Integration of mechanical and ECM microenvironment signals in the determination of cancer stem cell states

Abstract

Purpose of review:

Cancer stem cells (CSCs) are increasingly understood to play a central role in tumor progression. Growing evidence implicates tumor microenvironments as a source of signals that regulate or even impose CSC states on tumor cells. This review explores points of integration for microenvironment-derived signals that are thought to regulate CSCs in carcinomas.

Recent findings:

CSC states are directly regulated by the mechanical properties and extra cellular matrix (ECM) composition of tumor microenvironments that promote CSC growth and survival, which may explain some modes of therapeutic resistance. CSCs sense mechanical forces and ECM composition through integrins and other cell surface receptors, which then activate a number of intracellular signaling pathways. The relevant signaling events are dynamic and context-dependent.

Summary:

CSCs are thought to drive cancer metastases and therapeutic resistance. Cells that are in CSC states and more differentiated states appear to be reversible and conditional upon the components of the tumor microenvironment. Signals imposed by tumor microenvironment are of a combinatorial nature, ultimately representing the integration of multiple physical and chemical signals. Comprehensive understanding of the tumor microenvironment-imposed signaling that maintains cells in CSC states may guide future therapeutic interventions.

Introduction

Cancer stem cells (CSCs) are thought to be a subpopulation of tumor cells that have the capacity to self-maintain and give rise to the other cells that comprise the bulk of the tumor. CSCs are central players in therapeutic resistance, and they are imbued with the potential to spread and initiate growth in distant locations. CSC have been identified in many cancers, but it is not clear if all cancer types have CSC. Part of the confusion surrounding CSCs is a lack of consistency in the markers used to enrich for them, for even within one type of cancer there is little consensus as to the identity of the entity. An explanation of this could be that CSCs and more differentiated cancer cells represent transition states, and that any cancer cell can occupy those states given the correct set of equilibrium conditions [reviewed in (1)]. Thus, CSCs may be better conceptualized as having a functional state rather than as definable entities.

Tissue-specific stem cells are maintained in specialized microenvironments, often called niches, and this is a reasonable expectation also for CSCs. We have speculated that one impact of heterogeneity within a tumor is variability in microenvironments leading to some that maintain cells with CSC functions and others that impose more differentiated states (2). Such heterogeneity may underlie the wide variations of reported frequencies of CSCs which range from 0.0001% to 40% of cells in a tumor (3). The more traditionally held view that CSCs comprise a relatively rare sub-population of tumor cells is also challenged by a study of aggressive breast cancers in which it was found that adaptation of all tumor cell populations could mediate chemo resistance, not just one specific subpopulation (4). Similarly, in sporadic melanomas, expression of batteries of genes made some cells tolerant to targeted therapeutics, but this expression pattern was not limited to a specific sub-population (5). In a diverse collection of combinatorial microenvironments comprised of different combinations of tumor microenvironment components, epithelial to mesenchymal transition (EMT) and stem cell marker expression, and tolerance to anti-cancer drugs in cancer cells were determined by specific combinations of microenvironment components (6,7). Thus, tumor cells in multiple cancers exhibit properties of CSCs, and expression of genes and proteins that are often synonymous with CSC states are heavily influenced by the tumor ME.

The microenvironment is defined as the sum of cell-cell, cell-extracellular matrix (ECM), cell-soluble factor and chemical interactions, physical forces, and geometric constraints that are experienced by cells. That cell phenotypes and functions seem inextricably linked to their microenvironment suggests that it is essential to cells to sense and interact with their microenvironment. It is not well understood how cells integrate these very different types of microenvironment signals. In this review, we focus on two of the most stable microenvironment factors: mechanical forces and ECM.

ECM is a structural component of tissues and tumors, and it provides a scaffold where cells attach, however, the cell-ECM relationship is not one of inert interactions. ECM binds also to soluble ligands and acts as a reservoir for growth factors and cytokines. ECM can chemically and physically prevent cytotoxic drugs from penetrating deep within tumors (8). ECM is consists of structural proteins, such as collagens; cell-adhesion proteins, such as laminins and fibronectin; proteoglycans and polysaccharides, such as hyaluronan. Fibrillary collagens provide mechanical support, whereas proteoglycans and polysaccharides bind water and increase the viscosity of the matrix. These structural factors together determine the ECM stiffness, porosity and spatial organization, whereas the composition of cell adhesion proteins in ECM are central to cell-ECM interactions.

