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The prognostic biomarker TPGS2 is correlated with immune infiltrates in pan-cancer: a bioinformatics analysis

Zujun Ding1, Qing Ding2, Hang Li1

1Department of General Surgery, Affiliated Hospital of Hangzhou Normal University, Hangzhou, China; 2Department of Pharmacy, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, China

Contributions: (I) Conception and design: Z Ding; (II) Administrative support: H Li; (III) Provision of study materials: Z Ding, Q Ding; (IV) Collection and assembly of data: Z Ding; (V) Data analysis and interpretation: Z Ding, Q Ding; (VI) Manuscript writing: All authors; (VII) Final ☒ approval of manuscript: All authors.

Correspondence to: Hang Li, MD. Department of General Surgery, Affiliated Hospital of Hangzhou Normal University, 126 Wenzhou Road, Hangzhou 310015, China. Email: hanglhznu@126.com.

Background: Tubulin polyglutamylase complex subunit 2 (TPGS2) is an element of the neuronal polyglutamylase complex that plays a role in the post-translational addition of glutamate residues to C-terminal tubulin tails. Recent research has shown that TPGS2 is associated with some tumors, but the roles of TPGS2 in tumor immunity remain unclear.

Methods: The research data were mainly sourced from The Cancer Genome Atlas. The data were analyzed to identify potential correlations between TPGS2 expression and survival, gene alterations, the tumor mutational burden (TMB), microsatellite instability (MSI), immune infiltration, and various immune-related genes across various cancers. The Wilcoxon rank-sum test was used to identify the significance. A log- rank test and univariate Cox regression analysis were performed to assess the survival state of the patients. Spearman’s correlation coefficients were used to show the correlations.

Results: TPGS2 exhibited abnormal expression patterns in most types of cancers, and has promising prognostic potential in adrenocortical carcinoma and liver hepatocellular carcinoma. Further, TPGS2 expression was significantly correlated with molecular and immune subtypes. Moreover, the single-cell analyses showed that the expression of TPGS2 was associated with the cell cycle, metastasis, invasion, inflammation, and DNA damage. In addition, the immune cell infiltration analysis and gene-set enrichment analysis demonstrated that a variety of immune cells and immune processes were associated with TPGS2 expression in various cancers. Further, immune regulators, including immunoinhibitors, immunostimulators, the major histocompatibility complex, chemokines, and chemokine receptors, were correlated with TPGS2 expression in different cancer types. Finally, the TMB and MSI, which have been identified as powerful predictors of immunotherapy, were shown to be correlated with the expression of TPGS2 across human cancers.

Conclusions: TPGS2 is aberrantly expressed in most cancer tissues and might be associated with immune cell infiltration in the tumor microenvironment. TPGS2 could serve not only as a biomarker for predicting clinical outcomes, but also as a promising biomarker for evaluating and developing new approaches to immunotherapy in many types of cancers, especially colon adenocarcinoma and stomach adenocarcinoma.

Keywords: Tubulin polyglutamylase complex subunit 2 (TPGS2); pan-cancer; tumor microenvironment (TME); biomarker; immunotherapy

Submitted Jan 29, 2023. Accepted for publication Oct 20, 2023. Published online Mar 21, 2024. doi: 10.21037/tcr-23-113

View this article at: https://dx.doi.org/10.21037/tcr-23-113

Introduction

At present, cancer is a leading cause of disease morbidity and mortality worldwide. Unfortunately, the number of newly diagnosed cases continues to grow (1,2). Current mainstream treatment modalities, including surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, still do not provide a satisfactory prognosis for cancer patients (3). This pan-cancer study sought to apply diagnostic and therapeutic applications in gastric cancer to a broad range of tumors with characteristics of similarity. The identification of common key genes between different types of cancers can help in cancer diagnosis and treatment (4,5).

The application of immunotherapy has opened up a new era in tumor treatment, and it has achieved encouraging results in the treatment of several tumors (including lung cancer, melanoma, and liver cancer), greatly improving the prognosis of patients (6,7). Many immune checkpoint inhibitors (ICIs), such as programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitors, have been established as routine treatments for many types malignancies; however, their clinical efficacy is limited (8,9). Given the complexities in the efficacy of immunotherapy, it is of great significance to explore new and more effective immune biomarkers.

The tumor microenvironment (TME) refers to the pericellular environment including immune cells, blood vessels, extracellular matrices, fibroblasts, mast cells, and various signaling molecules around the tumor (10,11). In recent years, the important role of the TME in tumorigenesis and development has been recognized.

Highlight box

Key findings

· Tubulin polyglutamylase complex subunit 2 (TPGS2) is a potential prognostic and immunotherapeutic biomarker in many types of cancers, especially colon adenocarcinoma and stomach adenocarcinoma.

What is known and what is new?

· TPGS2 has been found to be associated with some tumors.

· TPGS2 plays a crucial role in tumor immunity.

What is the implication, and what should change now?

· TPGS2 is a promising tumor immune target, and more research on TPGS2 and tumor immunity should be conducted.

Tumor proliferation, infiltration, and metastasis depend not only on the tumor cells but also on the regulation of various cellular and signaling molecules in the TME (12). T cells, which are crucial in the anti-tumor immune response, have been relatively well studied among adaptive immune cells (13,14). Conversely, the second adaptive immune cell population (i.e., B cells) in the TME has not been well characterized.

However, in the last 5 years, several studies have reported an association between the presence of B cells in the TME and improved clinical outcomes (15-20). B cells can resist tumors by producing tumor-specific antibodies under certain conditions, but specific B cell subsets and antibody specificity can also suppress anti-tumor immunity and promote tumor growth (21). Meanwhile, infiltrating B cells are an important component of tertiary lymphoid structures (TLSs) in tumor tissues (15). Many studies have shown that the number and proportion of stromal cells and immune cells in tumor tissues are closely related to clinical features and prognosis (22-28). A thorough understanding of the TME is essential for accurate evaluation and treatment. To improve prognosis, more targeted molecules need to be identified for cancer diagnosis and treatment and to assess patient prognosis.

Few relevant studies have been conducted on tubulin polyglutamylase complex subunit 2 (TPGS2). We obtained partial information on TPGS2 from the Entrez Molecular Sequence Database Entrez (https://www.ncbi.nlm.nih.gov/ search/), which showed that TPGS2 encodes a protein that is an element of the neuronal polyglutamylase complex, which plays a role in the post-translational addition of glutamate residues to C-terminal tubulin tails, and alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. TPGS2 also appears to be associated with tumors and the TME (29,30).

In our preliminary analysis, we confirmed that TPGS2 has a special role in cancer immunity. Based on this finding, we then performed the pan-cancer analysis to explore the expression, prognostic function, and immune role of TPGS2 in various cancers. We comprehensively analyzed the relationship between TPGS2 expression and patient prognosis in 33 types of cancer. Additionally, we further evaluated the association between TPGS2 and tumor- infiltrating immune cells. Our findings revealed that TPGS2 has a potential role in the development and progression of cancers; thus, TPGS2 may serve as a potential prognostic and immunotherapeutic biomarker. We present this article in accordance with the REMARK reporting checklist (available at https://tcr.amegroups.com/article/

view/10.21037/tcr-23-113/rc).

Methods

The research data were downloaded from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and Human Protein Atlas (HPA). databases. Data were downloaded for the following tumors: adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain low-grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), mesothelioma (MESO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS), and uveal melanoma (UM or UVM). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Data source and processing

The gene expression and clinical data of normal human tissues and cancer tissues were downloaded from the GTEx database and TCGA by UCSC Xena (https://xenabrowser.net/) (31). For a multidimensional demonstration, the expression of TPGS2 was analyzed in various cancer cell lines with data from the CCLE. The transcripts per million (TPM) format and the log2(TPM+1) format were used for the expression profiles and subsequent analyses. Statistical significance was defined as follows: P<0.05, P<0.01, and P<0.001.

IHC of TPGS2

The protein expression of the tumor tissues and normal

tissues from the HPA (http://www.proteinatlas.org/) database was applied to verify the protein expression levels of TPGS2 (32). Data from the HPA database were also used to confirm the intensity of TPGS2 in immunohistochemical staining in six normal and cancer tissues, including LIHC, LUSC, COAD, STAD, PRAD, and TGCT.

Genomic alterations analysis of TPGS2

The cBioPortal (http://www.cbioportal.org) is a multipurpose cancer genomics database that can recognize the molecular information of cancer tissues and comprehend the associated genetics, epigenetics, gene expression, and proteome information (33,34). The cBioPortal was used to display the alteration frequency (including mutation, structural variation, amplification, deep deletion, and multiple alterations) across cancers, and the results were visualized in bar plots. We also obtained a landscape map of the gene mutation sites, a correlation diagram of the copy number alterations (CNAs) and TPGS2 expression, and Kaplan-Meier curves of the pan-cancer data using the cBioPortal webtool.

Prognostic analysis

The UCSC Xena database was used to download the related prognostic data, including overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) data. Next, we plotted the Kaplan-Meier model and univariate Cox regression results to assess the prognosis of various cancers. The TPGS2 expression median of each cancer was used to divide patients into high- and low- expression subgroups. Next, the Kaplan-Meier method was used to compute the log-rank P value and hazard ratio (HR) with a 95% confidence interval (CI). The “survival” package (3.2-10) was used for the statistical analysis of the survival data, and the “survminer” package (0.4.9) was used for the visualization.

Single-cell analysis of TPGS2

The Cancer Single-cell State Atlas (CancerSEA), a specialized single-cell sequencing database, provides various functional data on cancer cells at the single-cell level (35). The average correlations between the TPGS2 expression and functional states in different cancers were summarized and presented in a heatmap. The correlations between TPGS2 expression and several tumor functions

were investigated using single-cell sequencing data. The TPGS2 expression profiles of single cells are shown in the t-distributed stochastic neighbor embedding (t-SNE) diagrams.

Gene set enrichment analysis (GSEA)

A GSEA was conducted using the “clusterProfiler” package (3.14.3), and “ggplot2” (3.3.3) was used to graph the results (36,37). The reference gene set was c5.bp.v7.2.symbols.gmt (Gene Ontology), which was derived from the website of the Molecular Signatures Database (MSigDB, https://www. gsea-msigdb.org/gsea/index.jsp). It is generally accepted that the threshold of significant enrichment is a false discovery rate (FDR) <0.25 and a P adjusted value <0.05.

