, E-cadherin is retained in many invasive carcinomas and promotes collective cell invasion. To investigate how E-cadherin regulates metastasis, we examined the highly metastatic, E-cadherin-positive murine 4T1 breast cancer model, together with the less metastatic, 4T1-related cell lines 4T07, 168FARN and 67NR. We found that 4T1 cells display a hybrid epithelial/mesenchymal phenotype with co-expression of epithelial and mesenchymal markers, whereas 4T07, 168FARN, and 67NR cells display progressively more mesenchymal phenotypes in vitro that relate inversely to their metastatic capacity in vivo. Using RNA interference and constitutive expression, we demonstrate that the expression level of E-cadherin does not determine 4T1 or 4T07 cell metastatic capacity in mice. Mechanistically, 4T1 cells possess highly dynamic, unstable cell-cell junctions and can undergo collective invasion without E-cadherin downregulation. However, 4T1 orthotopic tumors in vivo also contain subregions of EMT-like loss of E-cadherin. Thus, 4T1 cells function as a model for carcinomas with a hybrid epithelial/mesenchymal phenotype that promotes invasion and metastasis.

Modelling the effect of cell motility on mixing and invasion in epithelial monolayers

Collective cell invasion underlies several biological processes such as wound healing, embryonic development, and cancerous invasion. Here, we investigate the impact of cell motility on invasion in epithelial monolayers and its coupling to cellular mechanical properties, such as cell-cell adhesion and cortex contractility. We develop a two-dimensional computational model for cells with active motility based on the cellular Potts model, which predicts that the cellular invasion speed is mainly determined by active cell motility and is independent of the biological and mechanical properties of the cells. We also find that, in general, motile cells out-compete and invade non-motile cells, however, this can be reversed by differential cell proliferation. Stable coexistence of motile and static cell types is also possible for certain parameter regimes.

MEK inhibitors and DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, prevent the migration and invasion of KRAS-mutant cancer cells.

The Ras-induced ERK pathway (Raf-MEK-ERK signaling cascade) regulates a variety of cellular responses including cell proliferation, survival, and migration. Activating mutations in RAS genes, particularly in the KRAS gene, constitutively activate the ERK pathway, resulting in tumorigenesis, cancer cell invasion, and metastasis. DA-Raf1 (DA-Raf) is a splicing isoform of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Consequently, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner and can serve as a tumor suppressor that targets mutant Ras protein-induced tumorigenesis. We show here that MEK inhibitors and DA-Raf interfere with the in vitro collective cell migration and invasion of human KRAS-mutant carcinoma cell lines, the lung adenocarcinoma A549, colorectal carcinoma HCT116, and pancreatic carcinoma MIA PaCa-2 cells. DA-Raf expression was silenced in these cancer cell lines. All these cell lines had high collective migration abilities and invasion properties in Matrigel, compared with nontumor cells. Their migration and invasion abilities were impaired by suppressing the ERK pathway with the MEK inhibitors U0126 and trametinib, an approved anticancer drug. Expression of DA-Raf in MIA PaCa-2 cells reduced the ERK activity and hindered the migration and invasion abilities. Therefore, DA-Raf may function as an invasion suppressor protein in the KRAS-mutant cancer cells by blocking the Ras-ERK pathway when DA-Raf expression is induced in invasive cancer cells.

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.

