Immune infiltrating cells in cholangiocarcinoma may become clinical diagnostic markers: based on bioinformatics analysis

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor originating from the secondary bile duct and its branch epithelium [1]. Among primary liver tumors, the incidence of ICC is second only to hepatocellular carcinoma, and the incidence of ICC in China is on the rise. Early diagnosis can give ICC patients better therapeutic effects, so it is very important to find more markers to diagnose ICC [2]. Recent studies have shown that the tumor microenvironment can regulate the occurrence and development of tumors. This study is dedicated to finding more markers that can diagnose ICC by finding the differential tumor microenvironment cells between tumors and normal tissues [3].

In this study, by searching the ICC-related data set in the GEO database, we established relevant selection and exclusion criteria: (1) must be paired intrahepatic cholangiocarcinoma and para-cancer tissue, (2) the quantity must be at least 10 pairs, and (3) these data sets have not previously been used to study immune cell infiltration in ICC [4]. It was found that GSE45001 [5] (https://​www.​ncbi.​nlm.​nih.​gov/​gds/​?​term=​GSE45001) met the research conditions (with 10 tumor samples and 10 normal tissue samples). We put it into the CIBERSORT (https://​cibersort.​stanford.​edu/​) [6] database to analyze their immune cell composition. Then, take histograms, principal component maps, heat maps [7], and other visualizations to show the differences in immune cells between tumor tissues and normal tissues. We found that there are indeed some differences in the content of immune cells between ICC tissues and normal tissues, and through co-expression heat maps, violin diagrams, etc., more accurately extract the difference between tumor tissues and normal tissues.

Intrahepatic cholangiocarcinoma (ICC) is a type of primary malignant tumor of the liver that originates from the bile duct and its branches. Because the clinical symptoms of ICC are not obvious early, it often causes patients to lose the opportunity for timely surgery. Therefore, it is extremely critical to find a better method for early diagnosis of ICC. The tumor microenvironment is closely related to the occurrence and development of tumors. Many studies have shown that the tumor microenvironment plays a role in promoting tumorigenesis and development, such as promoting angiogenesis, producing growth factors that promote tumors, and causing hypoxic environments and inflammatory reactions. However, ICC-related research has not explored the relationship between the tumor microenvironment and ICC. With the development of biochip technology, more and more bioinformatics databases and bioinformatics tools are available for our research and use. Therefore, we can analyze the cells that constitute the immune microenvironment of ICC through bioinformatics technology to find tumor markers that can better diagnose ICC.

In this study, we retrieved the ICC data set from the GEO database and found that GSE45001 met our research needs. Then we downloaded and grouped them reasonably, 10 samples were classified into tumor group, and 10 samples were classified into normal tissue group. Put these 20 samples into the CIBERSORT database to analyze the content of immune cells and draw the principal component map. The results show that the composition of immune cells between tumor tissues and normal tissues is not the same. Through the heat map we draw, we can find that some immune cells are expressed in tumors higher than normal tissues, but some immune cells are lower than normal tissues. From the co-expression heat maps we draw, we can better discover which immune cells are more or less relevant in this study, and drawing a violin chart can more intuitively find the expression of each immune cell in different tissue. Finally, we found that the expression of dendritic cells activated, and macrophages M2, T cells regulatory (Tregs) in tumor tissue was higher than that in normal tissue, while the expression of macrophages M1, monocytes, and T cells follicular helper in tumor tissue was lower than that in normal tissue. This difference provides us with more basis for clinical diagnosis of ICC.

Dendritic cells have long been considered to be anti-tumor immune cells, but studies have found that dendritic cells can promote tumor suppression of immunity. Some people have achieved the idea of inhibiting tumor growth by suppressing the number of dendritic cells and believe that dendritic cell-related pathways may become new therapeutic targets [22]. Macrophages M1 cells can accelerate the apoptosis process of tumor cells, in line with our research results, its expression in normal tissues is higher, and it has a certain effect on inhibiting tumor cells. However, macrophages M2 and macrophages M1 have opposite functions. They can delay the apoptosis of tumor cells and promote the progression of tumors [23]. Monocytes can not only promote tumor growth by increasing angiogenesis, but also inhibit tumor growth by identifying and clearing tumor escape signal pathways. In this study, although the content of monocytes was higher in normal tissues than in tumor tissues in general, there were obviously some of the opposite. According to our statistical analysis, monocytes inhibit tumor growth in ICC [24]. By querying in the TIMER 2.0 database, two immune cells (monocytes, T cells regulatory) were found to be related to the survival prognosis of ICC patients, and there was statistical significance. And these two cells also fit the logic of this study that both of these cells are protective factors for ICC.

In this study, we analyzed the components of the immune microenvironment of ICC and found that 6 types of immune cells are differentially expressed between ICC and normal tissues. These 6 types of immune cells are expected to become molecular markers for clinical diagnosis of ICC.