Immune cell infiltration analysis and TME

The immune cell infiltration analysis was largely performed using the Tumor Immune Estimation Resource (TIMER) (38). The TPGS2-associated immune cell infiltration correlations were downloaded from the TIMER 2.0 database (http://timer.cistrome.org/). Finally, we visualized the statistical Spearman correlations between TPGS2 messenger RNA (mRNA) expression and 20 immune cell subsets.

Correlation analysis of the TMB, MSI, and immune regulators

A Spearman correlation analysis of immune regulators and TPGS2 expression was performed to investigate the correlation between TPGS2 and the reported biomarkers of cancer immunotherapy, including immunostimulators, immunoinhibitors, the major histocompatibility complex (MHC) genes, chemokines, and chemokine receptors, for various cancer types. A Spearman correlation analysis was also conducted to analyze the relationship between the tumor mutational burden (TMB) (39), microsatellite instability (MSI), and TPGS2 expression (40) across various cancers.

Statistical analysis

The Wilcoxon rank-sum test was used to evaluate the statistical significance of and compare the TPGS2 expression levels between tumor and normal tissues. The survival analysis was performed using the Kaplan-Meier method

(log-rank test) and a univariate Cox regression analysis was also conducted. A Spearman correlation analysis was conducted to evaluate the correlations between TPGS2 and other factors, such as immune cell infiltration, the TMB, and MSI. A P value <0.05 was considered statistically significant.

Results

Expression of TPGS2 in cancer tissues

First, we integrated the mRNA expression levels of normal tissues in the GTEx database. The results showed that TPGS2 was highly expressed in the normal tissues of TGCT, BLCA, and OV, and most lowly expressed in LIHC (Figure 1A). According to the tumor cell data from the CCLE, compared to the other tumor cells, TPGS2 was the most highly expressed in small cell lung cancer and CESC, and the most lowly expressed in chronic lymphocytic leukemia (Figure 1B). We then combined the data from TCGA and GTEx databases to reflect the expression levels of TPGS2 mRNA in various malignancies (Figure 1C). The results showed that TPGS2 mRNA was more highly expressed in 22 kinds of tumors (BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, LGG, LIHC, LUAD, LUSC, OV, PAAD, PCPG, READ, SKCM, STAD, THCA, THYM, UCEC, and UCS) than their respective normal tissues. Conversely, TPGS2 was more lowly expressed in five tumors (ACC, KIRC, LAML, PRAD, and TGCT) than their normal tissues. Further, TPGS2 mRNA was significantly more highly expressed in BLCA, CHOL, COAD, ESCA, HNSC, LIHC, LUSC, STAD, and UCEC cancer tissues than matched normal tissues, and more highly expressed in KICH and PRAD tumor tissues (Figure 1D).

Moreover, we evaluated the protein expression of TPGS2 between normal and tumor tissues using the HPA database. As Figure 2 shows, compared to the weak staining of TPGS2 in the normal liver, lung, colon, and stomach tissues, stronger staining was observed in the LIHC, LUSC, COAD, and STAD tissues (Figure 2A-2D). Normal prostate and testes tissues had medium TPGS2 staining, while their tumor tissues had weaker staining (Figure 2E,2F). Thus, the immunohistochemistry (IHC) results re-confirmed our previous analyses. These results indicated that TPGS2 is aberrantly expressed across human cancers, and we speculated that TPGS2 may be able to inform the prognosis and treatment of various cancers.

Figure 1 TPGS2 mRNA expression levels in pan-cancer. (A) TPGS2 expression levels in normal tissues from the GTEx; (B) TPGS2 expression levels in tumor cells from the CCLE; (C) TPGS2 expression difference between tumor tissues from TCGA and normal tissues from the GTEx; (D) TPGS2 expression difference between tumor tissues and matched normal tissues from TCGA. ns, no significance; * , P<0.05; ** , P<0.01; *** , P<0.001. TPGS2, tubulin polyglutamylase complex subunit 2; TPM, transcripts per million; mRNA, messenger RNA; GTEx, Genotype-Tissue Expression; CCLE, Cancer Cell Line Encyclopedia; TCGA, The Cancer Genome Atlas.

A

TGCT

LIHC

PAAD

B

PAAD

STAD

CESC

SCLC

CLL

BLCA

8

7.5

ESCA

LGG

HNSC

OV

7

DLBC

ALL

7

COAD-READ

SKCM

6

THYM

NB

6

5

STAD

5

ACC

KIRC

GBM

6

LAML

4

5.5

LGG

LCML

3

KICH

SARC

5

MESO

Ewings

GBM

KIRP

DLBC

MM

LUSC

COAD

BLCA

LUAD

LUAD

READ

LUSC

BRCA

THCA

PRAD

KIRC

OV

CESC

BRCA

PRAD

LIHC

ESCA

UVM

UCEC

THCA

UCEC

MB

SKCM

NSC

Mean expression

Mean expression

C

10


ns


ns

**

ns

The expression of TPGS2 Log2 (TPM+1)

8

6

Normal

Tumor

4

2

0

ACC

BLCA

BRCA

CESC

CHOL

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LAML

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC SKCM

STAD

TGCT

THCA

THYM

UCEC

UCS

UVM

D

7

ns


**


ns

The expression of TPGS2 Log2 (TPM+1)

6

**

**


ns

ns

ns

5

**

*


ns

*

ns

Normal

4

Tumor

3

2

1

BLCA

BRCA

CHOL

COAD

ESCA

HNSC

KICH

KIRC

KIRP

LIHC

LUAD

LUSC

PAAD

PRAD

READ

STAD

THCA

UCEC

Association with molecular and immune subtypes

To explore the associations between TPGS2 expression and molecular and immune subtypes across human cancers, we performed a further analysis by the tumor-immune system interaction database (TISDB). As Figure 3A-3F

show, TPGS2 expression was significantly associated with the molecular stages of many cancers, such as BRCA, COAD, HNSC, LGG, LUSC, and PCPG. To explore the relationship between TPGS2 and cancer immunity, we analyzed the correlation between the immune subtypes and TPGS2 expression, and found that the expression of TPGS2

Figure 2 IHC results for various normal and tumor tissues from the HPA. The staining of the TPGS2 protein in (A) liver and LIHC tissues (https://www.proteinatlas.org/ENSG00000134779-TPGS2/pathology/liver+cancer); (B) lung and LUSC tissues (https://www.proteinatlas. org/ENSG00000134779-TPGS2/pathology/lung+cancer); (C) colon and COAD tissues (https://www.proteinatlas.org/ENSG00000134779- TPGS2/pathology/colorectal+cancer); (D) stomach and STAD tissues (https://www.proteinatlas.org/ENSG00000134779-TPGS2/ pathology/stomach+cancer); (E) prostate and PRAD tissues (https://www.proteinatlas.org/ENSG00000134779-TPGS2/pathology/ prostate+cancer); and (F) testis and TGCT tissues (https://www.proteinatlas.org/ENSG00000134779-TPGS2/pathology/testis+cancer). All images have a magnification of x80. LIHC, liver hepatocellular carcinoma; LUSC, lung squamous cell carcinoma; COAD, colon adenocarcinoma; STAD, stomach adenocarcinoma; PRAD, prostate adenocarcinoma; TGCT, testicular germ cell tumor; IHC, immunohistochemistry; HPA, Human Protein Atlas; TPGS2, tubulin polyglutamylase complex subunit 2.

A

B

80×

80×

80×

80×

80×

80×

Liver normal

LIHC

LIHC

Lung normal

LUSC

LUSC

C

D

80×

80×

80×

80×

80×

80×

Colon normal

COAD

COAD

Stomach normal

STAD

STAD

E

F

80×

80×

80×

80×

80×

80×

Prostate normal

PRAD

PRAD

Testis normal

TGCT

TGCT

was significantly related to immune subtypes in many cancers, including BRCA, KIRC, LIHC, STAD, OV, and SARC (Figure 3G-3L). These results indicated that TPGS2 has potential prediction and treatment functions in pan- cancer.

Genetic alteration of TPGS2

Given the abnormal expression of TPGS2 observed in cancer, we sought to examine whether genetic alterations in TPGS2 caused this change. Therefore, we conducted a genetic alteration analysis of TPGS2 using the cBioPortal database with data from TCGA, and PanCancer Atlas. As Figure 4A shows, PAAD had the highest alteration rate (6%)

with “amplification” and “deep deletion” as the primary types. Conversely, the “deep deletion” type of the CNAs was the primary altered type in the ESAD cases, which had an alteration frequency of ~4%. Notably, the main genetic alteration in UCEC and SKCM was “mutation” (Figure 4A). As Figure 4B shows, after examining putative CNAs from the significant targets in cancer (GISTIC) module, the CNAs were closely related to the mRNA expression of TPGS2.

Clinical prognostic significance of TPGS2

To examine the prognostic role of TPGS2 across human cancers, prognostic indicators in 33 cancers were

Figure 3 Distribution of TPGS2 expression across the molecular subtypes of BRCA (A), COAD (B), HNSC (C), LGG (D), LUSC (E), and PCPG (F); associations between TPGS2 expression and the main immune subtypes of BRCA (G), KIRC (H), LIHC (I), STAD (J), OV (K), and SARC (L). CPM, counts per million reads; TPGS2, tubulin polyglutamylase complex subunit 2; BRCA, breast carcinoma; COAD, colon adenocarcinoma; HNSC, head and neck squamous cell carcinoma; LGG, low-grade glioma; LUSC, lung squamous cell carcinoma; PCPG, pheochromocytoma and paraganglioma; KIRC, kidney renal clear cell carcinoma; LIHC, liver hepatocellular carcinoma; STAD, stomach adenocarcinoma; PRAD, prostate adenocarcinoma; OV, ovarian serous cystadenocarcinoma; SARC, sarcoma; C1, wound healing; C2, IFN- gamma dominant; C3, inflammatory; C4, lymphocyte depleted; C5, immunologically quiet; C6, TGF-b dominant.