Spatial heterogeneity in tumor adhesion qualifies collective cell invasion

Collective cell invasion (CCI), a canon of most invasive solid tumors, is an emergent property of the interactions between cancer cells and their surrounding extracellular matrix (ECM). However, tumor populations invariably consist of cells expressing variable levels of adhesive proteins that mediate such interactions, disallowing an intuitive understanding of how tumor invasiveness at a multicellular scale is influenced by spatial heterogeneity of cell-cell and cell-ECM adhesion. Here, we have used a Cellular Potts model-based multiscale computational framework that is constructed on the histopathological principles of glandular cancers. In earlier efforts on homogenous cancer cell populations, this framework revealed the relative ranges of interactions, including cell-cell and cell-ECM adhesion that drove collective, dispersed, and mixed multimodal invasion. Here, we constitute a tumor core of two separate cell subsets showing distinct intra- and inter-subset cell-cell or cell-ECM adhesion strengths. These two subsets of cells are arranged to varying extents of spatial intermingling, which we call the heterogeneity index (HI). We observe that low and high inter-subset cell adhesion favors invasion of high-HI and low-HI intermingled populations with distinct intra-subset cell-cell adhesion strengths, respectively. In addition, for explored values of cell-ECM adhesion strengths, populations with high HI values collectively invade better than those with lower HI values. We then asked how spatial invasion is regulated by progressively intermingled cellular subsets that are epithelial, i.e., showed high cell-cell but poor cell-ECM adhesion, and mesenchymal, i.e., with reversed adhesion strengths to the former. Here too, inter-subset adhesion plays an important role in contextualizing the proportionate relationship between HI and invasion. An exception to this relationship is seen for cases of heterogeneous cell-ECM adhesion where sub-maximal HI patterns with higher outer localization of cells with stronger ECM adhesion collectively invade better than their relatively higher-HI counterparts. Our simulations also reveal how adhesion heterogeneity qualifies collective invasion, when either cell-cell or cell-ECM adhesion type is varied but results in an invasive dispersion when both adhesion types are simultaneously altered.

Discrete and continuous mathematical models of sharp-fronted collective cell migration and invasion.

Mathematical models describing the spatial spreading and invasion of populations of biological cells are often developed in a continuum modelling framework using reaction-diffusion equations. While continuum models based on linear diffusion are routinely employed and known to capture key experimental observations, linear diffusion fails to predict well-defined sharp fronts that are often observed experimentally. This observation has motivated the use of nonlinear degenerate diffusion; however, these nonlinear models and the associated parameters lack a clear biological motivation and interpretation. Here, we take a different approach by developing a stochastic discrete lattice-based model incorporating biologically inspired mechanisms and then deriving the reaction-diffusion continuum limit. Inspired by experimental observations, agents in the simulation deposit extracellular material, which we call a substrate, locally onto the lattice, and the motility of agents is taken to be proportional to the substrate density. Discrete simulations that mimic a two-dimensional circular barrier assay illustrate how the discrete model supports both smooth and sharp-fronted density profiles depending on the rate of substrate deposition. Coarse-graining the discrete model leads to a novel partial differential equation (PDE) model whose solution accurately approximates averaged data from the discrete model. The new discrete model and PDE approximation provide a simple, biologically motivated framework for modelling the spreading, growth and invasion of cell populations with well-defined sharp fronts. Open-source Julia code to replicate all results in this work is available on GitHub.

Abstract 4297: Interrogating metabolic heterogeneity and cooperation in lung cancer collective cell invasion

Abstract Tumor heterogeneity contributes to tumor progression and a variable response to treatment. We have previously shown that tumor heterogeneity - including metabolic, transcriptional and mutational - underlies non-small cell lung cancer (NSCLC) collective invasion. Using stable subpopulations derived with our Spatiotemporal Cellular and Genomic Analysis (SaGA) technique, we found that two distinct subpopulations of the H1299 NSCLC line cooperate during collective tumor invasion in vitro; while H1299 pack leaders are heavily dependent on oxidative phosphorylation, H1299 pack followers are more reliant on glycolysis. To further dissect how intratumoral heterogeneity enables cancer progression to metastasis, we incorporate high resolution imaging, stable fluorescent labeling, gene editing, and in vitro tumor invasion models to interrogate mechanisms of metabolic cooperation between heterogeneous NSCLC subpopulations. We hypothesize that cooperation and communication between these distinct metabolic subpopulation drives metastatic tumor progression. To determine mechanisms of metabolic cooperativity between subpopulations, we stably transduced our subpopulations to fluorescently label either the mitochondria (through TOM20 labeling) or a key enzyme in glycolysis (through glucose 6-phosphate isomerase GPI labeling), cultured labeled subpopulations with unlabeled subpopulations, and observed metabolic changes and exchange via time-lapse, fluorescence microscopy and flow cytometry in 2D culture conditions. Next steps include visualizing the metabolic cooperation that occurs in conditions mimicking the tumor microenvironment (i.e., 3D invasion assays) using super-resolution microscopy. Taken together, we have developed a method of temporally visualizing metabolic exchange with high spatial resolution between NSCLC subpopulations to investigate the underlying mechanisms enabling successful cell invasion in a variety of tumor microenvironments. Thus far, we have discovered multiple broad mechanisms of exchange including tunneling nanotubes and extracellular vesicles, both of which are inducible with certain stressors common to the tumor microenvironment. Citation Format: Isaac E. Robinson, Veronika Matsuk, Mala Shanmugam, Janna K. Mouw, Adam I. Marcus. Interrogating metabolic heterogeneity and cooperation in lung cancer collective cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4297.