ECM composition and organization undergoes radical transformation during carcinoma progression. Remodeling of basement membrane proteins is among the first ECM transformations to occur during cancer progression and it may be crucial to enabling epithelial cell invasion into the stromal compartment. Further, collagen fibers are linearized and type I collagen and fibronectin are highly often abundant in tumor microenvironments. Most tumors are stiffer than normal parental tissues. Increased stiffness in tumors is generated by ECM crosslinking, elevated deposition, and altered organization. Solid tumors grow in confined spaces that also results in elevated pressure and interstitial fluid flow. Even though ECM is the principle mediator of mechanical forces in tissues, the mechanically generated and biochemically initiated signals are distinct, but must be integrated into a cellular response.

Regulation of CSC states by mechanical forces and ECM

CSCs exhibit activities that are reminiscent of stem cells; self-maintenance, differentiation and metabolic adaptation. Self-maintenance of CSCs is achieved through balancing proliferation, differentiation, quiescence, and apoptosis. Microenvironment stiffness is a key regulator of CSC proliferation in multiple tumor contexts (9–12). Interestingly, this regulation is tissue-specific and the tissue of origin of CSCs seems to establish the range of optimal stiffness for growth (13). Two of the most abundant ECM components of the tumor microenvironment, fibronectin and type 1 collagen, increase CSC proliferation and inhibit chemotherapy-imposed apoptosis (14–16). Thus, CSC self-maintenance can be regulated by ECM components of the tumor microenvironment.

A CSC hallmark characteristic is the ability to differentiate into the different types of cells within a tumor. A prominent tumor microenvironment ECM component, hyaluronan, supports CSC multipotent states in gliblastoma (17), and depletion of hyaluronan matrix in vivo by 4-methyl umbelliferon (4-MU) radically decreased CSC markers expression level in hepatocarcinogenesis (18). Profiling combinatorial microenvironments for their ability to impose CSC phenotypes in breast cancer cells showed that type I collagen, osteopontin and collagen VIA3 induced expression of CSC markers in breast cancer cells, the receptor tyrosine kinases Axl and cKit plus a number of EMT genes. Conversely, type IV collagen, a basement membrane component, repressed CSC marker expression (6). Vitronectin promoted CSC differentiation leading to decreased (CD44⁺/CD24⁻) CSC population in prostate and breast CSC cultures (19). These are examples whereby CSC cell fate decisions are dynamically regulated by tissue stiffness and ECM composition.

EMT is an example of epithelial plasticity, which is coopted during cancer development. Classically this cooptation is thought to pertain to a cancer cell’s ability to migrate and invade tissue, however the involvement of EMT-like states in stem cells raises the likelihood that it is involved in maintaining cancer cells in specific CSC states (e.g. epithelial vs. mesenchymal) (20). EMT is promoted by increasing tissue stiffness and by interaction with multiple ECM components, like fibronectin, proteoglycans and hyaluronan (21–23). Fibrillary type I collagen and fibronectin matrix promotes EMT by inhibiting epithelial differentiation during embryogenesis and in cancer (24). Thus, the equilibrium between epithelial and mesenchymal cancer cell phenotypes is regulated by mechanical and biochemical stimuli.

CSC growth and drug resistance properties are supported by aberrant metabolic states. CSCs across multiple tumor types show differing metabolic phenotypes, likely caused by differential effects from the tumor microenvironment, especially oxygen tension (25). Mechanical and ECM cues transmitted by signaling pathways also regulate metabolic processes (26). Stiff substrata activate, whereas compliant substrata inhibit, glycolysis in epithelial cells. Cancer cells sustained high glycolysis rates even in heterogeneous mechanical microenvironments (27). Altered CSC metabolic states combined with anti-apoptotic signals also lead to drug resistance. Tissue stiffness and fibronectin matrix promotes drug resistance and together they have a synergistic effect (7). Taken together, defining activities of stem cell states are imposed and regulated in CSCs by components and physical properties of tumor microenvironments.