A

BRCA:TPGS2_exp

Pv=4.97e-48

B

C

n=Basal 172,

COAD:TPGS2_exp

PV=8.72e-20

HNSC :: TPGS2_exp

Her2 73,

Pv=2.65e-06

LumA 508,

n=CIN 226, GS 49,

HM-SNV 6, HM-indel 60

n=Atypical 67,

LumB 191,

Basal 87,

Normal 137

Classical 48,

Mesenchymal 74

Expression (log2CPM)

9

Expression (log2CPM)

.

Expression (log2CPM)

9

8

6

i

B

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H

!

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Basal

Her2

LumA

LumB

Normal

CIN

GS

HM-SNV

HM-indel

Atypical

Basal

Classical

Mesenchymal

Subtype

Subtype

Subtype

D

LGG :: TPGS2_exp

E

F

PCPG:TPGS2_exp

Pv=1.07e-07

n=Classic-like 23, Codel 171,

LUSC :: TPGS2_exp Pv=1.41e-07 n=Basal 42, Classical 63, Primitive 26, Secretory 39

Pv=7.35e-10

n=Corticaladmixture 22,

G-CIMP-high 234, G-CIMP-low 12,

Kinasesignaling 68,

Expression (log2CPM)

Mesenchymal-like 45, PA-like 26

Pseudohypoxia 61, Wnt-altered 22

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Expression (log2CPM)

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Expression (log2CPM)

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Codel

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G-CIMP-low

Mesenchymal-like

PA-like

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Basal

Classical

Primitive

Secretory

Corticaladmixture

Kinasesignaling

Pseudohypoxia

Wnt-altered

Subtype

Subtype

Subtype

G

BRCA :: TPGS2_exp Pv=1.28e-19

H

KIRC:TPGS2_exp Pv=1.23e-04

I

n=C1 369, C2 390, C3 191, C4 92, C6 40

n=C1 7, C2 20, C3 445, C4 27, C5 3, C6 13

LIHC :: TPGS2_exp Pv=4.11e-08

8

n=C1 22, C2 45, C3 135, C4 159, C6 1

Expression (log2CPM)

9

Expression (log2CPM)

Expression (log2CPM)

8

8

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H

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C1

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C6

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C4

C6

Subtype

Subtype

Subtype

J

STAD:TPGS2_exp Pv=1.18e-04 n=C1 129, C2 210, C3 36, C4 9, C6 7

K

OV:TPGS2_exp Pv=3.38e-03

L

SARC:TPGS2_exp Pv=1.82e-04 n=C1 64, C2 38, C3 42, C4 59, C6 20

n=C1 46, C2 159, C3 3,C4 61

Expression (log2CPM)

Expression (log2CPM)

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Expression (log2CPM)

8

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C3

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C1

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C3

C4

C1

C2

C3

C4

C6

Subtype

Subtype

Subtype

Figure 4 Relationship between TPGS2 expression and gene alterations. (A) The genetic alteration types and frequency of TPGS2 in various cancers; (B) the association between CNA and the RNA expression of TPGS2. 1, structural variants are shown instead of CNA when a sample has both. TPGS2, tubulin polyglutamylase complex subunit 2; CNA, copy number alterations; TCGA, The Cancer Genome Atlas; VUS, variants of uncertain significance.

A

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Mutation

Structural variant

Amplification

Deep deletion

Multiple alterations

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Alteration frequency, %

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Pancreatic Adenocarcinoma (TCGA, PanCancer Atlas)

Esophageal Adenocarcinoma (TCGA, PanCancer Atlas)

Stomach Adenocarcinoma (TCGA, PanCancer Atlas)

Ovarian Serous Cystadenocarcinoma (TCGA, PanCancer Atlas)

Lung Squamous Cell Carcinoma (TCGA, PanCancer Atlas)

Uterine Corpus Endometrial Carcinoma (TCGA, PanCancer Atlas)

Sarcoma (TCGA, PanCancer Atlas)

Bladder Urothelial Carcinoma (TCGA, PanCancer Atlas)

Diffuse Large B-Cell Lymphoma (TCGA, PanCancer Atlas)

Lung Adenocarcinoma (TCGA, PanCancer Atlas)

Head and Neck Squamous Cell Carcinoma (TCGA, PanCancer Atlas)

Uterine Carcinosarcoma (TCGA, PanCancer Atlas)

Skin Cutaneous Melanoma (TCGA, PanCancer Atlas)

Colorectal Adenocarcinoma (TCGA, PanCancer Atlas) Liver Hepatocellular Carcinoma (TCGA, PanCancer Atlas)

Brain Lower Grade Glioma (TCGA, PanCancer Atlas)

Cervical Squamous Cell Carcinoma (TCGA, PanCancer Atlas)

Testicular Germ Cell Tumors (TCGA, PanCancer Atlas)

Breast Invasive Carcinoma (TCGA, PanCancer Atlas)

Kidney Renal Clear Cell Carcinoma (TCGA, PanCancer Atlas)

Prostate Adenocarcinoma (TCGA, PanCancer Atlas) Acute Myeloid Leukemia (TCGA, PanCancer Atlas) Glioblastoma Multiforme (TCGA, PanCancer Atlas) +

Thymoma (TCGA, PanCancer Atlas)

Thyroid Carcinoma (TCGA, PanCancer Atlas)

Kidney Chromophobe (TCGA, PanCancer Atlas)

Kidney Renal Papillary Cell Carcinoma (TCGA, PanCancer Atlas)

Uveal Melanoma (TCGA, PanCancer Atlas)

Cholangiocarcinoma (TCGA, PanCancer Atlas)

Adrenocortical Carcinoma (TCGA, PanCancer Atlas)

Pheochromocytoma and Paraganglioma (TCGA, PanCancer Atlas)

Mesothelioma (TCGA, PanCancer Atlas)

B

TPGS2: mRNA Expression, RSEM (batch normalized from

14

TPGS2

13

Splice (VUS)

Not mutated

· Gain

illumina HiSeq_RNASeqV2) [log2(value + 1)]

12

Deep deletion

11

· Truncating (VUS)

Not profiled for mutations

10

Diploid

9

· Structural variant1

Missense (VUS)

8

o Amplification

· Shallow deletion

7

6

5

Deep deletion

Shallow deletion

Diploid

Gain

Amplification

TPGS2: Putative copy-number alterations from GISTIC

investigated using the Kaplan-Meier method and a univariate Cox regression analysis. The results showed that TPGS2 was highly correlated with the prognosis of most cancers. As Figure 5A shows, the univariate Cox regression analysis of OS suggested that the high expression of TPGS2

was a risk factor for the poor prognosis of ACC, BLCA, LGG, LIHC, MESO, SARC, STAD, and UCS, while it was a protective factor for OV and THYM. The results of the Kaplan-Meier OS analysis, which were basically consistent with the results of the univariate Cox regression analysis,

A CancerP valueHazard Ratio(95% CI)
ACC0.00353.42787(1.49986,7.83422)
BLCA0.01401.45156(1.07835,1.95395)
BRCA0.96660.99324(0.72279,1.36488)
CESC0.21910.74506(0.46596,1.19133)
CHOL0.75181.16929(0.44362,3.08199)
COAD0.27400.80327(0.54249,1.18941)
DLBC0.57300.66075(0.15642,2.79112)
ESCA0.66741.11311(0.68281,1.81459)
GBM0.28301.21933(0.84901,1.75116)
HNSC0.07091.28282(0.979,1.68091)
KICH0.23762.30721(0.57615,9.23927)
KIRC0.08130.76544(0.56675,1.03378)
KIRP0.35520.75443(0.41513,1.37106)
LAML0.32771.23428(0.80976,1.88135)
LGG0.00071.89069(1.31078,2.72716)
LIHC0.00611.62865(1.14901,2.30851)
LUAD0.91280.98392(0.73614,1.31511)
LUSC0.26370.85718(0.65421,1.12313)
MESO0.00142.17943(1.35073,3.51654)
OV0.02590.74365(0.57306,0.96503)
PAAD0.07461.45428(0.96352,2.195)
PCPG0.40560.48661(0.08912,2.65709)
PRAD0.15492.69349(0.68768,10.54971)
READ0.16100.54709(0.23541,1.27144)
SARC0.04791.4957(1.00368,2.22892)
SKCM0.19261.19513(0.91402,1.56271)
STAD0.00701.57874(1.13307,2.19969)
THCA0.87240.92268(0.34566,2.46297)
THYM0.03370.10475(0.01305,0.84059)
UCEC0.40531.19142(0.78868,1.79984)
UCS0.03912.05947(1.03663,4.09156)
UVM0.60131.24823(0.54337,2.86746)
Figure 5 Relationship between TPGS2 expression and OS. (A) Forest map showing the univariate Cox regression analysis results for TPGS2 in pan-cancer samples from TCGA. (B-J) Kaplan-Meier curves for nine significant cancers. HR, hazard ratio; CI, confidence interval; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; LGG, low-grade glioma; LIHC, liver hepatocellular carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; SARC, sarcoma; STAD, stomach adenocarcinoma; THYM, thymoma; TPGS2, tubulin polyglutamylase complex subunit 2; OS, overall survival; TCGA, The Cancer Genome Atlas.

