Among the secondary target organs for metastatic breast cancer, brain metastasis is extremely aggressive in nature, resulting in lower survival rates. These metastatic cancer cells have the potential to enter a dormant state in the brain, allowing them to survive for extended time periods. The brain microenvironment plays a key role in controlling the dormant phenotype, yet how various components of this microenvironment influence dormancy is not well understood. In this work, we employed hyaluronic acid (HA)-based hydrogels as a mimetic of the brain tissue environment to study the role of biochemical cues, specifically, the impact of laminin and laminin-derived peptides IKVAV and YIGSR on the regulation of brain metastatic breast cancer cell dormancy versus proliferation. We applied varying protein/peptide concentrations and confirmed functionalization on HA hydrogel surfaces. We then seeded 10,000 cancer cells on the hydrogel surface and cultured them for 5 days. We found that in the presence of laminin or IKVAV, MDA-MB-231Br cells transitioned from a rounded to a spread morphology and exhibited enhanced proliferation as the laminin/IKVAV concentration increased. In contrast, in hydrogels functionalized with YIGSR, these cells maintained a rounded morphology, with no impact on proliferation with varying YIGSR concentrations. We confirmed the involvement of αVβ3 integrin in mediating tumor cell phenotype in hydrogels functionalized with laminin. By evaluating known markers of dormancy and proliferation, we found a direct correlation between the presence of laminin and IKVAV and increased phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) positivity, along with decreased phosphorylated p38 (p-p38) positivity, while in hydrogels functionalized with YIGSR, the levels of both p-ERK and p-p38 remained unaltered. Finally, we demonstrated that when cells were transferred from IKVAV-deficient to IKVAV-rich hydrogels, the hydrogel induced cellular dormancy was reversible. Collectively, our findings provide insights into how laminin and laminin-derived cues regulate brain metastatic breast cancer cell dormancy versus proliferation.

Tumor cells with organ-specific metastasis traits arise in primary lesions with substantial variations of local niche mechanics owing to intratumoral heterogeneity. However, the roles of mechanically heterogeneous primary tumor microenvironment in metastatic organotropism remain an enigma. This study reports that persistent priming in soft but not stiff niches that mimic primary tumor mechanical heterogeneity induces transcriptional reprogramming reminiscent of neuron and promotes the acquisition of brain metastatic potential. Soft-primed cells generate brain metastases in vivo through enhanced transendothelial migration across blood-brain barrier and brain colonization, which is further supported by the findings that tumor cells residing in local soft niches of primary xenografts exhibit brain metastatic tropism. Mechanistically, soft niches suppress cytoskeleton-nucleus-mediated mechanotransduction, which promotes histone deacetylase 3 activity. Inhibiting histone deacetylase 3 abolishes niche softness-induced brain metastatic ability. Collectively, this study uncovers a previously unappreciated role of local niche softness within primary tumors in brain metastasis, highlighting the significance of primary tumor mechanical heterogeneity in metastatic organotropism.

Breast cancer remains one of the leading causes of death in women around the world. A majority of deaths from breast cancer occur due to cancer cells colonizing distant organ sites. When colonizing these distant organ sites, breast cancer cells have been known to enter into a state of dormancy for extended periods of time. However, the mechanisms that promote dormancy as well as dormant-to-proliferative switch are not fully understood. The tumor microenvironment plays a key role in mediating cancer cell phenotype including regulation of the dormant state. In this review, we highlight cell-cell interactions in the tumor microenvironment mediating breast cancer dormancy at the primary and metastatic sites. Specifically, we describe how immune cells from the lymphoid lineage, tumor-associated myeloid lineage cells, and stromal cells of non-hematopoietic origin as well as tissue resident stromal cells impact dormancy vs. proliferation in breast cancer cells as well as the associated mechanisms. In addition, we highlight the importance of developing model systems and the associated considerations that will be critical in unraveling the mechanisms that promote primary and metastatic breast cancer dormancy mediated via cell-cell interactions.