Abstract 1780: Utilizing bisbiguanide compounds to disrupt metabolic heterogeneity in non-small cell lung cancer

Abstract Metastasis accounts for 90% of cancer-related deaths; however, the molecularmechanisms by which cancer cells invade and metastasize remain poorly understood.Around 80% of lung cancer patients present with metastatic disease and have only a 5%5-year relative survival. Metastases are often seeded by heterogeneous cells that invadecollectively in cellular packs. However, most studies and treatments focus on bulk cellularpopulations leaving subpopulations that drive metastasis masked. We previouslypublished on metabolic heterogeneity within collectively invading lung cancer cells andidentified the bisbiguanide compound alexidine as a disrupter of lung cancermitochondrial metabolism and invasion. To expand on these findings, we developed newbiguanides and identified promising candidates, AX-4 and AX-7. These analogs inducemitochondrial structural defects and cause metabolic rewiring. Treated lung cancer cellsexhibited reduced oxygen consumption and increased lactate production. Importantly,AX-4 and AX-7 reduced collective cell invasion, highlighting the potential of these newbiguanides to limit cancer metastasis. Taken together, we have developed new chemicaltools to disrupt mitochondrial metabolism. These tools will be useful to further parse theroles of metabolically heterogenous cancer cells on tumor growth and metastasis. Citation Format: Veronika Y. Matsuk, Christina M. Knippler, Jamie L. Arnst, Isaac E. Robinson, Tala O. Khatib, Mala Shanmugam, Thota Ganesh, Janna K. Mouw, Adam I. Marcus. Utilizing bisbiguanide compounds to disrupt metabolic heterogeneity in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1780.

Matrix confinement modulates 3D spheroid sorting and burst-like collective migration

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites. Statement of significanceThe mechanical properties of the tumor microenvironment significantly influence cancer cell migration within the primary tumor, yet how these properties affect intercellular interactions in heterogeneous tumors is not well understood. By utilizing calcium and calcium chelators, we dynamically alter collagen-alginate hydrogel stiffness and investigate tumor cell behavior within co-culture spheroids in response to varying degrees of matrix confinement. High confinement is found to trigger cell sorting while reducing confinement for sorted spheroids facilitates collective cell invasion. Notably, without prior sorting, spheroids do not exhibit burst-like migration, regardless of confinement levels. This work establishes that matrix confinement and intercellular adhesion regulate 3D spheroid dynamics, offering insights into cellular organization and migration within the primary tumor.

An entropy-based approach for assessing the directional persistence of cell migration

Cell migration, which is primarily characterized by directional persistence, is essential for the development of normal tissues and organs, as well as for numerous pathological processes. However, there is a lack of simple and efficient tools to analyze the systematic properties of persistence based on cellular trajectory data. Here, we present a novel approach, the entropy of angular distribution , which combines cellular turning dynamics and Shannon entropy to explore the statistical and time-varying properties of persistence that strongly correlate with cellular migration modes. Our results reveal the changes in the persistence of multiple cell lines that are tightly regulated by both intra- and extracellular cues, including Arpin protein, collagen gel/substrate, and physical constraints. Significantly, some previously unreported distinctive details of persistence have also been captured, helping to elucidate how directional persistence is distributed and evolves in different cell populations. The analysis suggests that the entropy of angular distribution-based approach provides a powerful metric for evaluating directional persistence and enables us to better understand the relationships between cellular behaviors and multiscale cues, which also provides some insights into the migration dynamics of cell populations, such as collective cell invasion.

Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models.

Cancers have a complex relationship with the surrounding environment that regulates everything from progression to response to treatment. Cell-cell and cell-matrix interactions are heavily influenced by protease biology. Studies on the tumor microenvironment have revealed a new complexity for proteases, describing novel substrates for classic proteases, and protease-independent roles for these enzymes. The rapid expansion of 3D in vitro model systems provides excellent tools to study the intricate influence of proteases on the tumor microenvironment. Here we describe a spheroid invasion assay, providing a platform to interrogate key protease-matrix interactions in the context of early-stage breast cancer. Incorporation of pharmacological inhibition and RNAi techniques enables the elucidation of key protease-dependent pathways and can be complemented with immunofluorescence analysis to visualize matrix cleavage events and visualize cell behavior during collective cell invasion.

Transcriptional induction of NF-κB-inducing kinase by E2F4/5 facilitates collective invasion of GBM cells

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway promotes the invasiveness of GBM cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK (MAP3K14) is upregulated at the transcriptional level in invading cell populations, with the highest NIK expression observed in the most invasive cells. GBM cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote GBM cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting GBM pathogenesis.

Matrix stiffening facilitates the collective invasion of breast cancer through the periostin-integrin mechanotransduction pathway

Matrix rigidity is a critical contributor to tumor progression; however, whether and how matrix stiffness modulates the collective invasion of tumor cells remain unknown. Here we demonstrate that increased matrix stiffness activates YAP to promote the secretion of periostin (POSTN) in cancer-associated fibroblasts, which in turn augments the matrix rigidity of mammary glands and breast tumor tissues by facilitating collagen crosslinking. Moreover, decreased tissue stiffening resulted from the POSTN deficiency impairs peritoneal metastatic potential of orthotopic breast tumors. Increased matrix stiffness also promotes three-dimensional (3D) collective breast tumor cell invasion via multicellular cytoskeleton remodeling. POSTN triggers the integrin/FAK/ERK/Cdc42/Rac1 mechanotransduction pathway during 3D collective invasion of breast tumor. Clinically, high POSTN expression correlates with high collagen levels in breast tumors and cooperatively determines the metastatic recurrence potential in breast cancer patients. Collectively, these findings indicate that matrix rigidity promotes 3D collective invasion of breast tumor cells via the YAP-POSTN-integrin mechanotransduction signaling.

Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma.

Cancers, such as squamous cell carcinoma, frequently invade as multicellular units. However, these invading units can be organised in a variety of ways, ranging from thin discontinuous strands to thick 'pushing' collectives. Here we employ an integrated experimental and computational approach to identify the factors that determine the mode of collective cancer cell invasion. We find that matrix proteolysis is linked to the formation of wide strands but has little effect on the maximum extent of invasion. Cell-cell junctions also favour wide strands, but our analysis also reveals a requirement for cell-cell junctions for efficient invasion in response to uniform directional cues. Unexpectedly, the ability to generate wide invasive strands is coupled to the ability to grow effectively when surrounded by extracellular matrix in three-dimensional assays. Combinatorial perturbation of both matrix proteolysis and cell-cell adhesion demonstrates that the most aggressive cancer behaviour, both in terms of invasion and growth, is achieved at high levels of cell-cell adhesion and high levels of proteolysis. Contrary to expectation, cells with canonical mesenchymal traits - no cell-cell junctions and high proteolysis - exhibit reduced growth and lymph node metastasis. Thus, we conclude that the ability of squamous cell carcinoma cells to invade effectively is also linked to their ability to generate space for proliferation in confined contexts. These data provide an explanation for the apparent advantage of retaining cell-cell junctions in squamous cell carcinomas.

Activation of an actin signaling pathway in pre-malignant mammary epithelial cells by P-cadherin is essential for transformation.

