Integrin-driven differences in extracellular matrix regulation of 3D-cultured tumor cells

Abstract

The differences in the regulation of 3D-cultured mesenchymal and epithelial cell lines are driven by a wide range of endogenous and exogenous factors. Recently published transcriptomic studies have demonstrated that these differences are, in part, associated with extracellular matrix (ECM) properties and ECM-related cellular factors. Based on these studies, we propose that the dysregulation of specific genes involved in epithelial-mesenchymal transition, including CD44, ALDH1A1, PROM1, CDH1, VIM, and ZEB1, may also influence interactions between cells and the newly synthesized ECM within tumor spheroids. These interactions may be directly affected by the density and spatial distribution of integrins on the cell membrane, potentially explaining differences between newly synthesized scaffolds in 3D spheroids and the native ECM. This review aims to discuss the factors underlying integrin-mediated regulatory differences among 3D-cultured cells with heterogeneous phenotypes.

Key Words: Cancer; Cell regulation; Epithelial-mesenchymal transition; Extracellular matrix; Integrins; Spheroids; Tumor microenvironment

Core Tip: Differences in gene regulation between 3D-cultured mesenchymal and epithelial cells extend beyond the epithelial-mesenchymal transition and influence how tumor cells interact with newly synthesized extracellular matrix. The regulation of integrin density and spatial distribution appears to play a central role in mediating cell-extracellular matrix interactions and may help explain the distinct behaviors observed among different tumor tissue models.

INTRODUCTION

Currently, 3D cell cultures are receiving significant attention because of their similarity to native solid tumor tissues. Recently published studies have provided detailed investigations into differences in the regulation of 3D-cultured mesenchymal and epithelial cells[1-5]. These findings extend beyond expected transcriptomic and phenotypic differences, offering new insights into the altered physiological relevance of tumor cell spheroids (tumoroids) compared with native tissues. We noted that the regulation of several genes associated with cellular phenotypes, including CD44, ALDH1A1, PROM1, CDH1, VIM, and ZEB1, may extend beyond epithelial-mesenchymal transition, a primary focus of cancer research, to include interactions with the newly synthesized extracellular matrix (ECM)[1,6-8].

Cancer development and progression are further promoted by dysfunction in integrin-mediated ECM adhesion within tumor cells and components of the tumor microenvironment[9,10]. ECM-derived mechanical forces regulate the transition between breast cancer stemness and quiescence through activation of integrin receptors[11]. In addition, interactions between cancer cells and ECM components can activate signaling pathways, including integrin signaling[12,13]. Therefore, integrin involvement in ECM regulation contributes to inflammation, migration, and tumorigenesis[14].

Current concepts of the physiological role of integrins in cancer growth include bidirectional signaling, integrin trafficking, and specialized functions within metastatic niches. Integrin-dependent bidirectional signaling in cancer can either promote or inhibit tumorigenesis through interactions with multiple signaling pathways[15]. Integrin trafficking refers to the continuous cycle of integrin internalization from the cell surface, followed by either recycling back to the plasma membrane or degradation[16]. Within metastatic niches, integrin regulation plays a critical role in modulating mechanical stiffness, thereby promoting cancer cell migration[17].

The purpose of this opinion review is to discuss the mechanisms underlying altered regulatory behavior in 3D-cultured cells, with a primary focus on integrin-mediated molecular signaling pathways.

EXTRACELLULAR MATRIX-MEDIATED PROPERTIES OF INTEGRINS

Integrins are heterodimeric receptors composed of two distinct subunits (α and β) that require divalent cations, such as Ca²⁺, Mg²⁺, and Mn²⁺, for ligand binding and activity. Several integrin subtypes are involved in interactions with the ECM. Arg–Gly–Asp-binding integrins recognize the Arg–Gly–Asp tripeptide motif present in ECM proteins such as fibronectin, vitronectin, and fibrinogen, and are associated with angiogenesis and tumor progression. Laminin-binding integrins mediate cell adhesion to basement membranes and are critical components of hemidesmosomes that anchor epithelial cells. Collagen-binding integrins interact with multiple collagen subtypes[18-20]. The specific properties that distinguish integrins from other adhesion molecules are summarized in Table 1.

Table 1 Properties of integrins and their involvement in cell-extracellular matrix interactions among other cell adhesion molecules.

	Integrins	Selectins	Ig superfamily cell adhesion molecules (ICAMs)
Cation dependence	Require divalent cations (Ca2+, Mg2+, Mn2+)	Require Ca2+	Independent of Ca2+
Ligand type	Bind ECM proteins or counter-receptors (e.g., ICAMs)	Bind carbohydrate ligands	Bind protein ligands (e.g., integrins)
Adhesion type	Cell-matrix and cell-cell	Transient cell-cell (rolling)	Stable cell-cell
Specific types involved into ECM-cell interactions	RGD-binding integrins, laminin-binding integrins, collagen-binding integrins, leukocyte-specific integrins	Specific selections can interact with integrins, facilitating cell adhesion and signaling	Some ICAMs can bind to integrins

ECM: Extracellular matrix; RGD: Arg-Gly-Asp; ICAMs: Intercellular adhesion molecules.

Integrin-ECM binding triggers the formation of heterogeneous multi-protein assemblies known as integrin adhesion complexes, which regulate multiple cellular functions, including tumor initiation, epithelial plasticity, metastatic reactivation, and resistance to therapies targeting oncogenic pathways and the immune system[21,22]. These findings support the hypothesis of an “archipelago” organization of focal adhesions as spatial patterns (Figure 1), in which cooperating integrin clusters concentrate adaptor recruitment and signaling outputs to facilitate mechanosensing and rapid responses to migratory cues[21].

