CMTM3 as a Potential New Immune Checkpoint Regulator

Objectives To evaluate the role of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) in tumor microenvironment and cancer immunotherapy and explore its potential mechanism. Method The cancer genome map was obtained from the UCSC Xena database. RNAseq data from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases were utilized for evaluating the expression and prognostic value of CMTM3 through survival data of clinical trials. The enrichment analysis of CMTM3 was performed using the R package “clusterProfiler.” The scores of immune cell infiltration in TCGA samples were downloaded from the ImmuCellAI database and TIMER2 database, and the relationship between both immune cell invasion and CMTM3 expression was investigated. Immunological activation and suppression genes, immune checkpoints, chemokines, and their receptors were all investigated in relation to CMTM3. Results Most tumor types had varied levels of CMTM3 expression and predicted poor survival status. The CMTM3 expression is closely associated with cancer-associated fibroblasts, macrophages, myeloid dendritic cells, endothelial cells, immune activation genes, immune suppressor genes, immune checkpoints, chemokines, and related receptors. Conclusion Our data reveal that CMTM3 might be used as a cancer biomarker. CMTM3 may work in conjunction with other immunological checkpoints to alter the immune milieu, which could lead to the establishment of new immunotherapy medicines.


Introduction
e buildup of various genetic changes resulting in the development of different neoantigens on the surface of tumor cells is one of the most essential characteristics of cancer [1,2]. Malignant cells often evolve to evade the attack by the immune system, which is the biggest hurdle in the immune therapies for cancer [3,4]. Till now, a number of immune evading processes have been discovered with regard to immunotherapy, and the endogenous "immune checkpoints," which regulate immune responses after antigen activation, are also expressed. Anticancer medicines based on immune checkpoint inhibitors, such as antiprogrammed cell death protein 1 (PD-1), antilymphocyte activation gene 3 (LAG3), and anticytotoxic T lymphocyte-associated protein 4, have been developed as a result of these discoveries (CTLA4) [5][6][7].
CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) is the main member of the chemokine-like factor family (CKLFS) and is also one of the chemokine-like factor genes located in a cluster on chromosome 16q22 [8], which is differentially expressed in various human malignant tumors and is directly linked to the malignant phenotype of cells. However, its upstream target genes and their related molecular regulatory mechanisms are not clear. CMTM3 is expressed on immune cells such as follicular helper T cells, activated CD4 memory T cells, and CD8 T cells [9]. rough its interaction with C-C chemokine receptor 4 (CCR4), CMTM3 plays a role in the growth of arthritis [10]. Previous research has discovered that CMTM3 inhibits cell migration and invasion and is linked to a favorable outcome in gastric cancer [11]. CMTM3 can participate in EMT induction by inhibiting the JAK2/STAT3 signaling pathway and may have a strong influence on the metastasis of liver cancer [12]. CMTM3 decreases EGFR expression and EGF-mediated tumorigenicity by promoting Rab5 activity in gastric cancer [13]. ese findings suggest that CMTM3 might be critical in the modulation of tumors and immune system diseases.
We explore the CMTM3 expression in a variety of cancers and its correlation with the prognosis of cancer patients. is research evaluates the link between CMTM3 and immune activation genes, immunosuppressive genes, immune cell infiltration scores, immune checkpoints, chemokines, and chemokine receptors. Our findings demonstrate the potential mechanisms by which CMTM3 affects tumor microenvironment and cancer immunotherapy. is research will shed light on the functional significance of CMTM3 in cancer.

Data Collection.
e cancer genome map was obtained from the UCSC Xena database (https://xenabrowser.net/ datapages/). e RNAseq data from the Genotype-Tissue Expression (GTEx) (https://commonfund.nih.gov/GTEx/) and e Cancer Genome Atlas (TCGA) (https://tcga-data.nci. nih.gov/) databases were utilized for evaluating the expression and the prognostic value of CMTM3 through survival data of clinical trials. Enrichment analysis of CMTM3 was performed using the R package "clusterProfiler."

Prognosis Analysis.
e overall survival (OS) of TCGA cohort patients was evaluated by the Kaplan-Meier analysis. e significance of CMTM3 in predicting OS, progressionfree interval (PFI), and disease-specific survival (DSS) in cancer patients was assessed by univariate Cox regression analysis.

Gene Enrichment Analysis.
e correlation of CMTM3 with genes was analyzed utilizing the TCGA data. e following settings were used to choose genes related with CMTM3 (p0.05) for gene set enrichment analysis (GSEA) using the R package "clusterProfiler": minGSSize � 10, nPerm � 1000, and maxGSSize � 1000 are valid values. 0.05 is the cutoff p value [14].

Statistical Analysis.
e data were analyzed and reported as mean ± SD (standard deviation). R.3.6.2 was availed for conducting the statistical analysis, and to examine the difference among all the groups, Student's t-test was utilized. P < 0.05 was defined as statistically significant.