In recent years, polymeric hydrogels have been employed to investigate cancer cell-extracellular matrix (ECM) interactions in vitro. In the context of breast cancer, cancer cells are known to degrade the ECM using matrix-metalloproteinases (MMPs) to support invasion resulting in disease progression. Polymeric hydrogels incorporating MMP-cleavable peptides have been employed to study cancer cell invasion, however, the approaches employed to incorporate these peptides often change other hydrogel properties. This underscores the need for decoupling hydrogel properties while incorporating MMP-cleavable peptides. Herein, we report structurally decoupled hyaluronic acid (HA) hydrogels formulated using varying ratios of a biologically sensitive MMP-cleavable peptide and an insensitive counterpart (Dithiothreitol (DTT) or polyethylene glycol dithiol (PEGDT)) to study MMP-mediated metastatic breast cancer cell invasion. Rheological, swelling ratio, estimated mesh size, and permeability measurements showed similar mechanical and physical properties for hydrogels crosslinked with different DTT (or PEGDT)/MMP ratios. However, their degradation rate in the presence of collagenase correlated with the ratio of MMP-cleavable peptide. Encapsulated metastatic breast cancer spheroids in HA hydrogels with MMP sensitivity exhibited increased invasiveness compared to those without MMP sensitivity after 14 days of culture. Overall, such structurally decoupled HA hydrogels provide a platform to study MMP-mediated breast cancer cell invasion in vitro.

Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.

Breast cancer is the most common malignancy accounting for 12.5% of all newly diagnosed cancer cases across the globe. Breast cancer cells are known to metastasize to distant organs (i.e., brain), wherein they can exhibit a dormant phenotype for extended time periods. These dormant cancer cells exhibit reduced proliferation and therapeutic resistance. However, the mechanisms by which dormant cancer cells exhibit resistance to therapy, in the context of brain metastatic breast cancer (BMBC), is not well understood. Herein, we utilized hyaluronic acid (HA) hydrogels with varying stiffnesses to study drug responsiveness in dormant vs. proliferative BMBC cells. It was found that cells cultured on soft HA hydrogels (∼0.4 kPa) that showed a non-proliferative (dormant) phenotype exhibited resistance to Paclitaxel or Lapatinib. In contrast, cells cultured on stiff HA hydrogels (∼4.5 kPa) that showed a proliferative phenotype exhibited responsiveness to Paclitaxel or Lapatinib. Moreover, dormancy-associated resistance was found to be due to upregulation of the serum/glucocorticoid regulated kinase 1 (SGK1) gene which was mediated, in part, by the p38 signaling pathway. Accordingly, SGK1 inhibition resulted in a dormant-to-proliferative switch and response to therapy. Overall, our study demonstrates that matrix stiffness influences dormancy-associated therapy response mediated, in part, via the p38/SGK1 axis.

Metastasis, the spread, and growth of malignant cells at secondary sites within a patient's body, accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10-16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses, which represents a critical barrier for the development of efficient therapies for affected breast cancer patients.

Previous research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM, we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP, other cell lines of MCF-7, HCC-1806, and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid.

We found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover, MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids.

Our co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture.

Here, we present a protocol to generate dormant brain metastatic breast cancer (BMBC) spheroids utilizing hyaluronic acid (HA) hydrogels. We describe the steps for construction of spheroids from human BMBC cell lines MDA-MB-231Br and BT474Br3, HA hydrogel preparation, and spheroid plating on HA hydrogels and in suspension culture. We then detail the impact of HA hydrogel on the dormant phenotype of spheroids by measuring spheroid cross-sectional area, cell numbers, and EdU staining. For complete details on the use and execution of this protocol, please refer to Kondapaneni et al.1.

After reading the review by An et al "Biological factors driving colorectal cancer metastasis", which covers the problem of the metastasis of colorectal cancer (CRC), I had a desire to discuss with readers one of the exciting problems associated with dormant metastases. Most deaths from CRCs are caused by metastases, which can be detected both at diagnosis of the primary tumor and several years or even decades after treatment. This is because tumor cells that enter the bloodstream can be destroyed by the immune system, cause metastatic growth, or remain dormant for a long time. Dormant tumor cells may not manifest themselves throughout a person's life or, after some time and under appropriate conditions, may give rise to the growth of metastases. In this editorial, we will discuss the most important features of dormant metastases and the mechanisms of premetastatic niche formation, as well as factors that contribute to the activation of dormant metastases in CRCs. We will pay special attention to the possible mechanisms involved in the formation of circulating tumor cell complexes and the choice of therapeutic strategies that promote the dormancy or destruction of tumor cells in CRCs.