The signalling toolbox for integrating mechanical and ECM cues

Understanding how mechanical properties and ECM composition regulate CSC states is a burgeoning field. Plasma membrane receptors and channels couple the microenvironment to intracellular signaling pathways. ECM composition sensing goes through receptors including integrins, syndecans and CD44. Cells sense mechanical forces via these same receptors, but mechanical forces can also stretch the plasma membrane to activate cadherin receptors and open mechanosensitive ion channels. Here we examine some of the receptors, signaling pathways and transcription factors that interact with both ECM and mechano-sensing processes in signal integration. A summary of the pathways is shown in Figure 1, in which the proteins that we found to have the most evidence of integration are colored in hues of red whereas other intermediates are in blue.

Figure 1. Integration of mechanical- and ECM-based tumor microenvironment-imposed signals.

Cancer stem cells sense mechanical forces and ECM to activate intracellular signaling through the indicated cell surface receptors. FAK, ILK, RhoA and hippo pathway (YAP/TAZ) are points of integration for these different types of signals that regulate CSC states.

Integrins

Integrins are heterodimeric transmembrane receptors that form a physical connection between the microenvironment and intracellular space. Integrins are comprised of 18α and 8β subunits that pair to form at least 24 different heterodimeric receptors. Each integrin subunit has unique binding sites to interact with different types of ECM, and the combination of subunits in each heterodimeric integrin determines a range of ECM binding specificities. Integrins are expressed in all human tissues, with each tissue having its own integrin subtype profile. Integrins play multiple roles in tumor progression as mediators of survival, migration and stemness (28). Integrin β1, β3, β4 and β6 subunits have been specifically implicated as CSC markers in breast, prostate, squamous cell, colorectal, lung and ovarian tumors, and mediate CSC self-maintenance and drug resistance (28,29).

When integrins bind to target ECMs they undergo conformational changes, which promote intracellular signaling via a physical linkage to the actin cytoskeleton. Their ability to respond to mechanical stimuli also is mediated through connections to the actin network. β subunits bind to α-actin or interact with linker proteins, like talin. Mechanical force unfolds the talin protein rod-domain and, depending on the unfolded state, talin will interact with different signaling molecules. For example, DLC1 attaches only within the folded rod-domain, whereas vinculin prefers to bind the unfolded rod-domain but detaches from the fully unloaded domain (30,31).

Integrin clustering within the plasma membrane amplifies the strength of an intracellular signal. Clustering is an active and spatially regulated process, and clusters form cell adhesion sites. Clusters are stabilized by cytoplasmic adhesion proteins that also link integrins to the actin cytoskeleton. Cluster stabilization leads to maturation of adhesion sites, and eventual formation of mature focal adhesions. Mature focal adhesions anchor cells to the substratum and functions as a signal carrier by activating intracellular signaling pathways (32). Integrin clustering also is promoted by ligand-independent mechanisms, such as by galectins (28), which are proteins that bind β-galactose sugars. Interestingly, galectin-3 is highly expressed by gastrointestinal CSC and promotes chemoresistance (33), suggesting a role for the glycocalyx more generally in integrin-mediated CSC regulation. Integrins are essential to so many mechanical- and ECM-triggered signaling pathways that we consider them to be essential to understanding how CSCs integrate different types of microenvironment cues.

Syndecans and CD44

Both syndecans and CD44 are associated with different CSC states. CD44 is used widely as a CSC biomarker, and syndecan-1 expression correlates with the presence of other CSC markers, including notch 1&3 and ALDH1 activity (34). Syndecan extracellular domains contain numerous glucosamine glycan chains that equip them to bind different ECMs and growth factors. Syndecans are linked closely to mechanical signaling (35, 36), and it is likely that syndecans have a role in CSC mechanosensing. Syndecan-1 knockdown reduced mammosphere and colony formation by breast cancer cells. Syndecan-1 and CD44 are co-expressed in breast CSCs, and syndecan-1 knockouts reduced CD44 expression (34) suggesting a regulatory relationship between the proteins.

CD44 is a receptor for hyaluronan polysaccharides and it also can bind the ECM protein osteopontin. CD44 has multiple transcript variants and is often subject to posttranslational modifications, and CD44 splice variants have determinative roles in breast CSC states. Deletion of the most common CD44 isoform (comprised of exons 1–5 and 16–20) impairs CSC self-renewal (37). CD44 spatial localization in cell membranes regulates its activity, colocalization and interaction with cell surface receptors like EGFR and ErbB2 leads to cancer cell migration and chemoresistance (38, 39). Homodimeric CD44 receptor clustering modulates signal transduction and increases leucocyte binding by causing extracellular hyaluronan matrix crosslinking (40). CD44 interacts with the actin cytoskeleton through ERM family proteins (ezrin, radixin and moesin). ERM protein conformations and functionality are regulated by phosphorylation states and modulate cell responses to extracellular stimuli (41). It is likely that CD44-ERM protein interactions modulate ECM- and mechanically activated signaling pathways.