GSEA Analysis of CMTM3.
e pathways by which CMTM3 may be involved were assessed using the GSEA in thirty-three tumor types from the TCGA database. e outcomes suggest that CMTM3 may be connected to the pathways related to immune functions. ese findings show that CMTM3 is involved in the regulation of the tumor immune microenvironment (Figures 6(a)-6(k)).
3.6. Analysis of Immune Cell Infiltration. We used immune cell infiltration data from the TIMER2 database to conduct a correlation study to see if there was an association between CMTM3 expression and immune cell infiltration. In analyzing clinical tumor biopsies, tumor quality has a significant impact on the extraction of immune cell infiltrates. After making the necessary adjustments for tumor purity/ quality, the CMTM3 expression was found to be considerably linked with different types of immune cells. e TIMER2 database results showed that CMTM3 was directly related to the invasion of cancer-associated fibroblasts, macrophages, myeloid dendritic cells, and endothelial cells in several TCGA tumor types (Figures 7(a)-7(d)). e correlation analysis by availing the data from the Immu-CellAI database demonstrated that CMTM3 was also directly proportional to the amount of infiltration of macrophages, dendritic cells, and others (Figure 7(e)). ese cells are all important components of the tumor microenvironment (TME). e TME plays a critical role in the tumorigenesis, development, and metastasis of tumors.
ese factors are not conducive to improving the effectiveness of tumor immunotherapy, suggesting that CMTM3 plays the role of an "oncogene" in most tumors.
Further research revealed that CMTM3 was directly connected to most of the immune activation genes, genes related to immune suppression status, chemokine receptor genes, and chemokine genes, and all of them are important elements of tumor microenvironment.

Discussion
Immune checkpoint inhibitors have transformed the face of cancer treatment in recent years, becoming a vital mode of immunotherapy for cancer treatment [15,16]. Immune   Journal of Oncology checkpoint inhibitors suppress immunity and induce a durable anticancer response [17][18][19]. Immune checkpoint proteins mainly include PD-L1, PD-1, and CTLA4 [20].
Previous studies have shown that CMTM3 can affect the efficacy of tumor immunotherapy by affecting the tumor microenvironment. e current research investigated the function of CMTM3 in various cancer types.
We evaluated the CMTM3 expression and its prognostic value in various types of cancers. We found that CMTM3 has high expression in 21  We then further assessed CMTM3 expression in tumors at separate WHO stages, and the outcome showed that its expression was low in most tumors at later stages, such as BLCA, BRCA, KIRC, STAD, and THCA. However, it was also observed in other tumors that CMTM3 has high expression in later stages, such as ACC, ESCA, and ESAD. For paired tumor and normal tissue analysis in TCGA tumors, we found that CMTM3 is highly expressed in BLCA, LUAD, COADREAD, COAD, CHOL, HNSC, KIRC, ESCA, KIRP, LIHC, STAD, THCA, OSaCC, and ESAD cancer tissue compared to normal tissue. However, CMTM3 was highly expressed in normal tissues rather than cancer tissue in KICH and UCEC.
OS analysis using Kaplan-Meier curves demonstrates that CMTM3 was a protective factor in patients with BLCA,   tumor biology, aggressiveness, or response to therapy. Furthermore, utilizing OS frequently necessitates more follow-up time. As a result, the use of DSS or PFI in clinical studies can adequately reflect the influence of variables on patients. In light of these findings, we conducted a univariate Cox regression analysis to determine the relationship between CMTM3 and DSS or PFI in cancer patients. e DSS analysis showed that CMTM3 was only a risk factor in PCPG patients, and the difference was not statistically significant in other tumors. Finally, PFI analysis demonstrated that CMTM3 is a protective factor in patients with LUAD, KICH, KIRP, SARC, SKCM, THCA, and UCEC, as well as in   T cells  T helper cells  Tcm  Tem  TFH  Tgd  1 cells  17cells  2 cells  TReg   OV  BLCA  LUAD  COADREAD  COAD  LUSC  ACC  BRCA  CESC  CHOL  DLBC  ESCA  GBM  HNSC  KICH  KIRC  KIRP  LAML  LGG  LIHC  MESO  PAAD  PCPG  PRAD  READ  SARC  SKCM  STAD  THCA  THYM  UCEC  UCS  UVM  GBMLGG  LUADLUSC  OSCC  ESAD  ese results suggest that the high CMTM3 expression is primarily a risk factor in most tumor types.
We further analyzed the signaling pathways by which CMTM3 may be involved using GSEA in 33 tumor types from the TCGA database. e results showed that CMTM3 was associated with immune-related pathways, such as the BIOCARTA_CTLA4_pathway, Reactomesignaling_by_ the_B_cell_receptor_BCR, KEGG_T_cell_receptor_ signaling_pathway, PID-IL8-cxcr2 pathway, PID_lym-ph_angiogenesis_pathway, and other pathways. ese results indicate that CMTM3 plays a significant role in the regulation of tumor immune microenvironment.
Cancer cells are surrounded by an abundant matrix composed of complex extracellular matrix (ECM) proteins, including collagen and fibronectin, and numerous cellular components such as immune cells, endothelial cells, pericytes, and cancer-associated fibroblasts. e favorable interaction between tumor cells, matrix, and cells is critical for tumor formation, progression, and metastasis [21][22][23]. Results of immune cell infiltration analysis in our study showed that CMTM3 was favorably linked with the invasion of cancer-associated fibroblasts, macrophages, myeloid dendritic cells, and endothelial cells in most tumors.
It has also been reported that CALD1, a key gene associated with cancer-associated fibroblasts, can promote the progression of bladder cancer by remodeling the tumor microenvironment [24]. By modifying the tumor immune milieu, macrophages regulate malignant capabilities of colorectal cancer, according to Zhang et al. [25].
ese findings support CMTM3's involvement as an immunological checkpoint regulator.
ere are some limitations to this study. An in vivo experiment was not performed to test the antitumor activity of targeting CMTM3. More clinical trials need to be conducted to confirm the role of CMTM3 as an immune checkpoint regulator.

Conclusion
Taken together, we evaluated the role of CMTM3 as a potential prognostic indicator and its role in regulating tumor immunotherapy by affecting the tumor microenvironment. CMTM3 could be a target for tumor immunotherapy and a novel immune checkpoint regulator.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.