Glioblastoma multiforme (GBM), a primary brain cancer, is one of the most aggressive forms of human cancer, with a very low patient survival rate. A characteristic feature of GBM is the diffuse infiltration of tumor cells into the surrounding brain extracellular matrix (ECM) that provide biophysical, topographical, and biochemical cues. In particular, ECM stiffness and composition is known to play a key role in controlling various GBM cell behaviors including proliferation, migration, invasion, as well as the stem-like state and response to chemotherapies. In this review, we discuss the mechanical characteristics of the GBM microenvironment at multiple length scales, and how biomaterial scaffolds such as polymeric hydrogels, and fibers, as well as microfluidic chip-based platforms have been employed as tissue mimetic models to study GBM mechanobiology. We also highlight how such tissue mimetic models can impact the field of GBM mechanobiology.

A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA-MB-231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle-like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.

The extracellular matrix (ECM) is a complex network of proteins and glycans, dynamically remodeled and specifically tailored to the structure/function of each organ. The malignant transformation of cancer cells is determined by both cell intrinsic properties, such as mutations, and extrinsic variables, such as the mixture of surrounding cells in the tumor microenvironment and the biophysics of the ECM. During cancer progression, the ECM undergoes extensive remodeling, characterized by disruption of the basal lamina, vascular endothelial cell invasion, and development of fibrosis in and around the tumor cells resulting in increased tissue stiffness. This enhanced rigidity leads to aberrant mechanotransduction and further malignant transformation potentiating the de-differentiation, proliferation and invasion of tumor cells. Interestingly, this fibrotic microenvironment is primarily secreted and assembled by non-cancerous cells. Among them, the cancer-associated fibroblasts (CAFs) play a central role. CAFs massively produce fibronectin together with type I collagen. This review delves into the primary interactions and signaling pathways through which fibronectin can support tumorigenesis and metastasis, aiming to provide critical molecular insights for better therapy response prediction.

Dormant, disseminated breast cancer cells resist treatment and may relapse into malignant metastases after decades of quiescence. Identifying how and why these dormant breast cancer cells are triggered into outgrowth is a key unsolved step in treating latent, metastatic breast cancer. However, our understanding of breast cancer dormancy in vivo is limited by technical challenges and ethical concerns with triggering the activation of dormant breast cancer. In vitro models avoid many of these challenges by simulating breast cancer dormancy and activation in well-controlled, bench-top conditions, creating opportunities for fundamental insights into breast cancer biology that complement what can be achieved through animal and clinical studies. In this review, we address clinical and preclinical approaches to treating breast cancer dormancy, how precisely controlled artificial environments reveal key interactions that regulate breast cancer dormancy, and how future generations of biomaterials could answer further questions about breast cancer dormancy.

The current research models of breast cancer are usually limited in their capacity to recapitulate the tumor microenvironment in vitro. The lack of an extracellular matrix (ECM) oversimplifies cell-cell or cell-ECM cross-talks. Moreover, the lack of tumor-associated macrophages (TAMs), that can comprise up to 50% of some solid neoplasms, poses a major problem for recognizing various hallmarks of cancer. To address these concerns, a type of direct breast cancer cells (BCCs)-TAMs coculture organoid model was well developed by a sequential culture method in this study. Alginate cryogels were fabricated with appropriate physical and mechanical properties to serve as an alternative ECM. Then, our previous experience was leveraged to polarize TAMs inside of the cryogels for creating an in vitro immune microenvironment. The direct coculture significantly enhanced BCCs organoid growth and cancer aggressive phenotypes, including the stemness, migration, ECM remodeling, and cytokine secretion. Furthermore, transcriptomic analysis and protein-protein interaction networks implied certain pathways (PI3K-Akt pathway, MAPK signaling pathway, etc.) and targets (TNF, PPARG, TLR2, etc.) during breast cancer progression in a TAM-leading immune microenvironment. Future studies to advance treatment strategies for BCC patients may benefit from using this facile model to reveal and target the interactions between cancer signaling and the immune microenvironment.