Integrins, syndecans, and CD44 represent essential tools used by CSCs to integrate molecular and mechanical information from the tumor microenvironment.

Signaling pathways

FAK

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase and it is one of the main signaling mediators in integrin-enriched cell adhesion sites. FAK kinase integrates extracellular signals initiated by growth factors, ECM, and mechanical stimuli (42). FAK directly binds to intracellular tails of β-integrins, and integrin mediated adhesions to ECM allows rapid auto phosphorylation of Y397-FAK. FAK is activated also by several growth factor receptors, CD44 and syndecans (42–44). ECM-integrin linkages work as mechanical sensors that can activate FAK (45). Direct mechanical propagation within the plasma membrane activates FAK, for as the plasma membranes stretch the FAK domain structure unfolds in the presence of PIP2 and reveals the Y397 auto-phosphorylation site, making kinase activation possible (46). Activated FAK associates with several SH2 domain containing proteins, including Src, PI3K and Grb7. Moreover, Src kinase binding to Y397-phosphorylated FAK promotes FAK activation and signaling by contributing several additional tyrosine phosphorylation sites (47). FAK activation is known to trigger several intracellular signaling pathways including Akt, MAPK/ERK, and cyclinD1. Through these pathways it is thought to act as a regulator of cancer cell proliferation, survival, and invasion (42).

FAK expression is upregulated in many epithelial cancers, and FAK regulates a number of CSC-related functions (42). Deletion of FAK in a conditional knock out mouse mammary tumor model reduced the frequency of CSC and suppressed tumor progression (48). Type 1 collagen-integrin mediated activation of FAK increased the frequency of pancreatic ductal adenocarcinoma CSCs and increased tumor initiation potential (49). ECM-triggered CD44 signaling through FAK mediated resistance against drug-induced apoptosis (43). Also, potentially relevant to the CSC differentiation states, TGFβ-induced EMT is mediated by FAK-Src signaling (50), and integrin-FAK-Src signaling inhibits epithelial differentiation by interruption of E-cadherin mediated epithelial adherens junctions (42). These reports support the concept that FAK signaling has a central role in maintenance of CSC states (51).

ILK

Integrin-linked kinase (ILK) is part of the Raf-like kinase family. ILK binds to integrins and works as a scaffold protein by coupling integrin with the actin cytoskeleton, but ILK transmits signals as well. ILK localizes mainly in focal adhesion sites, but also resides in cell-cell adhesion sites, centrosomes and in the nucleus (52). ILK transmits ECM and mechanically initiated signals from integrins to intracellular signaling pathways (52–54).

ILK binds directly to integrin β1 and β3 cytoplasmic domains or via the pinch-parvin complex (IPP). Human cells express several isoforms of pinch and parvin proteins and they form distinct IPP complexes. Each distinct IPP complex that forms with ILK results in different signaling outputs. IPP complexes mediate signals to the actin cytoskeleton, RhoA, Akt, and JNK (52). Whereas ILK clearly plays a role as a signal transducer (52), the exact mechanism is still not known, for it is not clear if ILK has kinase activity, since reports have shown that its kinase-domain lacks catalytic activity (52). ILK may function by controlling subcellular localization of signaling molecules and in that way, regulate their activity, for example by recruiting Akt to the plasma membrane (55). Furthermore, the IPP proteins that associate with ILK can bind to and inhibit the activity of protein phosphatases (56).

ILK expression is upregulated in stiff regions of tumors (57), and ILK expression is associated with CSC and EMT marker expression in vivo (58). In vitro ILK signaling through Akt is necessary for the induction of CSC marker expression and self-maintenance in breast cancer cells in response to matrix stiffness (57). Moreover, ILK expression was upregulated during EMT in colorectal and breast tumor CSCs, whereas ILK signaling inhibition reduced the expression of stem cell and EMT markers (58,59). The mechanism by which ILK signaling regulates CSC activities may become clearer when its signal transduction mechanisms are laid bare.

