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.

TRAIL specifically induces apoptosis in cancer cells without affecting healthy cells. However, TRAIL’s cancer cytotoxicity was insufficient in clinical trials. Circulatory-shear stress is known to sensitize cancer cells to TRAIL. In this study, we examine the mechanism of this TRAIL sensitization with the goal of translating it to static conditions. GsMTx-4, a Piezo1 inhibitor, was found to reduce shear stress-related TRAIL sensitization, implicating Piezo1 activation as a potential TRAIL-sensitizer. The Piezo1 agonist Yoda1 recreated shear stress-induced TRAIL sensitization under static conditions. A significant increase in apoptosis occurred when PC3, COLO 205, or MDA-MB-231 cells were treated with Yoda1 and TRAIL in combination, but not in Bax-deficient DU145 cells. Calpastatin inhibited apoptosis in Yoda1-TRAIL treated cells, indicating that calpain activation is necessary for apoptosis by Yoda1 and TRAIL. Yoda1 and TRAIL treated PC3 cells showed increased mitochondrial outer membrane permeability (MOMP), mitochondrial depolarization, and activated Bax. This implies that Piezo1 activation sensitizes cancer cells to TRAIL through a calcium influx that activates calpains. The Calpains then induce MOMP by enhancing Bax activation. From these experiments a computational model was developed to simulate apoptosis for cells treated with TRAIL and increased calcium. The computational model elucidated the proapoptotic or antiapoptotic roles of Bax, Bcl-2, XIAP, and other proteins important in the mitochondrial-apoptotic signaling pathway.

Previous studies have demonstrated that CTCs do not travel in the bloodstream alone, but rather are accompanied by clusters of stromal cells such as cancer associated fibroblasts (CAFs). Our laboratory has confirmed the presence of CAFs in the peripheral blood of prostate cancer (PC) patients. The observation that CAFs disseminate with CTCs prompts the examination of the role of CAFs in CTC survival under physiological shear stress during the dissemination process using a clinically relevant, three-dimensional (3D) co-culture model. In this study, we found that "reactive CAFs" induce shear resistance to prostate tumor cells via intercellular contact and soluble derived factors. In addition, these reactive CAFs conserve the proliferative capability of tumor cells in the presence of high magnitude fluid shear stress (FSS). This reactive CAF phenotype emerges from normal fibroblasts (NF), which take on the CAF phenotype when co-cultured with tumor cells. The reactive CAFs showed higher expression of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) compared to differentiated CAFs, when co-cultured with PC cells at the same experimental conditions. Together, we found that the activation mechanism of NF to CAF comprises different stages that progress from a reactive to quiescent cellular state in which these two states are differentiated by the fluctuation of intensity in CAF markers. Here we determined that a reactive state of CAFs proved to be important for supporting tumor cell survival and proliferation. These findings suggest the use of CAFs as a marker for cancer progression and a potential target for novel cancer therapeutics to treat metastatic disease.

While considerable progress has been made in studying genetic and cellular aspects of metastasis with in vitro cell culture and in vivo animal models, the driving mechanisms of each step of metastasis are still relatively unclear due to their complexity. Moreover, little progress has been made in understanding how cellular fitness in one step of the metastatic cascade correlates with ability to survive other subsequent steps. Engineered models incorporate tools such as tailored biomaterials and microfabrication to mimic human disease progression, which when coupled with advanced quantification methods permit comparisons to human patient samples and in vivo studies. Here, we review novel tools and techniques that have been recently developed to dissect key features of the metastatic cascade using primary patient samples and highly representative microenvironments for the purposes of advancing personalized medicine and precision oncology. Although improvements are needed to increase tractability and accessibility while faithfully simulating the in vivo microenvironment, these models are powerful experimental platforms for understanding cancer biology, furthering drug screening, and facilitating development of therapeutics.

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. Significance: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.