Thrombospondin-2 acts as a bridge between tumor extracellular matrix and immune in�ltration in pancreatic adenocarcinoma and stomach adenocarcinoma: An integrative pan-cancer analysis


 BackgroundThrombospondin-2 (THBS2) is a versatile glycoprotein that regulates numerous biological functions, including the apoptosis-proliferation balance in endothelial cells, and it has been linked to tumor angiogenesis. However, the role of THBS2 in human cancer remains unknown. This study aimed to determine THBS2 expression in pan-cancer, understand whether THBS2 is associated with its prognosis, and to identify possible roles of THBS2 in tumor immunity and the extracellular matrix (ECM).MethodsData about THBS2 expression in cancers and normal tissues were downloaded from the Genotype-Tissue Expression (GTEx) and UCSC Xena and analyzed using the ONCOMINE database, Perl programming language, and the Gene Expression Profiling and Interactive Analyses vision 2 (GEPIA2) web server. Survival prognosis was analyzed using the survival, survminer, and forestplot packages in R v. 4.0.3 and the limma package in R . Immune and matrix components were also analyzed using R v. 4.0.3. Most importantly, we partially validated the role and mechanism of THBS2 in pancreatic and gastric cancers in vitro using PANC1 and BGC-823 cell lines.ResultsThrombospondin-2 was significantly overexpressed in 17 of the 33 investigated cancers, and linked to a poor prognosis in pan-cancer survival analysis, especially in adrenocortical (ACC), kidney renal papillary cell (KIRP), and stomach (STAD) carcinomas, kidney chromophobe (KICH), and pancreatic adenocarcinoma (PAAD). Immune infiltration and THBS2 expression were also related. The expression of THBS2 has been linked to immune scores, stromal scores, and immune checkpoint markers in various cancers. The PPI network revealed that THBS2 is associated with multiple extracellular matrix proteins and immune proteins. Knockdown of THBS2 decreased the expression of CD47 and MMP-2 as well as the proliferation, migration, and invasion of PANC1 and BGC-823 cells in vitro. ConclusionsOur findings suggested that THBS2 could be a prognostic biomarker that acts as a bridge between the ECM and immune infiltration in various types of cancers, and that it promoted the progression of numerous types of cancers, especially PAAD and STAD.

Stromal broblasts, endothelial cells, and immune cells secrete THBS2, which functions in cell migration, angiogenesis, apoptosis, and cytoskeletal regulation [1].The signi cantly elevated expression of THBS2 in various types of cancers, including pancreatic and gastric cancers, is associated with their diagnosis, stage, and prognosis.Andreas et al. [2] found that early pancreatic cancer can be screened using serum markers, including THBS2.Li [3] found that combining THBS2 with CA19-9 improved the detection of early gastric cancer.Thrombospondin-2 is also a potential prognostic biomarker in colorectal, pancreatic, and gastric cancers [4].
Interplay between tumors and the immune system is complex.The tumor microenvironment (TME) is crucial for tumorigenesis and progression in human cancers and has recently become a hotspot in tumor research.In addition to cellular components, the TME contains non-cellular components that are dominated by in ltrating immune cells [5] comprising tumor-associated macrophages (TAMs), B cells, CD4+ T and CD8+ T cells, and neutrophils.In addition to mediating immune escape of tumor cells, tumor angiogenesis, and metastasis[6-8], TAMs can affect the prognosis of patients and the effect of immunotherapy [9,10].Compared with the limitations of traditional anticancer therapies, alternative immunotherapy has achieved good results in various cancers [11][12][13].However, the relevance of THBS2 in tumor immunity and the underlying mechanisms remain unclear.
We initially acquired information about the differential expression of THBS2 in normal and tumor tissues, then analyzed the relationship between THBS2 expression and patient prognosis in 33 tumors from various aspects.We also explored correlations between THBS2 expression and ECM and tumor immunity.
Finally, we knocked down THBS2 expression in PANC1 and BGC-823 cells in vitro to verify our results.Our ndings suggested that THBS2 affects the occurrence and development of cancers and the prognosis of patients via interaction with the ECM and immune cells, which might provide suggestions for cancer prevention and treatment targets.