RhoA

The Ras homolog family member A (RhoA) is a small GTPase protein, with inactive GDP-bound, and active GTP bound conformational states. RhoA is strongly associated with signaling responses to extracellular stimuli. RhoA activity can be increased by soluble molecules binding to cell surface receptors, cell-cell adhesion, mechanical stimuli (60) and by cell attachment to ECMs, like fibronectin and hyaluronan (61,62). Like iother GTPases, RhoA activation is regulated by three classes of molecules: guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). GAPs and GEFs are responsible for nucleotide exchange-dependent activation and inhibition of RhoA. GDIs maintain a cytosolic pool of inactive RhoA.

RhoA activation by ECM goes through integrins, syndecans or CD44 receptors. Mechanical stimuli can go through these same receptors but are also mediated by cadherins and ion channels. Initial cell-adhesion to fibronectin decreases RhoA activity, while in later stages of cell spreading integrin heterodimer composition determines if RhoA is activated or inactivated (63). Integrin receptor organization and heterodimer composition has a significant role in regulation of RhoA signaling. Syndecan-4 clustering in the plasma membrane leads to PKCα-mediated RhoA activation (64). Hyaluronan binding to CD44 activates RhoA through GEFs (62). Mechanical stimuli unfold the rod-(R8)-domain of integrin to bind talin. Talin unfolding releases and inactivates DLC1, which then loses the ability to inactivate RhoA (30). Additionally, mechanical stimuli opens piezo2 ion channels and allows Ca2+ influx, which leads to Ca2+ activated calpain and tyrosine kinase-Fyn mediated RhoA activation (60). Active GTP bound-RhoA binds to and stimulates downstream signaling factors. One of the most dynamic RhoA signals is ROCK activation. ROCK directly increases phosphorylation of myosin and activates myosin contractility: which is required for cell migration.

RhoA is overexpressed in many cancers, however there are reports showing that RhoA can act as a tumor suppressor or promoter in different tunor types (65–68). RhoA is required for efficient cell adhesion and migration, since active RhoA induces maturation of focal adhesions and actin cytoskeleton function. RhoA activation is linked to activities that are important for cells in a CSC state, like proliferation (69) and EMT (70). However, RhoA inactivation can enhance Wnt-signaling and chemokine signaling in breast and colorectal tumor CSCs, and both promote tumor progression (65,67). There is a dearth of information that directly connects CSC states with

RhoA signaling, however we felt it was important to mention in this context because RhoA is a key point of integration for microenvironment mechanical and ECM signals.

Hippo pathway and YAP/TAZ

The hippo pathway in mammalian cells consists of core kinases (MST1/2 & LATS1/2), other kinase cascade factors, transcription coactivators (YAP & TAZ) and transcription factors (TEAD1–4) - altogether there are over 30 known components. Several soluble ligands, G-protein-coupled receptors, ECM, as well as mechanical stimuli regulate the hippo pathway (reviewed in (71)). When the hippo pathway is activated core kinases MST and LATS phosphorylate YAP and TAZ, which causes them to remain in the cytoplasm. Hippo pathway inactivation leads to YAP/TAZ dephosphorylation and translocation to the nucleus where they associate with TEADs and activate expression of gene programs relevant to cell survival, basal differentiation, and EMT-related processes. Many of the same receptors and signaling proteins that were discussed previously in this review are known also to interact with the hippo pathway, suggesting that the hippo pathway is a signaling integrator for CSC function.