Alterations in the expression or function of cell adhesion molecules have been implicated in all steps of tumor progression. Among those, P-cadherin is highly enriched in basal-like breast carcinomas, playing a central role in cancer cell self-renewal, collective cell migration and invasion. To establish a clinically relevant platform for functional exploration of P-cadherin effectors in vivo, we generated a humanized P-cadherin Drosophila model. We report that actin nucleators, Mrtf and Srf, are main P-cadherin effectors in fly. We validated these findings in a human mammary epithelial cell line with conditional activation of the SRC oncogene. We show that, prior to promoting malignant phenotypes, SRC induces a transient increase in P-cadherin expression, which correlates with MRTF-A accumulation, its nuclear translocation and the upregulation of SRF target genes. Moreover, knocking down P-cadherin, or preventing F-actin polymerization, impairs SRF transcriptional activity. Furthermore, blocking MRTF-A nuclear translocation hampers proliferation, self-renewal and invasion. Thus, in addition to sustaining malignant phenotypes, P-cadherin can also play a major role in the early stages of breast carcinogenesis by promoting a transient boost of MRTF-A-SRF signaling through actin regulation.

Size-induced motion mode transitions in collective cell invasion toward free spaces.

Collective cell migration plays a vital role in various physiological and pathological processes, such as embryonic development and tumor metastasis. Recent experiments have shown that different from isolated cells, the moving cell groups exhibit rich emerging motion modes in response to external geometrical constraints. By considering the interactions between neighboring cells and internal biomechanical processes of each cell (i.e., cell sociality and cell individuality), we develop an active vertex model to investigate the emerging modes of collective cell migration in microchannels. Single-cell polarization is propelled by continuous protrusion of its leading edge and retraction of the rear. We here introduce the contribution of continuous protrusions and retractions of lamellipodia, named the protrusion alignment mechanism, to the cell individuality. Using the present model, it is found that altering the width of channels can trigger the motion mode transitions of cell groups. When cells move in narrow channels, the protrusion alignment mechanism brings neighboring groups of coordinated cells into conflicts and in turn induces the caterpillar-like motion mode. As the channel width increases, local swirls spanning the channel in width first appear as long as the channel width is smaller than the intrinsic correlation length of cell groups. Then, only local swirls with a maximum diameter of the intrinsic correlation length are formed, when the channel is sufficiently wider. These rich dynamical modes of collective cells originate from the competition between cell individuality and sociality. In addition, the velocity of the cell sheet invading free spaces varies with the channel size-induced transitions of migration modes. Our predictions are in broad agreement with many experiments and may shed light on the spatiotemporal dynamics of active matter.

Visualizing and Quantifying mRNA Localization at the Invasive Front of 3D Cancer Spheroids.

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.

Analysis of Energy-Driven Leader-Follower Hierarchy During Collective Cancer Cell Invasion.

Many solid tumors can invade the surrounding three-dimensional (3D) tissue in a collective manner, and increasing evidence suggests that collective migration makes cancer cell clusters more invasive and metastatic than individual cells. A cohesive cohort of cancer cells can have many advantages over individual cells, including more efficient bioenergetics that have been recently identified. Minimization of bioenergetic costs during collective cell migration drives leader-follower dynamics and contributes to enhanced cancer invasion. Hence, it is critical to understand the migratory and bioenergetic dynamics of cancer collective invasion. While analysis of structures and dynamics in a 3D space has been a challenging task, here we describe a widely applicable method to analyze the energy-driven leader-follower hierarchy during cancer collective invasion. An in vitro tumor spheroid model is employed to reproduce the in vivo collective behaviors of cancer cells while allowing high spatiotemporal resolution imaging, where the leader-follower dynamics can be analyzed by tracking nuclear positions. As glucose is one of the main energy sources that fuel cancer cell migration, the quantification of glucose uptake along the invading strands provides an estimate of the energy demand associated with collective invasion. Finally, we describe a method to quantify the dynamics of intracellular energy level using the PercevalHR ATP:ADP ratio biosensor.