B

C

D

ACC

BLCA

LGG

1.0

TPGS2

1.0

TPGS2

1.0

TPGS2

Low

Low

Low

High

High

High

Survival probability

0.8

Survival probability

0.8

Survival probability

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

Overall survival

0.2

Overall survival

0.2

HR =3.39 (95% CI, 1.48-7.75)

HR =1.39 (95% CI, 1.03-1.86)

Overall survival

HR =1.87 (95% CI, 1.31-2.65)

0.0

P=0.004

0.0

P=0.029

0.0

P=0.001

0

50

100

150

0

40

80

120

160

0

50

100

150

200

Time, months

Time, months

Time, months

E

LIHC

F

MESO

G

OV

1.0

TPGS2

1.0

TPGS2

1.0

TPGS2

Low

Low

High

++ Low

High

High

Survival probability

0.8

Survival probability

0.8

Survival probability

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

Overall survival

0.2

Overall survival

0.2

HR =1.72 (95% CI, 1.21-2.44)

HR =2.28 (95% Gl,1.41-3.69)

Overall survival

HR =0.74 (95% CI, 0.57-0.96)

0.0

P=0.002

0.0

P=0.001

0.0

P=0.024

0

30

60

90

120

0

25

50

75

0

50

100

150

Time, months

Time, months

Time, months

H

SARC

I

STAD

J

THYM

1.0

TPGS2

1.0

TPGS2

1.0

TPGS2

+ Low

Low

Low

High

High

Survival probability

0.8

High

Survival probability

0.8

Survival probability

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

Overall survival

0.2

0.2

HR =1.61 (95% CI, 1.08-2.40)

Overall survival

HR =1.49 (95% CI, 1.07-2.08)

Overall survival

HR =0.10 (95% CI, 0.01-0.83)

0.0

P=0.02

0.0

P=0.018

0.0

P=0.032

0

50

100

150

200

0

25

50

75

100

125

0

50

100

150

Time, months

Time, months

Time, months

0.0130501 2 3 4 5 6 7 8 9 10 11 Hazard Ratio

showed that the high expression of TPGS2 was significantly correlated with a poor prognosis in SARC, MESO, LIHC, LGG, BLCA, and ACC, but was significantly correlated with a better prognosis in OV and THYM (Figure 5B-5}). The results of the Cox regression analysis of PFS revealed that TPGS2 was a risk factor in ACC, HNSC, MESO, and UCS (Figure 6A). The results of the Kaplan-Meier analysis showed that patients with high TPGS2 expression had poorer PFS than those with low TPGS2 expression in ACC, CHOL, HNSC, LIHC, SARC, and UCS, but had better PFS in PCPG, LUCA (Figure 6B-6I). Additionally, the results of the Cox regression analysis of DSS revealed that TPGS2 acts as a risk factor for ACC, LGG, LIHC,

MESO, PAAD, and SARC, but acts as a protective factor for OV (Figure 7A). Further, the results of the Kaplan- Meier analysis of DSS showed that a high expression of TPGS2 was associated with a worse prognosis in ACC, LGG, LIHC, MESO, PAAD, and SARC (Figure 7B-7H). Overall, TPGS2 expression was significantly associated with prognostic parameters in many cancers.

Due to the complexity of tumor cells, the new technique of single-cell transcriptome sequencing is increasingly

A CancerP valueHazard Ratio(95% CI)
ACC<0.00014.51965(2.24091,9.1156)
BLCA0.23171.19994(0.89008,1.61766)
BRCA0.60160.91703(0.66246,1.26943)
CESC0.98660.99605(0.62716,1.5819)
CHOL0.10742.13312(0.84823,5.36433)
COAD0.2920.82516(0.57717,1.17971)
DLBC0.44050.61379(0.17756,2.12175)
ESCA0.21731.3202(0.84913,2.05261)
GBM0.46130.8734(0.60925,1.25207)
HNSC0.03651.35475(1.01931,1.80056)
KICH0.96610.97456(0.29698,3.19808)
KIRC0.0780.75293(0.54917,1.0323)
KIRP0.91691.02808(0.61116,1.72941)
LGG0.09611.27406(0.95783,1.6947)
LIHC0.081.29899(0.96919,1.74102)
LUAD0.17050.82579(0.62803,1.08582)
LUSC0.41280.87311(0.63103,1.20806)
MESO0.04021.74874(1.02537,2.9824)
OV0.26450.87307(0.68787,1.10813)
PAAD0.12631.35446(0.91805,1.99833)
PCPG0.05690.39618(0.15277,1.02745)
PRAD0.77510.94267(0.62871,1.41341)
READ0.67280.86948(0.45436,1.66389)
SARC0.0611.37683(0.98531,1.92392)
SKCM0.75041.03702(0.82893,1.29735)
STAD0.26981.21982(0.8571,1.73604)
TGCT0.88350.95135(0.48831,1.85349)
THCA0.40970.79838(0.46747,1.36354)
THYM0.18870.55272(0.22833,1.33795)
UCEC0.21151.25373(0.87934,1.78753)
UCS0.01122.36019(1.21515,4.5842)
UVM0.99020.99542(0.4769,2.0777)
Figure 6 Relationship between TPGS2 expression and PFI. (A) Forest map showing the univariate Cox regression analysis results for TPGS2 in pan-cancer samples from TCGA. (B-I) Kaplan-Meier curves for eight significant cancers. HR, hazard ratio; CI, confidence interval; PFI, progress free interval; TPGS2, tubulin polyglutamylase complex subunit 2; ACC, adrenocortical carcinoma; CHOL, cholangiocarcinoma; HNSC, head and neck squamous cell carcinoma; LIHC, liver hepatocellular carcinoma; PCPG, pheochromocytoma and paraganglioma; SARC, sarcoma; UCS, uterine carcinosarcoma; LUCA, lung carcinoma; TCGA, The Cancer Genome Atlas.

B

ACC

C

CHOL

D

HNSC

1.0

TPGS2

1.0-

TPGS2

1.0

TPGS2

++ Low

Low

+ Low

High

High

+ High

Survival probability

0.8

Survival probability

0.8

Survival probability

0.8

0.6

0.6

0.6

0.4

0.4.

0.4

0.2

Progress free interval

0.2

Progress free interval

0.2

HR =4.36 (95% CI, 2.16-8.81)

HR =2.63 (95% CI, 1.01-6.86)

Progress free interval

HR=1.38 (95% CI, 1.04-1.83)

0.0

P<0.001

0.0

P=0.048

0.0

P=0.027

0

50

100

150

0

20

40

60

0

50

100

150

200

Time, months

Time, months

Time, months

E

LIHC

F

PCPG

G

SARC

1.0

TPGS2

1.0

TPGS2

1.0

TPGS2

Low

++Low

++High

++ Low

High

High

Survival probability

0.8

Survival probability

0.8

Survival probability

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

Progress free interval

0.2

Progress free interval

0.2

HR =1.35 (95% CI, 1.01-1.81)

HR =0.34 (95% CI, 0.13-0.88)

Progress free interval

HR =1.49 (95% CI, 1.07-2.08)

0.0

P=0.041

0.0

P=0.027

0.0

P=0.018

0

30

60

90

120

0

50

100

150

200

0

40

80

120

160

Time, months

Time, months

Time, months

H

UCS

I

LUCA

1.0

TPGS2

1.0

TPGS2

+ Low

++ Low

0.8

High

0.8

High

Survival probability

Survival probability

0.6

0.6

0.4

0.4

0.2

Progress free interval HR =2.37 (95% CI, 1.2

0.2

Progress free interval

0.0

-4.67)

0.0

HR =0.74 (95% CI, 0.60-0.91)

P=0.013

P=0.004

0

50

100

0

50

100

150

200

250

0.15277 10

2

3

4

5

6

7

8

9

Hazard Ratio

Time, months

Time, months

being used to analyze a variety of cancer cells, immune cells, endothelial cells, and stromal cells. To explore the expression of TPGS2 in single-cell analyses in pan- cancer and its relationship with tumor functional status, we obtained tumor single-cell data on TPGS2 from the CancerSEA.

As Figure 8A shows, we found that many types of cancers, including UM, non-small cell lung cancer (NSCLC), and high-grade glioma (HGG), were associated with most tumor functional states. We also analyzed expression distribution with t-SNE plots, and the correlation between TPGS2 expression and functional states in different cancers based on the CancerSEA database, and found that TPGS2 expression in UM was significantly associated with DNA

repair, DNA damage, apoptosis, metastasis, invasion, and quiescence (Figure 8B); acute myelocytic leukemia (AML) was closely associated with invasion and differentiation (Figure 8C); retinoblastoma (RB) was correlated with differentiation and angiogenesis (Figure 8D); chronic myeloid leukemia (CML) was closely associated with proliferation (Figure 8E); and NSCLC was associated with epithelial-to-mesenchymal transition (Figure 8F). The t-SNE plots showed the expression distribution of TPGS2 in UM, AML, RB, CML and NSCLC cells (Figure 8G- 8K). In the plots, every point represents a single cell, and the color of the point represents the expression level of TPGS2 in the cell. The results suggest TPGS2 might play an important role in some functional states, and most of the

Figure 7 Relationship between TPGS2 expression levels and DSS. (A) Forest map showing the univariate Cox regression analysis results for TPGS2 in pan-cancer samples from TCGA. (B-H) Kaplan-Meier curves for seven significant cancers. HR, hazard ratio; CI, confidence interval; DSS, disease-specific survival; TPGS2, tubulin polyglutamylase complex subunit 2; ACC, adrenocortical carcinoma; LGG, low- grade glioma; LIHC, liver hepatocellular carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; SARC, sarcoma; TCGA, The Cancer Genome Atlast.

A

Cancer

P value

Hazard Ratio(95% CI)

B

ACC

C

LGG

D

LIHC

ACC

0.0041

3.587(1.5003,8.57604)

A

1.0

TPGS2

1.0

TPGS2

1.0.