Triple-Negative Breast Cancer is particularly aggressive, and its metastasis to the brain has a significant psychological impact on patients' quality of life, in addition to reducing survival. The development of brain metastases is particularly harmful in triple-negative breast cancer (TNBC). To date, the mechanisms that induce brain metastasis in TNBC are poorly understood.

Using a human blood-brain barrier (BBB) in vitro model, an in vitro 3D organotypic extracellular matrix, an ex vivo mouse brain slices co-culture and in an in vivo xenograft experiment, key step of brain metastasis were recapitulated to study TNBC behaviors.

In this study, we demonstrated for the first time the involvement of the precursor of Nerve Growth Factor (proNGF) in the development of brain metastasis. More importantly, our results showed that proNGF acts through TrkA independent of its phosphorylation to induce brain metastasis in TNBC. In addition, we found that proNGF induces BBB transmigration through the TrkA/EphA2 signaling complex. More importantly, our results showed that combinatorial inhibition of TrkA and EphA2 decreased TBNC brain metastasis in a preclinical model.

These disruptive findings provide new insights into the mechanisms underlying brain metastasis with proNGF as a driver of brain metastasis of TNBC and identify TrkA/EphA2 complex as a potential therapeutic target.

Circulating tumor cells (CTCs) are known to be prognostic for metastatic relapse and are detected in patients as solitary cells or cell clusters. Circulating tumor cell clusters (CTC clusters) have been observed clinically for decades and are of significantly higher metastatic potential compared to solitary CTCs. Recent studies suggest distinct differences in CTC cluster biology regarding invasion and survival in circulation. However, differences regarding dissemination, dormancy, and reawakening require more investigations compared to solitary CTCs. Here, we review the current state of CTC cluster research and consider their clinical significance. In addition, we discuss the concept of collective invasion by CTC clusters and molecular evidence as to how cluster survival in circulation compares to that of solitary CTCs. Molecular differences between solitary and clustered CTCs during dormancy and reawakening programs will also be discussed. We also highlight future directions to advance our current understanding of CTC cluster biology.

Tumour dormancy and recurrent metastatic cancer remain the greatest clinical challenge for cancer patients. Dormant tumour cells can evade treatment and detection, while retaining proliferative potential, often for years, before relapsing to tumour outgrowth. Cellular quiescence is one mechanism that promotes and maintains tumour dormancy due to its central role in reducing proliferation, elevating cyto-protective mechanisms, and retaining proliferative potential. Quiescence/proliferation decisions are dictated by intrinsic and extrinsic signals, which regulate the activity of cyclin-dependent kinases (CDKs) to modulate cell cycle gene expression. By clarifying the pathways regulating CDK activity and the signals which activate them, we can better understand how cancer cells enter, maintain, and escape from quiescence throughout the progression of dormancy and metastatic disease. Here we review how CDK activity is regulated to modulate cellular quiescence in the context of tumour dormancy and highlight the therapeutic challenges and opportunities it presents.

Human brain is characterized by extremely sparse extracellular matrix (ECM). Despite its low abundance, the significance of brain ECM in both physiological and pathological conditions should not be underestimated. Brain metastasis is a serious complication of cancer, and recent findings highlighted the contribution of ECM in brain metastasis development. In this review, we provide a comprehensive outlook on how ECM proteins promote brain metastasis seeding. In particular, we discuss (1) disruption of the blood-brain barrier in brain metastasis; (2) role of ECM in modulating brain metastasis dormancy; (3) regulation of brain metastasis seeding by ECM-activated integrin signaling; (4) functions of brain-specific ECM protein reelin in brain metastasis. Lastly, we consider the possibility of targeting ECM for brain metastasis management.

