The occurrence of angiogenesis inside a tumor mass represents a limiting factor for tumor growth over a few centimeters in diameter. Some tumor cells in the tumor mass can evolve to express membrane proteins, assume the characteristics of endothelial cells, and participate in a neovascularization process called VM. Several membrane proteins associated with VM have been identified, including CD31 [29] and VE-cadherin [12]. Stromal-enriched factors have been rarely studied, although they play a critical role in modulating the tumor microenvironment. VM can aggravate aggressive tumor progression and is inversely associated with patient survival rates in several cancer types, such as malignant melanoma and lung cancer. Recent studies have indicated that stromal CD248 expression in fibroblasts and pericytes plays a role in tissue fibrosis and tumor progression [17, 20, 30]. It has been shown that cancer VM phenotype is inversely associated with melanoma patient survival, whereas there is no report on the survival correlation with CD248 expression level in melanoma patients. Hong et al. demonstrated that CD248 expression level is inversely correlated with lung cancer patient survival [30]. CD248 expression has also been observed in some tumor cells [21, 31]. In this study, we found that tumor cells with autonomous CD248 expression exhibited VM expression potential to support blood supply in the tumor and promoted tumor growth and metastasis; therefore, CD248 expression might contribute to tumor malignancy.

We demonstrated that CD248 expression in melanoma tumor cells is correlated with tumor cell-fibronectin interaction, FAK activation, MMP9 expression, cell migration, and VM. The lectin domain at the N-terminal of CD248 may enhance the adherence of tumor cells to fibronectin, whereas its C-terminal cytoplasmic domain may anchor to F-actin, thereby forming a linkage between F-actin and ECM proteins. This linkage could promote cell anchoring to the ECM and cell migration. When the linkage function of CD248 is interrupted by the addition of decoy molecules such as rCD248 proteins in this study, the deletion of its cytoplasmic domain, or the knockdown of CD248 protein expression, cell migration activity is suppressed [25, 32]. Notably, VM is correlated with CD248 expression, indicating that membrane-anchoring proteins are essential for VM formation. Furthermore, melanoma cell adhesion to ECM proteins and cell migration activity can be inhibited by the exogenous addition of rCD248 proteins; VM on Matrigel and VM pattern in experimental lung metastasis mouse model were also inhibited by rCD248 proteins. Furthermore, the rCD248 protein suppressed tumor metastasis in mice. These results indicate that CD248 expression is associated with an aggressive phenotypic switch of melanoma, having better metastatic activity and VM function, partly by forming a linkage between tumor cells and ECM protein fibronectin.

It is still unclear how CD248 expression causes cells to assume VM function, although the lectin domain of CD 248 is essential for its function. In this study, we found that the lectin domain of CD248, not the EGF-like domain, was required for its interaction with fibronectin. Cell adhesion and FAK activity were compromised in cells with CD248-knockdown or rCD248D1-4 treatment during cell adhesion to fibronectin. Furthermore, we demonstrated that inhibition of cell migration by rCD248 was associated with its lectin domain but not the EGF-like domain. These results imply that the lectin domain of CD248 may be involved in melanoma cell–ECM interactions and cell migration. Though the current results did not directly test whether rCD248 suppresses tumor lung metastasis through interaction with fibronectin, our results, including protein-based and cell-based interaction assays and in vitro assays, all suggest that rCD248 could interfere with membrane-bound CD248 in melanoma migration and VM in part through the interference of cell-ECM interaction.

FAK signaling and MMPs have been associated with tumor VM [13]. In line with these observations, we demonstrated that CD248 expression modulates cell-ECM adhesion, FAK activation, and MMP9 expression in melanoma cells. In addition, exogenous expression of CD248 in the CD248-null HEK293 cells promotes cell migration, which was reduced in the presence of the rCD248 protein. These results suggest that CD248 is a molecule that can promote dynamic cell activity such as cell migration and VM, thereby transforming malignant melanoma tumors. Though we have demonstrated that CD248 expression level was associated with several cellular activities, whether CD248-mediated VM is responsible for tumor progression has not been directly tested in the current study. To address this question, constitutive activation of FAK signaling to overcome FAK suppression induced by knockdown of CD248 or rCD248 treatment in melanoma can be utilized.