TPGS2

BLCA

0.0754

1.3863(0.96716,1.98709)

Low

Low

+Low

High

++ High

High

BRCA

0.3660

0.82097(0.53531,1.25909)

Survival probability

0.8

Survival probability

0.8

Survival probability

0.8

CESC

0.7041

0.9024(0.53115,1.53313)

0.6

0.6

0.6

CHOL

0.4045

1.58079(0.53864,4.63922)

0.4

0.4

0.4

COAD

0.7096

0.91079(0.557,1.4893)

DLBC

0.3926

0.37042(0.03801,3.61026)

0.2

Disease specific survival

0.2

Disease specific survival

0.2

Disease specific survival HR =2.00 (95% CI, 1.27-3.16)

ESCA

0.3523

1.3146(0.73874,2.33932)

HR =3.54 (95% CI, 1.48-8.47)

HR =1.76 (95% CI, 1.22-2.54)

0.0

P=0.005

0.0

P=0.002

0.0

P=0.003

GBM

0.2001

1.28923(0.87413,1.90146)

0

50

100

150

0

50

100

150

200

0

30

60

90

120

HNSC

0.1117

1.32755(0.93634,1.88222)

Time, months

Time, months

Time, months

KICH

0.5475

1.58392(0.35396,7.08775)

E

MESO

F

OV

G

PAAD

KIRC

0.0975

0.72356(0.49349,1.06091)

1.0

TPGS2

1.0

TPGS2

KIRP

0.2897

0.66367(0.31071,1.4176)

Low

+Low

1.0

TPGS2

High

+ Low

LGG

0.0019

1.83527(1.25147,2.69141)

Survival probability

0.8

0.8

High

Survival probability

0.8

++ High

Survival probability

LIHC

0.0078

1.84747(1.17531,2.90403)

0.6

0.6

0.6

LUAD

0.7466

0.9407(0.64927,1.36294)

LUSC

0.1151

0.71209(0.46678,1.08633)

0.4

0.4

0.4

MESO

0.0004

3.31704(1.71104,6.43043)

0.2

Disease specific survival

0.2

Disease specific survival HR =0.71 (95% CI, 0.54-0.95)

0.2

HR =3.11 (95% CI, 1.62-5.97)

Disease specific survival

OV

0.0157

0.70708(0.53381,0.93657)

HR =1.71 (95% CI, 1.06-2.76)

0.0

P=0.001

0.0

P=0.019

0.0

P=0.028

PAAD

0.0169

1.78019(1.10916,2.85717)

0

25

50

75

0

50

100

150

0

25

50

75

PCPG

0.3319

0.32617(0.03393,3.13584)

Time, months

Time, months

Time, months

PRAD

0.5591

1.71142(0.28206,10.38404)

H

READ

0.3657

0.60322(0.20175,1.8036)

SARC

SARC

0.0123

1.76559(1.13135,2.75538)

1.0

TPGS2

Low

High

SKCM

0.3950

1.13211(0.85061,1.50677)

Survival probability

0.8

STAD

0.1495

1.35994(0.89529,2.06574)

0.6

THCA

0.8084

1.20375(0.269,5.38655)

THYM

0.2405

0.25638(0.02641,2.4888)

0.4

UCEC

0.1692

1.43377(0.85778,2.39652)

0.2

Disease specific survival

UCS

0.1394

1.71436(0.83888,3.50349)

HR =1.92 (95% CI, 1.23-3.01)

UVM

0.8278

1.0999(0.46627,2.59458)

0.0

P=0.004

0

50

100

150

200

0.02641

I

2

3

4

5

6

7

8

9

10

Time, months

Hazard Ratio

functional states have been linked to the occurrence and progression of cancers.

GSEA

To explore the biological processes related to TPGS2 expression across human cancers, we performed a differential expression analysis between the top 50% TPGS2 expression subgroup and the bottom TPGS2 expression subgroup for each type of cancer. We conducted a GSEA to evaluate the biological processes of 33 types of cancers from TCGA. The results showed that TPGS2 is likely involved in a great deal of immune regulation-related biological processes, especially immunoglobulin (Ig) production, the

humoral immune response mediated by circulating Ig, B cell-mediated immunity, the regulation of the humoral immune response, and the B cell receptor signaling pathway (Figure 9A-9F). This provides evidence that TPGS2 may be involved in the immune response and cancers, which prompted us to explore the role of TPGS2 in the cancer- immune process and immune microenvironment.

Immune cell infiltration analysis

To further explore the role of TPGS2 in tumor immunity, we explored the correlations between TPGS2 expression and immune cell infiltration levels across cancers. TIMER 2.0 was used to generate a heatmap associated with a variety

Figure 8 The expression levels of TPGS2 based on the single-cell analysis. (A) Average correlations between the expression levels of TPGS2 and functional states in various cancers; (B-F) the relationship between TPGS2 expression and various functional states in UM, AML, RB, CML, and NSCLC were explored by the CancerSEA; (G-K) the expression distribution of TPGS2 in cells of UM, AML, RB, CML and NSCLC are displayed in a t-SNE diagram. * , P<0.05; ** , P<0.01; *** , P<0.001. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CRC, colorectal cancer; BRCA, breast carcinoma; AST, astrocytoma; GBM, glioblastoma; HGG, high-grade glioma; ODG, oligodendroglioma; HNSCC, head and neck squamous cell carcinoma; RCC, renal cell carcinoma; LUAD, lung adenocarcinoma; NSCLC, non-small cell lung cancer; OV, ovarian carcinoma; MEL, melanoma; RB, retinoblastoma; UM, uveal melanoma; EMT, epithelial-to-mesenchymal transition; t-SNE, t-distributed stochastic neighbor embedding; TPGS2, tubulin polyglutamylase complex subunit 2.

A

ALL

B

*

AML

*

**

**

*

**

**

**

**

geneExp

CML

**

**

*

**

**

**

**

**

**

**

**

G

CRC

*

Correlation

P value

BRCA

Expression distribution with t-SNE plot

**

**

**

*

*

**

*P<0.05

DNArepair

-0.56

AST

**

**

**

**

*

**

** P<0.01

50

GBM

**

**

**

*

**

**

*

Correlation

Glioma

**

**

**

**

**

*

1.0

DNAdamage

-0.53


3.3

HGG

**

**

**

*

**

**

**

**

*

**

**

**

25

ODG

2.7

**

*

**

**

*

**

**

**

0.5

HNSCC

tSNE2

2.2

**

**

*

**

**

*

**

**

RCC

0.0

Apoptosis

-0.49


0

1.6

LUAD

*

-0.5

1.1

*

**

NSCLC

**

**

0.5

**

**

**

**

*

**

**

**

**

**

**

OV

**

**

-1.0

Metastasis

-0.42


-25

0.0

MEL

*

**

*

**

*

**

*

Expression

RB

**

**

**

**

**

**

**

**

**

**

**

**

UM

**

**

**

**

**

**

**

**

**

**

**

**

**

Invasion

CellCycle

DNArepair

Inflammation

Proliferation

-0.41

-50

Angiogenesis

Apoptosis

Differentiation

DNAdamage

EMT

Hypoxia

Invasion

Metastasis

Quiescence

Stemness

-60

-40

-20

0

20

40

60

tSNE1

Quiescence

-0.38


C

D

E

F

geneExp

geneExp

Invasion

Correlation P value

Correlation P value

0.36

Differentiation

0.35

*

geneExp


geneExp

Correlation P value

Correlation

Differentiation

Proliferation

-0.45

EMT

III

0.31

P value *

0.31

Angiogenesis

0.30


U

H

Expression distribution with t-SNE plot

I

Expression distribution with t-SNE plot

J

Expression distribution with t-SNE plot

K

Expression distribution with t-SNE plot

30

75

150

200

20

3.3

50

100

150

2.7

3.1

10.4

10

25

2.6

50

8.7

100

2.6

2.2

tSNE2

1.6

2.1

6.9

2.2

1.7

0

1.1

0.5

INSI

0

1.6

tSNE2

50

1.0

0

5.3

tSNE2

3.5

92

1.3

..

-10

0.0

-25

0.5

-50

1.7

0

0.9

Expression

0.0

0.0

-50

0.4

0.0

-20

-50

Expression

-100

Expression

-100

Expression

-30

-75

-150

-150

-100

-50

0

50

100

150

-75

-50

-25

0

25

50

-75

-50

-25

0

25

50

75

100

-200

-100

0

100

200

tSNE1

ISNE1

tSNE1

ISNE1

of immune cell infiltration, which was performed on a variety of quantitative immuno-infiltration platforms. The results were then categorized based on different immune score categories (Figures S1-S6). These analyses showed the infiltration levels of cluster of differentiation (CD)8+ T cells, CD4+ T cells, T regulatory cells (Tregs), B cells, neutrophils, macrophages, progenitors, dendritic cells (DCs), mast cells, cancer-associated fibroblasts (CAFs), endothelial cell (endos), natural killer (NK) cells, T cell follicular helper (Tfh) cells, T cell gamma delta (yö’T) cells, NK T cells, monocytes, myeloid-derived suppressor cells (MDSCs), and eosinophils (Eos). As Figure 10 shows, TPGS2 was positively related to the level of many cells, such as macrophages, CD4+ T cells, CD8+ T cells, B cells, neutrophils, and CAFs. We also found that TPGS2

had a higher correlation with immune cell infiltration in THYM and HNSC than other tumors, especially in CD4+ T cells, CD8+ T cells, macrophages, and B cells. However, their trend of correlation was slightly different, which might be related to the immune specificity of these cancers.

Co-expression of TPGS2 with immune-associated genes

To further explore the role of TPGS2 in cancer immunity, we performed a Spearman correlation analysis to reveal the correlation between TPGS2 expression and immune-related genes (Figure 11A-11E). The heatmaps illustrated that the gene encoding immunoinhibitor, immunostimulator, MHC, chemokine, and chemokine receptor proteins were significantly correlated with the expression of TPGS2 in

Figure 9 GSEA of TPGS2 in various cancers. (A-F) Top biological processes of the GSEA in indicated tumor types. GO, Gene Ontology; PAAD, pancreatic adenocarcinoma; LIHC, liver hepatocellular carcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; SKCM, skin cutaneous melanoma; THCA, thyroid carcinoma; GSEA, gene-set enrichment analysis; TPGS2, tubulin polyglutamylase complex subunit 2.

A

PAAD

B

LIHC

C

COAD

GO HUMORAL IMMUNE RESPONSE MEDIATED BY CIRCULATING

GO B CELL RECEPTOR SIGNALING PATHWAY

IMMUNOGLOBULIN

I

GO IMMUNOGLOBULIN PRODUCTION

GO ADAPTIVE IMMUNE RESPONSE BASED ON SOMATIC RECOMBINATION OF IMMUNE RECEPTORS BUILT FROM IMMUNOGLOBULIN SUPERFAMILY

GO B CELL MEDIATED IMMUNITY -

.