Cancer progression is associated with extensive remodeling of the tumor microenvironment (TME), resulting in alterations of biochemical and biophysical cues that affect both cancer and stromal cells. In particular, the mechanical characteristics of the TME extracellular matrix undergo significant changes. Bioengineered polymer hydrogels can be instrumental to systematically explore how mechanically changed microenvironments impact cancer cell behavior, including proliferation, survival, drug resistance, and invasion. This article reviews studies that have explored the impact of different mechanical cues of the cells' 3D microenvironment on cancer cell behavior using hydrogel-based in vitro models. In particular, advanced engineering strategies are highlighted for tailored hydrogel matrices recapitulating the TME's micrometer- and sub-micrometer-scale architectural and mechanical features, while accounting for its intrinsically heterogenic and dynamic nature. It is anticipated that such precision hydrogel systems will further the understanding of cancer mechanobiology.

Clinical evidence supports a role for the extracellular matrix (ECM) in cancer plasticity across multiple tumor types. The lack of in vitro models that represent the native ECMs is a significant challenge for cancer research and drug discovery. Therefore, a major motivation for developing new tumor models is to create the artificial ECM in vitro. Engineered biomaterials can closely mimic the architectural and mechanical properties of ECM to investigate their specific effects on cancer progression, offering an alternative to animal models for the testing of cancer cell behaviors. In this review, we focused on the biomaterials from different sources applied in the fabrication of the artificial ECM and their biophysical cues to recapitulate key features of tumor niche. Furthermore, we summarized how the distinct biophysical cues guided cell behaviors of cancer plasticity, including morphology, epithelial-to-mesenchymal transition (EMT), enrichment of cancer stem cells (CSCs), proliferation, migration/invasion and drug resistance. We also discuss the future opportunities in using the artificial ECM for applications of tumorigenesis research and precision medicine, as well as provide useful messages of principles for designing suitable biomaterial scaffolds.

Approximately 90% of breast cancer related mortalities are due to metastasis to distant organs. At the metastatic sites, cancer cells are capable of evading death by exhibiting cellular or mass dormancy. However, the mechanisms involved in attaining dormancy at the metastatic site are not well understood. This is partly due to the lack of experimental models to study metastatic site-specific interactions, particularly in the context of brain metastatic breast cancer (BMBC). Herein, an in vitro hyaluronic acid (HA) hydrogel-based model is developed to study mass dormancy in BMBC. HA hydrogels with a stiffness of ≈0.4 kPa are utilized to mimic the brain extracellular matrix. MDA-MB-231Br or BT474Br3 BMBC spheroids are prepared and cultured on top of HA hydrogels or in suspension for 7 days. HA hydrogel induced a near mass dormant state in spheroids by achieving a balance between proliferating and dead cells. In contrast, these spheroids displayed growth in suspension cultures. The ratio of %p-ERK to %p-p38 positive cells is significantly lower in HA hydrogels compared to suspension cultures. Further, it is demonstrated that hydrogel induced mass dormant state is reversible. Overall, such models provide useful tools to study dormancy in BMBC and could be employed for drug screening.

The reactivating of disseminated dormant breast cancer cells in a soft viscoelastic matrix is mostly correlated with metastasis. Metastasis occurs due to rapid stress relaxation owing to matrix remodeling. Here, we demonstrate the possibility of promoting the permanent cell cycle arrest of breast cancer cells on a viscoelastic liquid substrate. By controlling the molecular weight of the hydrophobic molten polymer, poly(ε-caprolactone-co-D,L-lactide) within 35-63 g/mol, this study highlights that MCF7 cells can sense a 1000 times narrower relaxation time range (80-290 ms) compared to other studies by using a crosslinked hydrogel system. We propose that the rapid bulk relaxation response of the substrate promotes more reactive oxygen species generation in the formed semi-3D multicellular aggregates of breast cancer cells. Our finding sheds light on the potential role of bulk stress relaxation in a viscous-dominant viscoelastic matrix in controlling the cell cycle arrest depth of breast cancer cells.