Materials And Methods
Abbreviations and names of 33 types of cancer.
The details see Table 1.

Gene expression analysis
The mRNA expression of THBS2 in different cancers was analyzed using the ONCOMINE database (www.oncomine.org).The expression of THBS2 in different normal tissues and cancers was acquired from GEPIA2 (http://gepia2.cancer-pku.cn/).

Pan-cancer dataset source and processing
Gene-expression data and full clinical annotation of 33 cancer datasets were obtained from UCSC Xena (https://xenabrowser.net.).We then used Perl software (version 5.34.0) (https://www.perl.org/) to sort out the matrix data of gene expression values.

Survival prognosis
Survival and prognosis were analyzed using the survival, survminer, and forestplot packages in R v. 4.0.3 and the Bioconductor package limma in R.

Immune in ltration
We analyzed immune and matrix components in the TME using the estimate package in R (version 4.0.3), and ImmuneScore, StromalScore, and EstimatScore.We analyzed associations between THBS2 and ImmuneScore, and StromalScore in 33 types of cancers.We calculated the putative proportions of immune cells from gene expression pro les using the online analytical platform CIBERSORT (https://cibersort.stanford.edu/)[14].We estimated the relationship between THBS2 and the relative abundance of tumor-in ltrating immune cells in 33 types of cancers using a reference set with 22 sorted immune cell subtypes (LM22) and CIBERSORT.

Cell culture
We cultured human stomach cancer (BGC-823) and human pancreatic cancer (PANC1) cells (China Center for Type Culture Collection, Wuhan, China) in RPMI 1640 medium, and cultured in Dulbecco Modi ed Eagle medium (DMEM).The cells were routinely maintained and supplemented with 10% fetal bovine serum.
Thereafter, cancer cells were transfected with siTHBS2 or NC using Lipofectamine2000 (Thermo Fisher Scienti c Inc., Waltham, MA, USA).

Cell migration, invasion, and proliferation assay
We evaluated the migration and proliferative capacity of PANC1 and BGC-823 cells using CCK-8, transwell, and wound healing assays.

Statistical analysis
Differences between normal and cancer tissues were compared using Student t-tests.Associations between THBS2 expression and patient survival were investigated using univariate survival analysis and Kaplan-Meier curves.Values with P < 0.05 were considered statistically signi cant.The statistical signi cance of the data in vitro was determined using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA).

Expression of THBS2 mRNA in pan-cancer
We evaluated THBS2 expression in 34 normal tissues from the GTEx database.The results showed that THBS2 was signi cantly higher in the cervix, uterus, endometrium, and adipose tissue, and signi cantly lower in the skeletal muscle, cerebellum, pancreas, and stomach (Figure 1B).We further analyzed the mRNA expression of THBS2 in the Oncomine database to explore THBS2 expression in pan-cancer.The ndings revealed that cancer groups, such as bladder, colorectal, breast, esophageal, gastric, leukemia, liver, lung, lymphoma, myeloma, ovarian, pancreatic, and brain and central nervous system cancer, as well as head and neck cancer, showed higher THBS2 expression than normal groups (Figure 1C).We used TIMER2 to analyze RNA sequencing data from the TCGA database to evaluate THBS2 expression in pancancer.The differential expression of THBS2 in tumor and normal tissues is shown in Figure 1D.THBS2 expression was signi cantly lower in CESC, KICH, and UCEC than in normal tissues.However, in BRCA, COAD, CHOL, ESCA, KIRC, KIRP, LUAD, LUSC, PRAD, PEAD, and STAD, THBS2 expression was signi cantly higher than that in normal tissues.Then, we analyzed THBS2 expression in pan-cancer and normal tissues using GEPIA2, which contains data of 31 tumor tissues from the TCGA database, as well as the data of their corresponding normal tissues in the GTEx database.The results indicated that THBS2 expression was signi cantly higher in 17 cancers, mainly in BRCA, PAAD, and SARC, and signi cantly lower in 14 cancers, especially in CESC, UCEC, and UCS (Figure 1A).These ndings reveal that THBS2 is aberrantly expressed in various cancers.
Expression of THBS2 is correlated with immune in ltration and immune checkpoint markers Immune cells play important roles in tumorigenesis and cancer development [15] and are closely associated with patient survival and prognosis [16].Therefore, we obtained scores for 15 types of in ltrative immune cells in 21 types of cancer using R.The results are shown as heat maps.(Figure 7).