GO FC RECEPTOR MEDIATED STIMULATORY SIGNALING PATHWAY

GO B CELL RECEPTOR SIGNALING

DOMAINS

PATHWAY

GO MEIOTIC CELL CYCLE PROCESS

GO RESPONSE TO CHEMOKINE

TT

GO COMPLEMENT ACTIVATION -

GO B CELL MEDIATED IMMUNITY

GO MEMBRANE INVAGINATION

T

GO PHAGOCYTOSIS RECOGNITION

GO POSITIVE REGULATION OF B CELL ACTIVATION

GO B CELL RECEPTOR SIGNALING PATHWAY

GO FC RECEPTOR MEDIATED

GO HUMORAL IMMUNE RESPONSE MEDIATED BY CIRCULATING IMMUNOGLOBULIN

STIMULATORY SIGNALING PATHWAY

P value

GO IMMUNOGLOBULIN PRODUCTION

GO MEIOTIC CELL CYCLE

TT

P value

GO PRODUCTION OF MOLECULAR MEDIATOR OF IMMUNE RESPONSE

P value

0.007

0.006

0.00115

0.04

GO POSITIVE REGULATION OF B CELL ACTIVATION

0.005

GO PHAGOCYTOSIS RECOGNITION

0.00110

GO ANTIGEN RECEPTOR MEDIATED SIGNALING PATHWAY

0.03

0.02

0.004

0.00105

0.01

GO REGULATION OF B CELL

ACTIVATION

GO REGULATION OF B CELL

0.00100

ACTIVATION

T

GO PHAGOCYTOSIS RECOGNITION

GO FC EPSILON RECEPTOR SIGNALING PATHWAY

GO MEIOSIS I CELL CYCLE

GO RESPONSE TO INTERFERON GAMMA

PROCESS

TT

TT

GO FC RECEPTOR SIGNALING PATHWAY

GO CHROMOSOME SEPARATION

GO MEMBRANE INVAGINATION -

GO MEMBRANE INVAGINATION

GO MITOTIC SISTER CHROMATID SEGREGATION

GO FC RECEPTOR MEDIATED STIMULATORY SIGNALING PATHWAY

GO REGULATION OF HUMORAL IMMUNE RESPONSE

GO CORNIFICATION

GO COLLAGEN FIBRIL

GO IMMUNOGLOBULIN PRODUCTION

GO POSITIVE REGULATION OF B CELL ACTIVATION

ORGANIZATION

GO REGULATION OF CHROMOSOME SEPARATION

GO CELL RECOGNITION

GO COMPLEMENT ACTIVATION

GO RESPONSE TO CHEMOKINE

GO METAPHASE ANAPHASE TRANSITION OF CELL CYCLE

GO POSITIVE REGULATION OF LYMPHOCYTE ACTIVATION

0.0

0 0.5

1.0

1.5

2.0

0

1

2

3

4

5

0

1

2

3

Enrichment score

Enrichment score

Enrichment score

D

ESCA

E

SKCM

F

THCA

GO HUMORAL IMMUNE RESPONSE MEDIATED BY CIRCULATING IMMUNOGLOBULIN

GO CORNIFICATION

I

GO HUMORAL IMMUNE RESPONSE MEDIATED BY CIRCULATING IMMUNOGLOBULIN

GO ANTIMICROBIAL HUMORAL IMMUNE RESPONSE MEDIATED BY ANTIMICROBIAL PEPTIDE

GO COMPLEMENT ACTIVATION

I

GO IMMUNOGLOBULIN PRODUCTION

GO PHAGOCYTOSIS RECOGNITION

GO KERATINIZATION

THỊ II I III

GO B CELL MEDIATED IMMUNITY .

GO REGULATION OF HUMORAL IMMUNE RESPONSE

GO ANTIMICROBIAL HUMORAL RESPONSE

GO COMPLEMENT ACTIVATION

GO B CELL RECEPTOR SIGNALING PATHWAY

GO SENSORY PERCEPTION OF SMELL

GO PHAGOCYTOSIS RECOGNITION

GO B CELL MEDIATED IMMUNITY -

GO KERATINOCYTE DIFFERENTIATION

GO B CELL RECEPTOR SIGNALING PATHWAY

P value

P value

P value

GO HUMORAL IMMUNE RESPONSE

GO ANTIBACTERIAL HUMORAL RESPONSE

0.00110

0.003

GO ORGAN OR TISSUE SPECIFIC IMMUNE RESPONSE

GO POSITIVE REGULATION OF B CELL ACTIVATION

GO IMMUNOGLOBULIN PRODUCTION -

0.001

0.002

0.00105

0.001

0.00100

GO MEMBRANE INVAGINATION

GO DEFENSE RESPONSE TO GRAM POSITIVE BACTERIUM

GO FC RECEPTOR MEDIATED STIMULATORY SIGNALING PATHWAY

GO REGULATION OF B CELL ACTIVATION

GO DEFENSE RESPONSE TO GRAM NEGATIVE BACTERIUM

GO LYMPHOCYTE MEDIATED IMMUNITY

TT

GO FC RECEPTOR MEDIATED STIMULATORY SIGNALING PATHWAY GO ADAPTIVE IMMUNE RESPONSE BASED ON SOMATIC RECOMBINATION

GO KILLING OF CELLS OF OTHER ORGANISM

GO REGULATION OF HUMORAL IMMUNE RESPONSE

GO MEMBRANE INVAGINATION

OF IMMUNE RECEPTORS BUILT FROM

GO REGULATION OF TYPE 2 IMMUNE RESPONSE

IMMUNOGLOBULIN SUPERFAMILY

DOMAINS

GO LYMPHOCYTE MEDIATED

IMMUNITY

GO DEFENSE RESPONSE TO FUNGUS

GO REGULATION OF B CELL ACTIVATION

GO POSITIVE REGULATION OF B CELL ACTIVATION

GO NEGATIVE REGULATION BY HOST

GO PRODUCTION OF MOLECULAR MEDIATOR OF IMMUNE RESPONSE

OF VIRAL PROCESS

GO PHAGOCYTOSIS

GO INNATE IMMUNE RESPONSE IN MUCOSA

GO PHAGOCYTOSIS

GO FC EPSILON RECEPTOR SIGNALING PATHWAY

GO CELL KILLING

II

GO FC EPSILON RECEPTOR SIGNALING PATHWAY

I

-3

-2

-1

0

-5 -4 -3 -2 -1

0

-4-3-2-1 0 Enrichment score

Enrichment score

Enrichment score

Figure 10 The correlations between TPGS2 expression and the infiltration levels of B cells, CD4+ T cells, CD8+ T cells, macrophages, Endos, monocytes, neutrophils, Tregs, DCs, CAFs, NK T cells, NK cells, MDSCs, Tfh cells, progenitor, yoT cells, eosinophils (Eos), and mast cells in cancers. Positive correlations in red, and negative correlations blue. TPGS2, tubulin polyglutamylase complex subunit 2; Endos, endothelial cells; Tregs, T regulatory cells; DCs, dendritic cells; CAFs, cancer-associated fibroblasts; NK, natural killer; MDSCs, myeloid- derived suppressor cells; Tfh, T cell follicular helper; yoT, T cell gamma delta.

B cell memory_CIBERSORT

8 cell memory_CIBERSORT-ABS

8 cell naive_CIBERSORT-ABS

B cell plasma_CIBERSORT

B cell plasma_CIBERSORT-ABS 8 cell plasma_XCELL

Class-switched memory B cell_XCELL

T cell CD4+ (non-regulatory)_QUANTISEQ

T cell CD4+ (non-regulatory)_XCELL

T cell CD4+ naive CIBERSORT

T cell CD4+ naive_CIBERSORT-ABS

T cell CD4+ naive XCELL

T cell CD4+ memory_XCELL

T cell CD4+ central memory_XCELL

T cell CD4+ effector memory_XCELL

T cell CD4+ memory activated_CIBERSORT

T cell CD4+ memory activated_CIBERSORT-ABS

T cell CD4+ memory resting_CIBERSORT

T cell CD4+ memory resting_CIBERSORT-ABS

B cell_QUANTISEQ

T cell CD4+ Th1_XCELL

T cell CD4+ Th2_XCELL

T cell CD8+ MCPCOUNTER

T cell CD8+_CIBERSORT-ABS

T cell CD8+ central memory_XCELL

T cell CDS+ effector memory_XCELL

Macrophage MO_CIBERSORT

Macrophage MO_CIBERSORT-ABS

Macrophage MI_CIBERSORT

Macrophage MI_CIBERSORT-ABS

Macrophage MI_QUANTISEQ

Macrophage M2_CIBERSORT

Macrophage M2_CIBERSORT-ABS Macrophage M2_QUANTISEQ

Macrophage/Monocyte_MCPCOUNTER

Endothelial cell_MCPCOUNTER Endothelial cell_XCELL

Macrophage/Monocyte_MCPCOUNTER,

Monocyte_CIBERSORT-ABS

B cell_TIMER

8 cell_EPIC

B cell_XCELL

B cell_MCPCOUNTER

B cell memory_XCELL

B cell naive_CIBERSORT

B cell naive_XCELL

T cell CD4+_EPIC T cell CD4+ TIMER

T cell CD8+_TIMER

T cell CD8+_EPIC

T cell CD8+_CIBERSORT

T cell CD8+_QUANTISEQ

T cell CD8+_XCELL

T cell CD8+ naive_XCELL

Macrophage_EPIC

Macrophage_TIMER Macrophage_XCELL

Macrophage MI_XCELL

Macrophage M2_XCELL

Macrophage M2_TIDE

Endothelial cell_EPIC

Monocyte_CIBERSORT

Monocyte_MCPCOUNTER

Monocyte_QUANTISEQ Monocyte_XCELL

ACC (n=79)

BLCA (n=408)

BRCA (n=1100)

BRCA-Basal (n=191)

BRCA-Her2 (n=82)

BRCA-LumA (n=568)

BRCA-LumB (n=219)

CESC (n=306)

CHOL (n=36)

X

X

COAD (n=458)

DLBC (n=48)

ESCA (n=185)

GBM (n=153)

HNSC (n=522)

HNSC-HPV- (n=422)

HNSC-HPV+ (n=98)

KICH (n=66)

KIRC (n=533)

KIRP (n=290)

LGG (n=516)

LIHC (n=371)

LUAD (n=515)

LUSC (n=501)

MESO (n=87)

OV (n=303)

PAAD (n=179)

PCPG (n=181)

PRAD (n=498)

READ (n=166)

SARC (n=260)

SKCM (n=471)

SKCM-Metastasis (n=368)

SKCM-Primary (n=103)

STAD (n=415)

TGCT (n=150)

THCA (n=509)

THYM (n=120)

UCEC (n=545)

