With the gradually increasing incidence of lung cancer, humans have had to endure great social and economic burdens resulting from lung cancer. According to a report from China, lung cancer has been the fastest growing malignancy in the last 30 years and is the leading cause of cancer death (Chen et al., 2022). Non-small cell lung cancer (NSCLC) is the main tissue type of lung cancer and includes lung adenocarcinoma (LUAD), lung squamous (LUSC) and large cell lung cancer (Thai et al., 2021). In recent years, great progress has been made in the treatment of lung cancer. The advent of immunotherapy and targeted therapy has greatly prolonged the survival of lung cancer patients, while the efficacy is limited due to drug resistance and recurrence (Hirsch et al., 2017). Therefore, it is necessary to explore potential therapeutic targets for lung cancer.

The mode of tumor blood vessel formation is complex; vasculogenesis and angiogenesis are the two main modes, while vasculogenic mimicry (VM) is a new mode of tumor circulation (Luo et al., 2020). VM is a novel angiogenic modality based on the melanoma model proposed by Maniotis et al. (1999) When the tumor volume is larger than 2 mm³, access to oxygen and nutrients by diffusion is not sufficient for the growth of tumor cells, and it is in this context that VM is formed (Treps et al., 2021). VM is considered a unique vascular model for tumors, unlike conventional angiogenesis, which is not dependent on endothelial cell production (Wechman et al., 2020). Studies have indicated that the presence of VM is characteristic of highly aggressive and metastatic tumor cells, which also suggests that VM is associated with poor prognosis in tumor patients (Cao et al., 2013; Zhang et al., 2019). In addition to melanoma, VM is also present in solid malignancies such as lung, breast and bladder cancers (Passalidou et al., 2002; Delgado-Bellido et al., 2017), which may explain the poor effectiveness of traditional antiangiogenic drugs. VM usually occurs in a hypoxic tumor microenvironment, and the main signaling pathways associated with VM are the VE-cadherin (VE-Cad), Notch and HIF pathways (Delgado-Bellido et al., 2017; Valdivia et al., 2019). These signaling pathways are all interconnected, with VE-Cad being the factor that intersects most with other signaling pathways (Paulis et al., 2010). Some progress has been made in recent years on the mechanisms of VM formation in lung cancer. VE-cadherin (VE-Cad) has been reported to promote VM formation in lung cancer and to provide the blood supply required for lung cancer cell proliferation and invasion (Ding et al., 2018). In addition, previous studies have confirmed the role of DKK1, MMP2, MMP13 and ERβ in promoting VM formation in NSCLC (Yao et al., 2016; Li et al., 2017; Yu et al., 2019). Encouragingly, a growing number of studies have identified relevant genes and pathways involved in VM in lung cancer (Xia et al., 2019; Li et al., 2021; Shi et al., 2022; Zhang et al., 2022), which provides more evidence to clarify the mechanism of VM. However, no studies have reported the interaction of VM-related genes in lung cancer.

In this study, we obtained VM-related genes by a literature review and explored the relevant functions and expression of VM-related genes in LUAD samples based on The Cancer Genome Atlas (TCGA) database. Furthermore, we established VM scores based on univariate Cox regression analysis and Lasso regression analysis and evaluated the correlation of VM scores with prognosis and immune cell infiltration. Finally, we compared the correlation of the VM score with the sensitivity of some common antitumor drugs to provide new ideas for the clinical treatment of lung cancer.