Figure 1 Factors involved in integrin-mediated cellular mechanotransduction. Created with BioRender.com. ECM: Extracellular matrix.

MOLECULAR INSIGHTS FROM THE PROPERTIES OF TUMOR EXTRACELLULAR MATRIX

Epithelial and mesenchymal cells cultured in 2D exhibit distinct metabolic and adhesive characteristics depending on whether they are grown on flat surfaces or microfibrous scaffolds[23,24]. However, 3D spheroids substantially alter the cell-scaffold interactions by enabling multiple points of contact throughout the scaffold volume and promoting bidirectional ECM-cell-responses. For example, the matrix synthesized within 3D cell spheroids differs from tumor-associated ECM, reflecting the dynamic and stable nature of these interactions[25,26]. Therefore, the properties of cell-generated 3D scaffolds may influence integrin density and establish a positive feedback loop that reinforces cellular regulatory programs[27,28].

Unlike 2D culture systems, in which cellular regulation primarily depends on the biochemical and morphological properties of the material, 3D scaffolds respond not only to these properties but also to the density of external cell-scaffold contacts[24,29,30]. This complex phenomenon is associated with sensitivity to small perturbations that disrupt physiological robustness[31,32], highlighting an additional layer of bidirectional regulation. Interestingly, this collective integrin behavior has been compared to the complexity of the “three-body problem”[33]. This biophysical analogy may help explain why cell-ECM interactions are particularly difficult to interpret within the proposed “archipelago” organization of integrin-mediated focal adhesions.

Yoshimura et al[1] reported upregulation of CD44, ALDH1A1, and PROM1 in HCT-15 (epithelial) cells, as well as increased CD44 and ALDH1A1 expression and reduced PROM1 expression in SW480 (mesenchymal) cells. In the same study, expression levels of CDH1, VIM, and ZEB1 increased in HCT-15 cells, whereas SW480 cells showed upregulation of CDH1 and VIM but downregulation of ZEB1[1], consistent with epithelial-mesenchymal transition and mesenchymal-epithelial reversion processes.

Although the authors primarily focused on phenotypic changes associated with ALDH1A1, PROM1, and CDH1, attention should also be given to the subgroup of genes including CD44, VIM, and ZEB1. These genes share related molecular regulatory mechanisms because they contribute to integrin-associated changes and cell-ECM interactions[34-36]. In this context, the opposing pattern of ZEB1 expression is of particular interest.

ZEB1 expression is associated with downregulation of epithelial adhesion genes, such as CDH1, and upregulation of genes involved in mesenchymal cell-ECM interactions[37,38]. In addition, ZEB1 can induce transcription of mesenchymal integrins, including α5-integrin and β1-integrin, either directly or through downstream effectors, thereby shifting the adhesion profile from an epithelial toward a mesenchymal state. ZEB1 also promotes increased expression of VIM and CD44[39,40].

Vimentin facilitates integrin recycling to the plasma membrane and regulates integrin abundance, thereby influencing cellular adhesion strength and migratory potential[41]. Furthermore, co-expression of CD44 and vimentin reinforces signaling pathways and promotes alignment of cytoskeletal networks in motile cells[42,43]. Therefore, ZEB1, VIM, and CD44 proteins directly influence integrin engagement, and their regulatory networks integrate gene expression, cytoskeletal organization, and mechanical feedback to enable effective ECM sensing. Loss of critical integrin density disrupts cell-matrix adhesion, weakens cellular attachment, and increases susceptibility to apoptosis[44,45].

Identification of key adhesion-related signaling pathways may provide new therapeutic opportunities targeting integrins and integrin-mediated regulatory mechanisms[18,46,47]. However, these findings alone are insufficient to establish definitive physiological relevance, although they suggest conditions that alter the natural interactions between cells and the ECM.

The properties of the tumor microenvironment extend beyond those of its cellular and biomolecular components. In addition to these factors, the tumor ECM (oncomatrix) differs from that of normal healthy tissue in both antigen composition and tissue microarchitecture, thereby influencing the balance of cell-ECM interactions[48,49]. Regulation of CD44, VIM, and ZEB1 is closely associated with coordinated cellular programs that govern interactions between cells and the ECM[50-52]. These observations further support the differences identified between epithelial and mesenchymal 3D cell spheroids (tumoroids)[53-57].

In living tissues, alterations in ECM properties immediately affect not only resident cells but also the immune microenvironment and intercellular immune communication[58-60]. These changes can limit the effectiveness of advanced therapy medicinal products, including CAR-T cells and mRNA vaccines[61,62], and may also promote senescence and inflammatory responses[63-67].

These findings highlight an underexplored area of cell-ECM research related to the loss of the native integrin landscape in cultured cells and aberrant integrin activation. In particular, the spatial distribution of integrins on the cell membrane and the organization of “archipelagos” of integrin clusters shift the focus from individual adhesion molecules to broader patterns of integrin spatial organization.

CONCLUSION

Inspired by recent studies describing differences in the responses of epithelial and mesenchymal 3D cell cultures, as well as the heterogeneity of 3D tumoroids, we sought to examine the molecular mechanisms underlying integrin density and spatial distribution. These mechanisms may help explain differences not only between 2D and 3D cell cultures, but also within the tumor microenvironment itself, particularly in relation to cell-ECM interactions.