XIX

UCS (n=57)

X

X

Y

UVM (n=80)

X

X

X

B cell

CD4+ T cell

CD8+ T cell

Macrophage

Endo

Monocyte

P>0.05

Neutrophil_CIBERSORT

Neutrophil_CIBERSORT. ABS Neutrophil_MCPCOUNTER

Neutrophil_QUANTISEQ

T cell regulatory [Tregs)_CIBERSORT

T cell regulatory [Tregs)_CIBERSORT-ABS T cell regulatory (Tregs)_QUANTISEQ

T cell regulatory (Tregs)_XCELL

Myeloid dendritic cell_TIMER

Myeloid dendritic cell_XCELL

Myeloid dendritic cell_MCPCOUNTER

Myeloid dendritic cell_QUANTISEQ

Myeloid dendritic cell activated_CIBERSORT

Myeloid dendritic cell activated_CIBERSORT-ABS

Myeloid dendritic cell activated_XCELL

Myeloid dendritic cell resting_CIBERSORT

Myeloid dendritic cell resting_CIBERSORT-ABS

Plasmacytold dendritic cell_XCELL

Cancer associated fibroblast_EPIC

Cancer associated fibroblast_MCPCOUNTER

Cancer associated fibroblast_XCELL

Ps0.05

Cancer associated fibroblast_TIDE

NK cel_MCPCOUNTER

NK cel_QUANTISEQ

NK cell activated_CIBERSORT

NK cel activated_CIBERSORT-ABS

NK cel resting_CIBERSORT

NK cel resting_CIBERSORT-ABS

T cell follicular helper_CIBERSORT

T cell follicular helper_CIBERSORT-ABS

Hematopoletic stem cell_XCELL

Common lymphoid progenitor_XCELL

Common myeloid progenitor_XCELL

Granulocyte-monocyte progenitor_XCELL

Neutrophil_XCELL

T cell gamma delta_CIBERSORT

T cell gamma delta_CIBERSORT-ABS T cell gamma delta_XCELL

Neutrophil_TIMER

Eosinophil_CIBERSORT

Eosinophil_CIBERSORT ABS

Mast cell activated_CIBERSORT ABS Mast cell resting_CIBERSORT

Eosinephil_XCELL

Mast cell activated_CIBERSORT

Mast cell resting_CIBERSORT-ABS

Partial_Cor

1

T cell NK XCELL

NK cel_EPIC

NK cel_XCELL

0

MOSC_TIDE

Mast cell_XCELL

-1

ACC (n=79)

X

X

BLCA (n=408)

XI

XIX

BRCA (n=1100)

BRCA-Basal (n=191)

BRCA-Her2 (n=82)

X

XIXIXIX

XX

BRCA-LumA (n=568)

XX

BRCA-LumB (n=219)

CESC (n=306)

CHOL (n=36)

XXXXX

X

X

X

X

X

XX [X]

COAD (n=458)

XXX

XX

DLBC (n=48)

XIXIXIX

XIXIX

X

XXIX

X

X

ESCA (n=185)

XIX

X

X

XXX

X

GBM (n=153)

X

XIXIX

XIX

XX

X

X

XX

XIX

HNSC (n=522)

HNSC-HPV- (n=422)

HNSC-HPV+ (n=98)

XIX

X

KICH (n=66)

XIX

X

XXX

KIRC (n=533)

KIRP (n=290)

X

XIX

LGG (n=516)

LIHC (n=371)

XIXIX

LUAD (n=515)

X

LUSC (n=501)

21521

MESO (n=87)

XIX

XIXIXIX

IXIX

IXIXIX

XIX

XXIXI

XXX

OV (n=303)

PAAD (n=179)

XIXIX

PCPG (n=181)

XXIX

XIX

PRAD (n=498)

READ (n=166)

XIX

XXI

XX

SARC (n=260)

X

SKCM (n=471)

SKCM-Metastasis (n=368)

SKCM-Primary (n=103)

X

XIXIXIX

X

XIXI

STAD (n=415)

TGCT (n=150)

XIXIX

XIX

XIXIX

XIX

THCA (n=509)

THYM (n=120)

UCEC (n=545)

UCS (n=57)

XI

X

XIXIX

X

X

UVM (n=80)

4

Neutrophil

Tregs

DC

CAF

NKT

NK cell

MDSC

Tfh

Hsc

Progenitor

γoT

Eos

Mast cell

Figure 11 Co-expression between TPGS2 and immune-associated genes. Co-expression between TPGS2 and gene encoding chemokines (A), MHCs (B), immunoinhibitors (C), chemokine receptors (D), and immunostimulators (E). * , P<0.05; ** , P<0.01. TPGS2, tubulin polyglutamylase complex subunit 2; MHC, major histocompatibility complex.

A

ACC

B

ACC

BLCA

BLCA

BRCA

BRCA

CESC

CESC

CHOL

CHOL

COAD

COAD

DLBC

DLBC

ESCA

ESCA

GBM

GBM

HNSC

*P<0.05

HNSC

**

*P<0.05

KICH

KICH

KIRC

** P<0.01

KIRC

** P<0.01

KIRP

KIRP

Correlation

LAML

Correlation

LAML

LGG

1.0

LGG

1.0

LIHC

LIHC

LUAD

0.5

LUAD

0.5

LUSC

LUSC

MESO

0.0

MESO

0.0

OV

-0.5

OV

PAAD

-0.5

PAAD

PCPG

-1.0

PCPG

-1.0

PRAD

PRAD

READ

READ

SARC

SARC

SKCM

SKCM

STAD

STAD

TGCT

TGCT

THCA

THCA

THYM

THYM

UCEC

UCEC

UCS

UCS

UVM

**

UVM

**

**

CCL1

CCL2

CCL3

CCL4

CCL5

CCL7 CCL8

CCL 11

CCL13

CCL14

CCL15

CCL16

CCL 17

CCL 18

CCL19

CCL20

CCL21

CCL22

CCL23

CCL24

CCL25

CCL26

CCL27

CCL28

CX3CL1

CXCL 1

CXCL2

CXCL3

CXCL5 CXCL6

CXCL8

CXCL9

CXCL10

CXCL 11

CXCL12

CXCL13 CXCL14

CXCL 16

CXCL17

XCL1

XCL2

B2M

HLA-A

HLA-B

HLA-C

HLA-DMA

HLA-DMB

HLA-DOA HLA-DOB

HLA-DPA1 HLA-DPB1

HLA-DQA1

HLA-DQA2 HLA-DQB1

HLA-DRA

HLA-DRB1

HLA-E

HLA-F HLA-G

TAP1

TAP2

TAPBP

C

ACC

**

**

D

BLCA

ACC

**

**

BLCA

**

BRCA

CESC

BRCA

**

**

CHOL

CESC

COAD

CHOL

DLBC

**

COAD

**

**

**

**

ESCA

DLBC

**

GBM

ESCA

HNSC

*P<0.05

GBM

KICH

HNSC

** *P<0.05

KIRC

** P<0.01

KICH

** P<0.01

KIRP

**

LAML

Correlation

KIRC

KIRP

**

1.0

LAML

Correlation

LGG

**

LIHC

LGG

1.0

LUAD

0.5

LIHC

**

LUSC

LUAD

**

0.5

MESO

0.0

LUSC

0.0

OV

PAAD

-0.5

MESO

OV

-0.5

PCPG

**

PRAD

-1.0

PAAD

PCPG

**

-1.0

READ

PRAD

SARC

READ

SKCM

SARC

STAD

SKCM

TGCT

STAD

THCA

TGCT

THYM **

THCA

**

UCEC

THYM

**

UCS

UCEC

**

UVM

*

**

UCS

ADORA2A

BTLA

CD160

CD244

CD274

CD96

CSF1R

CTLA4

HAVCR2

IDO1

IL10

IL 10RB

KDR

KIR2DL1

KIR2DL3

LAG3

LGALS9

PDCD1

PDCDILG2

NECTIN2

TGFB1

TGFBR1

TIGIT

VTCN1

UVM

**

CCR1

CCR2

CCR3

CCR4

CCR5

CCR6

CCR7

CCR8

CCR9

CCR10

CXCR1

CXCR2

CXCR3

CXCR4 CXCR5

CXCR6

XCR1

CX3CR1

E

ACC

**

**

**

**

**

**

**

BLCA

BRCA

CESC

CHOL

**

COAD

DLBC

ESCA

GBM

HNSC

*P<0.05

KICH

KIRC

** P<0.01

KIRP

LAML

Correlation

LGG

1.0

LIHC

LUAD

0.5

LUSC

MESO

0.0

OV

**

PAAD

-0.5

PCPG

PRAD

-1.0

READ

SARC

SKCM

STAD

TGCT

**

THCA **

THYM

**

UCEC

**

UCS

UVM

**


**

**

**

**

BTNL2

VSIR

CD27

CD276

CD28

CD40

CD40LG

CD48

CD70

CD80

CD86

CXCL12

CXCR4

ENTPD1

HHLA2

ICOS

ICOSLG

IL2RA

IL6

IL6R

KLRC1 KLRK1

**

LTA

MICB

NTSE

PVR

RAET1E

STING1

TMIGD2

TNFRSF13B

TNFRSF13C

TNFRSF14

TNFRSF17

TNFRSF18

TNFRSF25

INFRSF4 TNFRSF8

**

TNFRSF9

TNFSF13

TNFSF13B

TNFSF14

TNFSF15

TNFSF18

TNFSF4

TNFSF9

ULBP1

Figure 12 The correlation between TPGS2 expression and the TMB, and MSI. (A) Radar map showing the correlation between TPGS2 expression and the TMB. (B) Radar map showing the correlation between TPGS2 expression and MSI. The red lines represent the correlation coefficients. Spearman correlation test, *, P<0.05; ** , P<0.01; and *** , P<0.001. TPGS2, tubulin polyglutamylase complex subunit 2; TMB, tumor mutational burden; MSI, microsatellite instability.

A

STAD


COAD


THYM


ESCA **

B

0.4

KIRC


COAD


STAD

DLBC *

PCPG *


0.3

ACC

ACC

CHOL

CHOL

KIRP

SKCM


*

0.2

THCA


MESO

0.2

0.1

UCS

UCS

0

CESC

OV

0

THYM

TGCT

-0.2

MESO

KIRC

-0.