Lung cancer remains a serious health hazard and the leading cause of cancer deaths in China (Wu et al., 2021). LUAD is the most common type of lung cancer, accounting for approximately 40% of all types (Siegel et al., 2019). The advent of immunotherapy and targeted therapies has disrupted the classic platinum-containing treatments for lung cancer; unfortunately, drug resistance remains an inevitable problem (Lim and Ma, 2019). VEGF is the main signaling pathway for tumor angiogenesis, but antiangiogenic drugs targeting VEGF have not achieved the desired results (Ren et al., 2021). VM is another modality of tumor angiogenesis that has the potential to be a target for antitumor therapy (Luo et al., 2020). Previous studies confirmed that HIF-1α, VEGF, Wnt/β-catenin, and MAPK are possible signaling pathways for VM; however, their specific roles in VM formation are not clear (Wechman et al., 2020). Wang et al. (2022) evaluated the prognostic value of VM-related genes and demonstrated that VM may be a potential target for evaluating immunotherapy in gastric cancer. Previous studies confirmed the regulatory effects of ARHGAP25, RhoA, PP2A, MMP2, and sema4D on VM based on lung cancer cells (Li et al., 2017; Xia et al., 2019; Shi et al., 2022; Zhang et al., 2022), suggesting the involvement of these molecules in the VM formation process. Here, we identified VM-related hub genes associated with the prognosis of LUAD patients and developed a VM score based on 3 prognostic genes (EPHA2, LAMC2, and LOXL2). We confirmed that a nomogram based on the VM score could predict the survival probability of LUAD patients; in addition, the VM score has important value in assessing the immune landscape immunotherapy response in LUAD patients.

In the hypoxic microenvironment, the HIF signaling pathway induces cancer stem cell (CSC) differentiation, and CSCs acquire endothelial-like features and eventually participate in VM (Wei et al., 2021). Our study also identified VM-related genes significantly enriched in the HIF-1 signaling pathway, suggesting a critical role of HIF-1 in the VM process. In addition, PGs in cancer, the VEGF signaling pathway and the relaxin signaling pathway are also pathways of VM-related genes. PGs are components of the extracellular matrix (ECM) that promote tumor angiogenesis and provide favorable conditions for tumor invasion and metastasis (Wei et al., 2020). VEGF is a major effector of tumor angiogenesis, and VEGF induces VM formation in melanoma through activation of the PI3K/Akt signaling pathway (Xu et al., 2019). A recent review suggested that the process of tumor development is a multidimensional ecological disease; VM is formed to adopt multiple cellular phenotypes due to the external environment, which contributes to understand the mechanism of VM formation in lung cancer (Luo, 2023). Collectively, the results of the enrichment analysis showed that the VM-related signaling pathways were the VEGF signaling pathway, HIF-1 signaling pathway and PGs in cancer, which is consistent with previous studies (Xu et al., 2019; Wei et al., 2020).

The vascular lining structure in VM is devoid of endothelial cells, and the formation of VM is not dependent on vascular endothelial cells (Xiang et al., 2018). Therefore, tumor cells are prone to metastasize through the bloodstream under this condition, which also suggests that VM is often found in highly aggressive and metastatic tumors and is associated with a poor prognosis (Yang et al., 2016). Our results demonstrated that the VM score is associated with poor prognosis in LUAD patients based on EPHA2, LAMC2, and LOXL2. EPHA2, a receptor tyrosine kinase, promotes the proliferation and migration of lung cancer cells and is associated with poor prognosis of lung cancer (Ishikawa et al., 2012). Wang et al. reported that EPHA2 contributes to VM in prostate cancer and could be a potential therapeutic target (Wang et al., 2016). LAMC2, a member of the extracellular matrix glycoproteins, is an important part of the epithelial basement membrane (Moon et al., 2015). LAMC2 promotes the migration and invasion of LUAD cells and is associated with LUAD prognosis (Moon et al., 2015). Subsequently, Okada et al. identified that LAMC2 not only correlates with OS in pancreatic cancer but also modulates gemcitabine sensitivity (Okada et al., 2021), suggesting that LAMC2 is a promising target in pancreatic cancer. LOXL2, a member of the lysyl oxidase (LOX) family, regulates epithelial-mesenchymal transition (EMT) through the Snail pathway. It was found that LOXL2 is highly expressed in tumor cells and promotes tumor cell invasion and metastasis (Wu and Zhu, 2015). The function of EPHA2, LAMC2 and LOXL2 in tumor cells may explain the correlation between the VM score and poor prognosis of LUAD patients. Previous studies have also demonstrated that the presence of VM is associated with poor prognosis of tumors, such as in patients with gastric cancer, melanoma, hepatocellular carcinoma, and breast cancer (Luo et al., 2020). VM correlated with clinical parameters (tumor size, lymph node metastasis, and poor differentiation) (Jafarian et al., 2019), indicating that the presence of VM is often found at an advanced stage of the tumor. Our results indicated that the VM score is associated with worsening T stage, which also indicated that high expression of VM-related genes is associated with poor prognosis in LUAD patients. Overall, the VM score was a risk factor for LUAD patients and was associated with poor prognosis in LUAD patients. In addition, a nomogram constructed by combining clinical information could realistically predict the survival probability of LUAD patients.