The extracellular matrix (ECM) is pivotal in modulating tumor progression. Besides chemically stimulating tumor cells, it also offers physical support that orchestrates the sequence of events in the metastatic cascade upon dynamically modulating cell mechanosensation. Understanding this translation between matrix biophysical cues and intracellular signaling has led to rapid growth in the interdisciplinary field of cancer mechanobiology in the last decade. Substantial efforts have been made to develop novel in vitro tumor mimicking platforms to visualize and quantify the mechanical forces within the tissue that dictate tumor cell invasion and metastatic growth. This review highlights recent findings on tumor matrix biophysical cues such as fibrillar arrangement, crosslinking density, confinement, rigidity, topography, and non-linear mechanics and their implications on tumor cell behavior. We also emphasize how perturbations in these cues alter cellular mechanisms of mechanotransduction, consequently enhancing malignancy. Finally, we elucidate engineering techniques to individually emulate the mechanical properties of tumors that could help serve as toolkits for developing and testing ECM-targeted therapeutics on novel bioengineered tumor platforms. STATEMENT OF SIGNIFICANCE: Disrupted ECM mechanics is a driving force for transitioning incipient cells to life-threatening malignant variants. Understanding these ECM changes can be crucial as they may aid in developing several efficacious drugs that not only focus on inducing cytotoxic effects but also target specific matrix mechanical cues that support and enhance tumor invasiveness. Designing and implementing an optimal tumor mimic can allow us to predictively map biophysical cue-modulated cell behaviors and facilitate the design of improved lab-grown tumor models with accurately controlled structural features. This review focuses on the abnormal changes within the ECM during tumorigenesis and its implications on tumor cell-matrix mechanoreciprocity. Additionally, it accentuates engineering approaches to produce ECM features of varying levels of complexity which is critical for improving the efficiency of current engineered tumor tissue models.

Cell stiffness is an important characteristic of cells and their response to external stimuli. In this review, we survey methods used to measure cell stiffness, summarize stimuli that alter cell stiffness, and discuss signaling pathways and mechanisms that control cell stiffness. Several pathological states are characterized by changes in cell stiffness, suggesting this property can serve as a potential diagnostic marker or therapeutic target. Therefore, we consider the effect of cell stiffness on signaling and growth processes required for homeostasis and dysfunction in healthy and pathological states. Specifically, the composition and structure of the cell membrane and cytoskeleton are major determinants of cell stiffness, and studies have identified signaling pathways that affect cytoskeletal dynamics both directly and by altered gene expression. We present the results of studies interrogating the effects of biophysical and biochemical stimuli on the cytoskeleton and other cellular components and how these factors determine the stiffness of both individual cells and multicellular structures. Overall, these studies represent an intersection of the fields of polymer physics, protein biochemistry, and mechanics, and identify specific mechanisms involved in mediating cell stiffness that can serve as therapeutic targets.

Dormant, disseminated tumor cells (DTCs) can persist for decades in secondary tissues before being reactivated to form tumors. The properties of the premetastatic niche can influence the DTC phenotype. To better understand how matrix properties of premetastatic niches influence DTC behavior, three hydrogel formulations are implemented to model a permissive niche and two nonpermissive niches. Poly(ethylene glycol) (PEG)-based hydrogels with varying adhesivity ([RGDS]) and degradability ([N-vinyl pyrrolidinone]) are implemented to mimic a permissive niche with high adhesivity and degradability and two nonpermissive niches, one with moderate adhesivity and degradability and one with no adhesivity and high degradability. The influence of matrix properties on estrogen receptor positive (ER⁺ ) breast cancer cells (MCF7s) is determined via a multimetric analysis. MCF7s cultured in the permissive niche adopted a growth state, while those in the nonpermissive niche with reduced adhesivity and degradability underwent tumor mass dormancy. Complete removal of adhesivity while maintaining high degradability induced single cell dormancy. The ability to mimic reactivation of dormant cells through a dynamic increase in [RGDS] is also demonstrated. This platform provides the capability of inducing growth, dormancy, and reactivation of ER⁺  breast cancer and can be useful in understanding how premetastatic niche properties influence cancer cell fate.

Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogenous, being comprised of cellular and matrix components, which contribute to tumor cell invasion, cancer stem cell maintenance, and drug resistance. Here, we developed a heterotypic 3D spheroid model integrating GBM cells with astrocytes and endothelial cells (ECs) to better simulate the cellular components of the tumor microenvironment and investigate their impact on the stemness marker expression of GBM cells, which has not been previously investigated.

We used U87 GBM cells, C8-D1A mouse astrocytes, and human umbilical vein ECs to construct co- and tri-culture spheroid models in low-attachment U-well plates. We characterized the expression of known stemness markers NESTIN, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using qRT-PCR and immunostaining.

We incorporated GBM cells and astrocytes/ECs in 1:1, 1:2, 1:4, and 1:9 ratio and observed spontaneous self-assembled spheroids in all coculture conditions. We observed changing spheroid size dynamics over 7 days and an increased expression in stemness markers in GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively. In a triculture model employing GBM cells, astrocytes, and ECs in a 1:4:9 ratio, we found an increased expression of all the stemness markers.

We elucidated the impact of astrocytes and ECs on GBM stemness marker expression. This multicellular spheroid model may provide an important tool for investigating the crosstalk between cell types in GBM.

The online version contains supplementary material available at 10.1007/s12195-021-00691-y.

Modification of the extracellular matrix (ECM) is a critical aspect of developing a metastasis-supportive organ niche. Recent work investigating ECM changes that facilitate metastasis has revealed ways in which different metastatic organ niches are similar as well as the distinct characteristics that make them unique. In this review, we present recent findings regarding how ECM modifications support metastasis in four frequent metastatic sites: the lung, liver, bone, and brain. We discuss ways in which these modifications are shared between metastatic organs as well as features specific to each location. We also discuss areas of technical innovation that could be advantageous to future research and areas of inquiry that merit further investigation.

Hyaluronic acid (HA) is a natural polysaccharide and a key component of the extracellular matrix (ECM) in many tissues. Therefore, HA-based biomaterials are extensively utilized to create three dimensional ECM mimics to study cell behaviors in vitro. Specifically, derivatives of HA have been commonly used to fabricate hydrogels with controllable properties. In this review, we discuss the various chemistries employed to fabricate HA-based hydrogels as a tunable matrix to mimic the cancer microenvironment and subsequently study cancer cell behaviors in vitro. These include Michael-addition reactions, photo-crosslinking, carbodiimide chemistry, and Diels-Alder chemistry. The utility of these HA-based hydrogels to examine cancer cell behaviors such as proliferation, migration, and invasion in vitro in various types of cancer are highlighted. Overall, such hydrogels provide a biomimetic material-based platform to probe cell-matrix interactions in cancer cells in vitro and study the mechanisms associated with cancer progression.

The role of hydrogel properties in regulating the phenotype of triple negative metastatic breast cancer is investigated using four cell lines: the MDA-MB-231 parental line and three organotropic sublines BoM-1833 (bone-tropic), LM2-4175 (lung-tropic), and BrM2a-831 (brain-tropic). Each line is encapsulated and cultured for 15 days in three poly(ethylene glycol) (PEG)-based hydrogel formulations composed of proteolytically degradable PEG, integrin-ligating RGDS, and the non-degradable crosslinker N-vinyl pyrrolidone. Dormancy-associated metrics including viable cell density, proliferation, metabolism, apoptosis, chemoresistance, phosphorylated-ERK and -p38, and morphological characteristics are quantified. A multimetric classification approach is implemented to categorize each hydrogel-induced phenotype as: 1) growth, 2) balanced tumor dormancy, 3) balanced cellular dormancy, or 4) restricted survival, cellular dormancy. Hydrogels with high adhesivity and degradability promote growth. Hydrogels with no adhesivity, but high degradability, induce restricted survival, cellular dormancy in the parental line and balanced cellular dormancy in the organotropic lines. Hydrogels with reduced adhesivity and degradability induce balanced cellular dormancy in the parental and lung-tropic lines and balanced tumor mass dormancy in bone- and brain-tropic lines. The ability to induce escape from dormancy via dynamic incorporation of RGDS is also presented. These results demonstrate that ECM properties and organ-tropism synergistically regulate cancer cell phenotype and dormancy.