This study found that the TME-related gene, IGLL5 has potential as a robust indicator for TME status and a strong predictor for the ccRCC population in the TCGA database. The ESTIMATE algorithm was used to calculate the immune and stromal scores of ccRCC tumour samples. The Kaplan–Meier survival curve showed that the patients with high immune score had worse prognosis. IGLL5 was the only common gene identified by PPI network and Cox regression analyses. Internal and external validation were performed to verify that IGLL5 is highly expressed in ccRCC tumour tissues showed that IGLL5 > 16.33 conferred a higher risk of developing high Stage and high T stage in clinical application; thus, it might serve as an indicator for the early detection of ccRCC development and recurrence.
TME is the complex cellular milieu where the tumour is located. The importance of TME in tumour formation, progression, invasion and recurrence has been well shown by a wealth of evidence[17, 29, 30]. In the present study, the Kaplan–Meier survival curve revealed that patients with high immune score had significantly shorter survival time than those with low immune score (P < 0.05, Fig. 2A). Moreover, high immune score led to a higher tumour grade and higher Stage than low immune score (Fig. 3). These results indicated that patients with higher immune score had considerably worse overall survival. This finding also corroborated with previous studies[31, 32]. This finding demonstrated that the immune component of the ccRCC TME may be double-edged sword. The immune system, as a defensive mechanism, could inhibit tumour development to protect the host. However, immune components might promote tumour cell growth and progression once cancer immunogenicity is reduced. Furthermore, several cytokines and chemokines produced by tumour cells, such as TGF-β and VEGF, promote the recruitment and activation of immune-inhibitory cells and lead to an immunosuppressive state, which results in the growth of the tumour, whose bidirectional interactions may function as an ‘amplification loop’ to enhance ccRCC tumour progression.
Based on the analyses of the PPI network, univariate Cox regression and Venn diagram, IGLL5 was identified as a potential prognostic biomarker (Figs. 4 and 5) that is negatively correlated with the prognosis of patients with ccRCC. This finding was consistent with previous reports[31, 32] and was internally and externally validated. The mRNA expression of IGLL5 was remarkably higher in the tumour tissues compared with the adjacent normal tissues (Figs. 6A and 6B). Additionally, the protein expression of IGLL5 was remarkably up-regulated in Caki-1, 786-O and 769-P cell lines (Fig. 6C). The 293T cell line, derived from human embryonic kidney cells, displays a cancer stem cell-like properties. Compared to the HK2 cells lines, the expression of IGLL5 was significantly increased in 293T cell line, which may indicate the role of IGLL5 in tumorigenesis and development. Multiple studies reported that IGLL5 plays a key role in tumour pathogenesis, especially in haematological malignancies. As oncogenic drivers, the coding and non-coding mutations of IGLL5 have been observed in chronic lymphocytic leukaemia, Burkitt lymphoma, diffuse large B-cell lymphoma and multiple myeloma and lead to rapid tumourigenesis. IGLL5 is also a biomarker for the early progression of multiple myeloma and serves as a predictor for the relapse-free survival of patients with breast cancer. One possible mechanism is that IGLL5 expression might remodel an immunosuppressed microenvironment via the infiltration of immune-inhibitory cells or the loss of anti-tumour T cell activity by pro-inflammatory cytokines and various immune cells pathways. These interactions would be enhanced further as the expression of IGLL5 exceeds 16.33, which results in the advanced tumour stage and worse prognosis of patients with ccRCC.
Indeed, GSEA and immune infiltration analyses were performed to further unveil the potential mechanism of IGLL5. IGLL5 had a strong association with inflammatory and immune responses (Figs. 7A, Fig. 7C and Supplementary Table 1). According to the analysis results of the spatial transcriptomics of patients with periodontitis, IGLL5 is remarkably up-regulated in the inflamed areas of the gingival connective tissue, which suggests the unique role of IGLL5 in chronic inflammatory response. KEGG enrichment analysis also showed that IGLL5 was substantially enriched in cytokine– cytokine receptor interaction (Fig. 7C), which implies that high IGLL5 expression might activate various pro-inflammatory cytokines. In fact, ccRCC is largely considered to be a pro-inflammatory tumour. Pro-inflammation mediators,such as IL-1β, IL-6 and TNF, are produced at high levels in ccRCC and stimulate immune-inhibitory cell activation to promote tumour growth and metastasis. Moreover, as a member of the immunoglobulin gene superfamily, IGLL5 was suggested to be closely related to various immune responses. Pathway enrichment analysis revealed that IGLL5 was remarkably enriched in the NK cell-mediated cytotoxicity pathway, T cell receptor signalling pathway and B cell receptor signalling pathway (Fig. 7C). Besides, the immune infiltration analysis demonstrated that IGLL5 was positively correlated with 7 kinds of TICs (B cell memory, plasma cells, CD8 T cells, CD4 memory activated T cells, follicular helper T cells, Tregs and gamma delta T cells; Fig. 8), which fully illustrated a high immune infiltration state in ccRCC. This result was in accordance with previous reports[31, 32]. The study of Chevrier et al. showed that half of infiltrating immune cells in the TME are the T cells of ccRCC tumours and have immunosuppressed properties. This finding implies that high levels of infiltrating T cells leads to an immunosuppressed microenvironment. This finding is in line with our data and that is most of the cell types tightly linked to IGLL5 are T cells. Tregs belong to the immune-inhibitory cells and mediate immune dysfunction by inhibiting the secretion of immunosuppressed signals. When T cells are persistently exposed to antigen and/or inflammatory cytokines, they could lose their effector functions. Thus, one possible mechanism is that the expression of IGLL5 might remodel an immunosuppressed microenvironment via the infiltration of immune-inhibitory cells or the loss of anti-tumour T cell activity by pro-inflammatory cytokines and various immune cell pathways, which result in the poor prognosis of patients with ccRCC.
Finally, we explored the relation between the expression level of IGLL5 and clinicopathological characteristics to verify the clinical application of IGLL5. Firstly, the optimal cut-off value of IGLL5 was identified as 16.33 using the X-tile software, and IGLL5 was split into two groups based on this threshold (Fig. 9). Secondly, the comparison of the two groups showed that the risk of developing high Stage and high T stage in the IGLL5 > 16.33 group was two times higher than that in the IGLL5 ≤ 16.33 group (all P < 0.001). Thus, IGLL5 > 16.33 may suggest a later tumour stage and worse prognosis.
The strength of our study is that the protein expression of IGLL5 in ccRCC cells was externally verified by Western blotting, which further proves the role of IGLL5 in tumorigenesis and development. The expression level of IGLL5 was split into two groups depending on the optimal cut-off value calculated by the X-tile software to confirm clinical application of IGLL5. However, some limitations of our study should be acknowledged. Randomised controlled studies are needed to confirm the clinical value of IGLL5 to improve the outcome of patients with ccRCC in our future study.