The hippo pathway is responsive to multiple ECM and mechanical cues. Increased matrix stiffness inactivates the hippo pathway and YAP/TAZ expression levels themselves also are responsive to microenvironment stiffness (13). Differential ECM composition modulates hippo pathway activation, for example cell attachment to fibronectin and arginine rich ECMs inactivate the hippo pathway (71–73), whereas attachment to high molecular weight hyaluronan or laminin-111 activates the hippo pathway, inhibiting YAP and TAZ nuclear localization (74,75). Experiments that examined the role of laminins in YAP/TAZ activation also implicated integrins and CD44 as hippo control elements. Stiff or fibronectin-rich microenvironments inactivate LATS through β1-integrin (71). Laminin-511 through integrin α6Bβ1 triggered TAZ nuclear localization, whereas laminin-111 binding favored TAZ cytoplasmic localization, independent of LATS (75). High molecular weight hyaluronan drove CD44 clustering to activate hippo pathway, whereas low molecular weight hyaluronan cannot stimulate receptor clustering and this leads to inactivation of hippo pathway (74). Responses to mechanical stimuli or ECM signaling via FAK activates the Src-PI3K-PDK1 signaling cascade, which inhibits LATS phosphorylation (72). Compliant matrices inactivate RhoA through GAPs leading to LATS activation (76). Conversely, activated RhoA mediates LATS inactivation by binding angiomotin (AMOT), which together with NF2 is a LATS activator (77). ILK inhibits MST and LATS phosphorylation through inactivation of NF2 (78). Clustered CD44 receptors bind MST kinase inhibitor PAR1b and this leads to MST phosphorylation and hippo pathway activation, whereas non clustered CD44 receptors release PAR1b and this inhibits MST phosphorylation (74). Ridgid matrices increase vinculin association with the cytoskeleton triggering nuclear translocation of YAP and TAZ in a LATS independent manner (79). It is also possible that mechanical stimuli alone can trigger YAP/TAZ import into the nucleus by stretching actin and myosin fibers which flattens the nucleus and opens nuclear pores (71).

The hippo pathway may present another example of a dynamic and reciprocal relationship with the microenvironment that has specific implications for CSC regulation. While mechanical forces and ECM composition regulate hippo-pathway activation, the reverse also is known to happen. YAP/TAZ driven fibronectin and laminin-511 expression induced stiffening of the surrounding matrix. Laminin-511 and fibronectin-rich, stiff microenvironments then supported maintenance of CSCs (71,75). Further, YAP and TAZ together with TEADs promotes the transcription of multiple integrin and other focal adhesion-related genes (80) and in this way, establishes a dynamic and reciprocal cell-microenvironment sensing and modification circuit.

The hippo pathway is known for its role in modulating organ size by regulating cell proliferation, apoptosis, and stem cell self-maintenance -the pathway ostensibly exists to constrain growth (81). Inactivation of the hippo pathway, causing YAP and TAZ translocation to the nucleus, is related to enhanced expression of stem cell-related genes in CSCs (82,83). YAP and TAZ promote tumor spheroid growth in both in vitro and in vivo models (84–86) and facilitates resistance to anoikis in CSCs (87). The timing of hippo activation also may be a key consideration in CSC self-renewal because LATS1-mediated phosphorylation just before cell division was essential for tumorsphere formation in a mode of aggressive oral cancer (88). YAP mediated transcription caused de-differentiation of adult hepatocytes and resulted in accumulation of CSCs (89). Taken together, YAP and TAZ are transcriptional drivers of genes that are essential to the CSC state, which are regulated by microenvironmental signals that are both mechanical and biomolecular in nature. That YAP and TAZ are examples of transcription factors that are both regulated by and are producers of regulatory ECMs suggests these factors are key sculptors of the CSC niche microenvironments.

Conclusions

Physical and biochemical features of tumor microenvironments have a key role as managers of the CSC state. Here we reviewed several receptors and intracellular signaling agents that transduce and integrate mechanical and ECM-triggered signals and in this way, regulate CSC states. CSC and their progeny are thought to transit between different states (20), and perhaps the integration of mechanical and ECM signals determines the equilibrium of the states that are achieved. While dynamic signaling pathways integrate acute microenvironmental signals, probably equally important is the signal integration that happens at the epigenetic level. Epigenetic cell memory guides CSCs to adapt and survive in changing tumor microenvironments and may, for instance establish, the mechanical rheostats in CSCs that seem to be determined by the tissue of origin. It is not yet comprehensively studied, but shreds of evidence support the contention that microenvironment cues underlie epigenetic states (90).

There is a great challenge ahead to reveal how combinatorial and highly heterogeneous tumor microenvironments regulate CSC states. High throughput cell-based microenvironment assays (6,91) provide tools for addressing this challenge and by combining these assays with computational models (92) it may be possible to make in silico reconstructions of complex tissue microenvironments in order to more efficaciously establish testable hypotheses. If CSCs are indeed representative of a transient cell state, then therapeutic targeting of CSCs as entities will be extremely challenging. However, if the CSC state is regulated by specific microenvironment components then perhaps targeting the key integrators of microenvironment directives will lead to durable decreases in CSC activities.