PRAD

-0.2

BLCA *

-0.4

KIRP

GBM

-0.3

LUAD

LAML

-0.6

LUSC

BRCA **

-0.4

ESCA

BRCA *

UVM

LIHC

KICH

KICH

PAAD

PAAD

LUSC

LGG

LIHC

READ

THCA

PCPG

PRAD

TGCT

SKCM

HNSC

DLBC

LAML

SARC

LUAD

UCEC

SARC

LGG

BLCA

OV

READ

GBM

CESC

UCEC

HNSC

UVM

Correlation (TMB)

Correlation (MSI)

most cancers. The correlation analyses revealed strong connections between TPGS2 and specific cancer types, such as HNSC, LIHC, PAAD, PRAD, SARC, THYM, and UM. Additionally, TPGS2 was positively correlated with most of the immunomodulatory factors in COAD, HNSC, KIRP, LIHC, PAAD, PRAD, READ, and UVM, but negatively correlated with ESCA and SARC.

TMB and MSI analysis

The TMB and MSI are two well-known biomarkers that predict immune therapy responses across different cancers (41,42). Most scholars believe that patients with a high TMB and MSI have increased response rates to immunotherapy and display better outcomes to immunotherapy treatments. Therefore, we assessed the correlation with TMB and MSI to evaluate the efficacy of TPGS2 in predicting ICIs therapy outcomes in pan-cancer. As Figure 12 shows, TPGS2 expression was positively correlated with the TMB in BRCA, BLCA, ACC, SKCM, STAD, and COAD, and TPGS2 expression was negatively correlated with the TMB in ESCA, KIRC, THCA, and THYM (Figure 12A). Additionally, TPGS2 expression was positively correlated with MSI in BRCA, COAD, and STAD, and TPGS2 expression was negatively correlated with MSI in DLBC and PCPG (Figure 12B). Thus, our analyses indicate that TPGS2 could have a potential role in predicting the effectiveness of ICIs in a number of cancers.

Discussion

Currently, many immune checkpoint molecules have been applied to pharmacotherapy, such as CTLA-4, PD-1, PD-L1, T cell immunoreceptors with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT), and lymphocyte activating 3 (43-47). CTLA-4 and PD-L1/PD-1 have received the greatest attention; thus, anti-PD-1/PD-L1 and anti-CTLA-4 techniques have been applied to the immunotherapy of a number of cancers (46). However, at present, the most serious challenges for such treatments are related to their inapparent efficacy and side effects (48). As a result, there is a pressing need to investigate novel immunological checkpoints and methods to estimate the effects of cancer immunotherapy (48,49). Our results showed that TPGS2 is a promising biomarker of cancer, which provides a vital clue for further research on the potential role of TPGS2 in prognosis and tumor immunity.

The analysis of TPGS2 expression based on the GTEx and TCGA databases revealed that TPGS2 was abnormally upregulated in 22 cancers and downregulated in five cancers (Figure 1A). Moreover, the expression of TPGS2 was notably downregulated in TGCT tissue compared with normal testicular tissue. Data about RNA was used to cluster genes based on their expression in single-cell types. Based on the single-cell type expression cluster from HPA, we found that TPGS2 was mainly expressed in spermatids.

Thus, we speculate that the specific expression in normal testicular tissue is connected with the lower expression of TPGS2 in TGCT. However, it is not yet known why TPGS2 is unevenly expressed across cancers.

Next, the IHC results from the HPA were consistent with our preliminary conclusions (Figure 2). According to the analysis of the association between TPGS2 and various subtypes, TPGS2 may be associated with molecular and immune subtypes across human cancers (Figure 3). This suggests that TPGS2 can be used to differentiate among molecular and immune types of tumors. Since most genetic alteration proportions of TPGS2 in cancers are less than 5%, there appears to be no significant correlation between TPGS2 expression and genomic alterations (Figure 4A).

In addition, we evaluated the clinical prognosis of patients who were grouped according to TPGS2 expression levels. There were differences among the various survival measures (i.e., OS, PFI, and DSS); however, the expression of TPGS2 was still significantly associated with survival (Figures 5-7). According to the Kaplan-Meier and univariate Cox regression analyses, the upregulated expression of TPGS2 was associated with a poor prognosis in patients with SARC, LIHC, and LGG, while the high expression of TPGS2 was associated with a better OS prognosis in patients with OV and THYM. Thus, TPGS2 is likely to be an important biomarker for predicting the prognosis of cancer patients.

The single-cell analysis showed that TPGS2 expression was associated with a number of functional states, including the cell cycle, metastasis, invasion, inflammation, DNA damage, and stemness, in various cancers (Figure 8). These results suggest that TPGS2 is associated with multiple cancer functional states in many human cancers.

According to the GSEA, TPGS2 was closely related to some immune response processes, such as Ig production, B cell-mediated immunity, the humoral immune response mediated by circulating Ig, the regulation of the humoral immune response, and the B cell receptor signaling pathway (Figure 9). Therefore, it is very likely that TPGS2 is involved in the functions related to B cells.

Recently, the importance of B cells has been found in tumor immunity. Since 2020, three research teams from the United States, France, and Sweden have analyzed a large sample of clinical cohort studies, and reported a positive correlation between B-cell infiltration and the formation of TLSs and the response to immunotherapy in a variety of cancer types (50-52). B cells also express a number of checkpoint molecules, including PD-1, PD-L1/2, and

CTLA-4B (15). Patients who responded to ICIs therapy were reported that more memory B cells, C-X-C motif chemokine receptor 3+ cells, and germinal center-like B cells were found in their TMEs than patients who did not response to ICIs therapy (52).

In addition, studies (21,53) have shown that the Igs in many antibody-secreting B cells, which mainly secrete IgG and IgA, are tumor-dependent. These Igs are correlated with the tumorigenesis site, and higher proportions of IgG have been observed in thyroid, testicular, and skin tumors, while higher proportions of IgA been observed in kidney, ovarian, pancreatic, and colorectal cancers. This is consistent with our GSEA finding that a higher number of Ig-related biological states were enriched.

Another important finding of our research is that TPGS2 plays a pivotal role in cancer immunity. In recent years, many studies have shown that the immune status of cancers is closely correlated to the cell composition of and infiltration concentration around the tumor (54-56). TPGS2 was found to be positively correlated with the infiltrating levels of multiple immune cells, such as macrophages, CD4+ T cells, CD8+ T cells, B cells, and neutrophils (Figure 10), which suggests that TPGS2 is likely to influence development and prognosis of various cancers by affecting the TME.

Pro-inflammatory mediators, including chemokines, cytokines, and prostaglandins, have been found in the TME, which affects tumor initiation, progression, and metastasis (57-59). Our analyses of TPGS2 expression and pan-cancer immunomodulatory factors suggested that TPGS2 is co-expressed with genes that encode the immunoinhibitor, immunostimulator, MHC, chemokine, and chemokine receptor proteins, especially in THYM and HNSC (Figure 11). These results suggest that TPGS2 is likely to be involved in the progression and prognosis of cancers by interacting with the TME.

Further, the TMB is a promising prognostic and predictive biomarker for immunotherapy in human cancers (60-63). Research has demonstrated that patients, suffering from melanoma (64,65) or urothelial carcinoma (66,67) with a high TMB achieve better clinical outcomes from ICIs. Similarly, MSI also plays a vital role as a predictive biomarker for tumor immunotherapy (68). The Food and Drug Administration has authorized MSI-high status or deficient mismatch repair as prognostic biomarkers for directing the therapeutic application of ICIs in certain malignancies (69). According to our analyses, the TMB in 10 types of cancers and MSI in five types of cancers were

significantly correlated with the expression of TPGS2. Thus, TPGS2 is likely to act as a predictor of the efficacy of immunotherapy in many cancers.

Wang’s study demonstrated that the cir-TPGS2 (derived from TPGS2)-related axis promoted breast cancer cell motility by the TME (30). Another study demonstrated that TPGS2 could be a potential gene in renal cell carcinoma (29). Together with our findings, such results suggest that TPGS2 could serve as a potential biomarker in anti-tumor immunity treatments.

However, our research had a number of limitations. First, while we showed that TPGS2 is a promising predictor of prognosis and immunotherapy in many cancers, the mechanism by which this occurs remains unknown, and we have no evidence of any direct interaction. Second, our data were mainly obtained from open databases, and no clinical cohort was used for verification, which inevitably led to various biases and decreased the credibility of the results. Third, TPGS2 has rarely been studied in human tumors, and in-depth studies need to be conducted to verify its role in cancer prognosis prediction and immunotherapy. Fourth, our research revealed a promising direction for tumor research in TPGS2; however, this study was a descriptive study based on bioinformatics, and the mechanism related to both TPGS2 and the development and therapy of specific tumors need to be further explored, and experimental verification in vitro and in vivo is required. In the future, we intend to conduct further experiments to examine the mechanism of TPGS2 across human cancers.

Conclusions

In summary, we performed a comprehensive pan-cancer analysis that demonstrated that the aberrant expression of TPGS2 was associated with the prognosis, immune cell infiltration, TME, some immune response processes, and various function states across human cancers. Thus, TPGS2 could serve not only as a biomarker for predicting clinical outcomes, but also as a promising biomarker for evaluating and developing new approaches to immunotherapy in many types of cancers, especially COAD and STAD.

Acknowledgments

We greatly appreciate the TCGA database for providing useful data, as well as bioinformatics tools for data analyses. Funding: This work was supported by Zhejiang Province Medicine and Health Science and Technology Plan Project

(Nos. 2022KY966 and 2021KY250).

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tcr. amegroups.com/article/view/10.21037/tcr-23-113/rc

Peer Review File: Available at https://tcr.amegroups.com/ article/view/10.21037/tcr-23-113/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups. com/article/view/10.21037/tcr-23-113/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non- commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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Cite this article as: Ding Z, Ding Q, Li H. The prognostic biomarker TPGS2 is correlated with immune infiltrates in pan-cancer: a bioinformatics analysis. Transl Cancer Res 2024;13(3):1458-1478. doi: 10.21037/tcr-23-113