Protein-protein interaction network of THBS2 in cancer
We created a PPI network for THBS2 using STRING to identify probable processes through which THBS2 contributes to cancer carcinogenesis.Figure 10 shows that THBS2 is closely associated with ECM proteins such as MMP-2 and immune proteins, such as CD47.

Thrombospondin-2 promoted proliferation and metastasis in PAAD and STAD
To verify our results and better understand the biological effects of THBS2 on PAAD and STAD, we knocked down THBS2 expression by transfecting PANC-1 and BGC-823 cells with THBS2 small interfering (si) RNA.Western blotting results showed decreased expression of CD47, MMP-2 and THBS2 expression (Figure 11A-D).The results of CCK8 assays showed that THBS2 downregulation inhibited PANC1 and BGC-823 cell proliferation (Figure 11E).Finally, transwell and wound healing assays showed that THBS2 knockdown also inhibited the metastasis and invasion of PANC1 and BGC-823 cells.(Figure 12).These results indicated that THBS2 promotes the growth and metastasis of PAAD and STAD.

Discussion
Pan-cancer analysis has recently attracted increasing interest because it can easily and economically analyze tumor similarities and differences, and thus provide suggestions for cancer prevention and treatment targets [17].Thrombospondin-2 protein belongs to the thrombospondin family, and is considered as a vital regulator of tumorigenesis.A clinical study of patients with cancer in China has found signi cant heterogeneity in the distribution of serum THBS2 in various types of cancer compared with healthy control individuals [18].Thrombospondin-2 might be a diagnostic marker of pancreatic, gastric, non-small-cell lung, and colorectal cancers [2,3,[19][20][21].
Our analysis of data from TCGA and the GTEx database revealed the abnormal expression of THBS2 in 17 types of cancer compared with healthy tissues.Therefore, we further investigated the importance of THBS2 expression to the prognosis of patients and found that increased THBS2 expression was associated with a worse prognosis (OS, DSS, DFI, and PFI), especially those with ACC, KICH, KIRP, PAAD, and STAD.These results indicated that THBS2 could be a prognostic marker for ACC, KICH, KIRP, PAAD, and STAD.
The TME has become a prominent and important area in tumor research.The TME consists of immune cells, stromal cells, blood vessels, and the ECM [22].Immune cells comprise in ltrating cells such as B, T, and other types of immune cells.Immune cells play dual roles in tumors.On the one hand, they can detect and kill tumor cells [22,23], whereas on the other hand, they can allow tumor cells to evade immune surveillance and metastasize via various mechanisms [24][25][26].For example, CD8+ T cells can recognize and kill cancer cells that express aberrant tumor antigens.In response to the actions of various cytokines and environments, CD4+T cells can differentiate into various cell subtypes and participate in the adaptive immune response.Although the TME contains fewer B than T cells, they are nevertheless important to tumor development [27].In addition to immune-in ltrating cells, other types of immune cells have speci c functions in the TME.Elevated levels of macrophage in ltration are related to a poor prognosis among patients with breast, lung, and gastric cancers [11,28,29].Neutrophils are recruited to the TME early in tumor development and promote tumor cell apoptosis by releasing cytokines and reactive oxygen species (ROS).However, neutrophils can later stimulate angiogenesis by producing matrix metalloproteinases (MMPs) that ultimately promote tumor progression, local invasion, and consequently enhanced tumor growth [30].In conclusion, immune cells play crucial roles in tumorigenesis and progression.The present study found that THBS2 expression was signi cantly related to the abundance of various immune cells in various types of tumors.We also determined the THSB2 expression was associated with > 30 immune checkpoint genes in 20 types of cancer.ImmuneScore and StromalScore are associated with the amount of immune and matrix components in the tumor microenvironment [31].Our results showed that THBS2 expression positively correlated with ImmuneScore and StromalScore in 30 and 19 types of cancer, respectively.The ECM plays an important role in the TME.In addition to being a physical scaffold for cells, it is also a vital element in driving tumor cell spread.Matrix metalloproteinases comprise a family of zinc-and calcium-dependent proteases that digest ECM proteins and are essential for reshaping the ECM [27].Furthermore, MMPs not only degrade ECM, they play crucial roles in mediating tumor angiogenesis, metastasis, and invasion.Among the MMP family, MMP-2 promotes tumor growth, tissue invasion, angiogenesis, and metastasis [32][33][34], and its overexpression might be associated with tumor progression and a poor of ovarian epithelial carcinoma, oral cavity cancers, and non-small cell lung cancer [35][36][37][38].Therefore, MMP-2 is considered a potential biomarker of tumorigenesis, a key effector of ECM remodeling, and a potential target for antitumor therapy.We showed that THBS2 in the tumor microenvironment was signi cantly associated with ECM proteins, including MMP-2, and immune-related proteins such as CD47, in the PPI network.Finally, our ndings in vitro con rmed that THBS2 knockdown inhibited the proliferation, migration and invasion of PANC1 and BGC-823 cells and decreased the expression of the immune-related protein CD47 and the ECM proteinase MMP-2.These results suggest that THBS2 acts as a bridge between ECM and immune in ltration, thus in uencing tumor progression.