The functions of CD248 in tumors might not be limited to the cell-matrix interaction as its expression level has been correlated with the activities of the cell in different cell types, including the activity of cell adhesion to the matrix, migration, proliferation, and modulation of signal transduction. For instance, we demonstrated that CD248 expression plays a role in activated myofibroblast proliferation and migration and modulates PDGF receptor signaling [25]. Consequently, CD248 expression in myofibroblasts is closely associated with cutaneous wound healing [25]. CD248 in pericytes regulates cell proliferation and modulates pericyte–endothelial cell interaction [33, 34]. In contrast, CD248 expression in CD8+ T cells suppressed cell proliferation [35]. It is unclear whether CD248 has a proliferative effect on macrophages. Our recent study demonstrated that macrophages with lectin domain-deleted CD248 exhibit less pro-inflammatory reactions induced by LPS, and mice with this genetic background respond less to LPS-induced septic shock [36], suggesting that CD248 has the potential to regulate toll-like receptor signaling similar to thrombomodulin [37]. Here, we demonstrated that CD248 in melanoma cells also plays an important role in cell adhesion, migration, and VM. However, CD248 expression had less of an impact on melanoma cell proliferation. Based on these observations, we can conclude that CD248 plays a critical role in cell-matrix adhesion and cell migration in most studied cell types. However, the expression of CD248 may not have similar effects on cell proliferation in different cell types. Since CD248 could act as a co-receptor to modulate membrane receptor activity, as demonstrated in regulating PDGF signaling [25, 33, 38, 39], the cell-type specific effects of CD248 on cell proliferation may be attributed to the nature of different membrane receptors for growth factors existing in different cell types.

CD248 has been proposed as a valuable biomarker for evaluating tumor progression in renal cell carcinoma, soft tissue sarcoma, glioblastoma, colorectal cancer, bladder cancer, and melanoma [23, 40,41,42,43]. CD248 promotes tumor progression through both tumor cell-autonomous and non-cell-autonomous effects. Through a non-autonomous effect, stromal CD248 can promote tumor progression [32] by modulating the tumor microenvironment. For example, CD248 expressed in tumor-associated pericytes facilitates distal dissemination in a contact-dependent manner, thereby increasing circulating tumor cell numbers [20]. Moreover, CD248 regulates Wnt signaling in pericytes to promote angiogenesis and tumor growth in lung cancer [30]. CD248 expression in cancer-associated fibroblasts promotes hepatocellular carcinoma progression through interaction with CD68 on macrophages and consequent polarization to the tumor-promoting M2 phenotype [44]. In contrast, CD248 expression in some cancer cells may contribute to tumor progression through autonomous effects. For example, CD248 expression in osteosarcoma may contribute to cancer invasion and metastasis [21]. Our results show that melanoma CD248 contributes to cell migration and VM in part through modulating cell-ECM adhesion and MMP9 expression, which could promote tumor metastasis. These studies indicate that CD248 may be a promising therapeutic target for cancer treatment.

Owing to its oncofetal gene-like expression pattern, targeting CD248 has been suggested for treating several disorders, such as tissue fibrosis and cancer [17, 45]. Either by drug conjugation or by inducing internalization and degradation, antibodies against CD248 have been intensively developed [46]. Recent studies indicate that humanized monoclonal antibodies against CD248 show a significant pre-clinical effect on the suppression of tumor progression partly through the reduction of membrane CD248 expression [47] and maybe because of the inhibition of cell-ECM interaction [16]; however, the beneficial effects on cancer patients with CD248 antibody treatment was limited [48, 49]. CD248 may have multiple ECM-binding sites because it has various interacting partners, which has been demonstrated in several studies [16, 25, 44, 50, 51]. Therefore, the good and bad of being a monovalent monoclonal antibody can only block one site, which limits the use of the existing CD248 antibody. In this regard, molecular mimics and receptor decoys are promising alternatives for treating various disorders. Accordingly, molecular decoys that can fine-tune endogenous protein functions have been proposed as novel therapeutics for fighting cancer and inflammatory disease [52]. Treatment with rCD248 leads to a pronounced reduction in tumor in vitro and in vivo, suggesting that decoy molecules, such as rCD248D1-4, may have the potential to interfere with tumor progression. Although we have identified that the lectin domain of CD248 could be the functional region of CD248 that interferes with melanoma VM and metastasis, more specific sequences/regions in the lectin domain might be clearly defined in the future. Thus, synthetic peptides of active CD248 molecular decoys hold promise for treating cancer and fibrotic diseases.