The TME and immune cell infiltration play key roles in tumor development and the tumor immune response (Mao et al., 2021). We demonstrated that the VM score is a valuable indicator to assess immune cell infiltration in LUAD patients. The expression of naïve B cells, activated memory CD4 T cells, follicular helper T cells, activated NK cells, eosinophils, and neutrophils was significantly correlated with the VM score based on the CIBERSORT results. We discovered that neutrophil levels were positively correlated with the VM score, suggesting that neutrophils may promote tumor growth, while neutrophils were reported to have both promoting and inhibiting effects on tumor growth (Quail et al., 2022). Naïve B cells, a member of the TME, inhibit tumor growth (Zhang et al., 2021), and we discovered a negative correlation between naïve B cells and the VM score, suggesting that VM may be a factor in promoting tumor growth. In addition, we confirmed that the TME score in the high VM score group was higher than that in the low VM score group (stromal score and ESTIMATE score). The TME plays an important role not only in tumor development and metastasis but also in tumor treatment response and drug resistance (Wu and Dai, 2017). In fact, the purpose of the TME is mainly to safeguard the growth of tumor cells. In the hypoxic and acidic TME, tumor cells need to obtain oxygen and nutrients through VM (Andreucci et al., 2022), which may explain the correlation between the TME and VM scores. Our study confirmed the importance of the VM score in assessing the TME in LUAD patients. The evaluation of immune checkpoints is a critical step in immunotherapy for LUAD patients (Li et al., 2019). We identified a positive correlation between the VM score and the expression of common immune checkpoint genes (CD274, IDO1, HAVCR2, CTLA4, PDCD1 and PDCD1LG2), which suggested that LUAD patients with a high VM score may be more sensitive to immunotherapy. The VM score may be a valuable indicator for assessing the response to immunotherapy in LUAD patients. Finally, we found that patients with high VM scores were more sensitive to common antitumor drugs (docetaxel, cisplatin, gemcitabine, paclitaxel and vinblastine) than those with low VM scores. Of note, we screened two antiangiogenic drugs, pazopanib and sorafenib, which may be more suitable for high VM score patients with LUAD. We confirmed that the VM score is a valuable indicator for assessing the immune landscape of LUAD patients. Furthermore, the VM score was significantly correlated with the expression levels of immune checkpoint-related genes, and patients with high VM score LUAD were more sensitive to immunotherapy and antiangiogenic drugs.

The VM score is a valuable indicator in evaluating the prognosis and immune profile of LUAD patients; however, there are several limitations. First, our study was retrospective and subject to selection bias. Second, we failed to confirm the relationship between VM score and immune cell infiltration in lung cancer tissues. Third, our study was not able to complete in vivo or in vitro experiments to confirm the specific function of VM-related genes in lung cancer. These are the directions of our future research.

In conclusion, we developed a VM score based on EPHA2, LAMC2 and LOXL2 in LUAD patients. We confirmed that VM may be a valuable indicator to evaluate the prognosis, immune cell infiltration and TME of LUAD patients and can contribute to guiding immunotherapy.