Conclusions
In general, our ndings indicated that THBS2 overexpression correlates with a poor prognosis and increased immune cell in ltration in numerous types of cancer.Furthermore, THBS2 was closely associated with various ECM and immune proteins, and the expression of immune checkpoint markers in various types of cancer.Our result in vitro con rmed that THBS2 knockdown inhibited CD47 and MMP-2 expression and the progression of pancreatic and gastric cancers.Thus, THBS2 might promote cancer progression, function as a bridge between the ECM and immune in ltration in cancer, and serve as a potential prognostic biomarker of several cancers, especially pancreatic and gastric adenocarcinoma.Correlations between THBS2 expression and overall survival.
A Relationship between HRs and THBS2 expression in 33 types of cancer.B-J Kaplan-Meier curves show relationships between differential expression of THBS2 and overall survival of ACC, BLCA, KIRC, KIRP, LGG, MESO, PAAD, STAD, SKCM and UVM.
Correlation between THBS2 expression and disease-speci c survival.
A Relationships between hazard ratios and THBS2 expression in 32 cancers.B-H Kaplan-Meier curves show relationships between differential expression of THBS2 and disease-speci c survival of ACC, KIRC, KIRP, LGG, MESO, PAAD and UVM.
Correlations between THBS2 expression and disease-free interval.
A Relationships between hazard ratios and THBS2 expression in 28 types of cancer.B-C Kaplan-Meier curves show relationships between differential expression of THBS2 and disease-free intervals in CESC and PAAD.
Correlations between THBS2 expression and platinum-free interval.
A Relationships between hazard ratios and THBS2 expression in 32 types of cancer.B-I Kaplan-Meier curves show relationships between differential expression of THBS2 and platinum-free intervals in BRCA, COAD, KICH, KIRC, MESO, PAAD, PRAD, and UVM.
Association between THBS2 expression and stages of 10 types of cancer.
Correlations between THBS2 expression and abundance of in ltrating immune cells among types of cancer.Correlations between THBS2 expression and 48 common immune checkpoint genes in 33 types of cancer.
Protein-protein interaction network for THBS2.

Figure 11 Expression
Figure 11

Table 1
Abbreviations and names of 33 types of cancer.