- Academic Editor
Background: Triple-negative breast cancer (TNBC) is an aggressive form
of breast cancer (BC), and it is often associated with a high tumor grade, a
younger age at diagnosis, and a low survival rate. Conventional
endocrine and anti-HER-2 therapies are usually ineffective against TNBC, creating
treatment challenges and resulting in a poor prognosis. Hence, new targets and
treatment strategies for TNBC are urgently required. Methods: The
GSE102818 dataset was used to identify differentially expressed genes (DEGs)
between primary BC and metastatic BC lesions. The Cancer Genome Atlas and the
cBioPortal platform were employed to explore mutations in candidate genes.
Utilizing the Tumor IMmune Estimation Resource (TIMER), the relationship between
the expression of candidate genes and immune cell infiltration was assessed.
Additionally, the cell-specific expression of the candidate genes was examined in
the immune microenvironment of primary BC and metastatic BC lesions using the
single-cell RNA sequencing (scRNA-seq) datasets GSE118389 and GSE202695. Finally,
the protein expression of the candidate genes in clinical TNBC samples was
evaluated. Results: CD8A was identified as a hub gene in the
DEG network and was found to be down-regulated in metastatic BC lesions.
CD8A expression was highly correlated with the infiltration of CD8
Triple-negative breast cancer (TNBC) is a highly heterogeneous type of breast cancer (BC) [1]. Compared with other subtypes of BC, TNBC is aggressive and often has a high tumor grade and poor prognosis [2]. Notably, TNBC is associated with a high risk of metastasis, with the lungs, liver, and brain being the three most common sites of metastasis [3].
Owing to a lack of target receptors, TNBC is typically unresponsive to endocrine therapy and targeted therapy. Hence, chemotherapy is often employed as the primary treatment strategy for TNBC. At present, adjuvant chemotherapy with anthracyclines plus taxanes is typically used to treat TNBC. Further, it is recommended as a first-line therapeutic option for TNBC per international guidelines. However, some patients with BC do not respond to anthracyclines and taxanes, which results in a poor prognosis [4]. Owing to limited treatment options and drug resistance in TNBC, research on this malignancy has become a key priority in the field of oncology. However, so far, studies on the treatment of TNBC have largely focused on the discovery of new targets and the use of endocrine therapy and immunotherapy.
Tumor immunotherapy aims to enhance the body’s immune response against tumors, thereby reducing tumor immunosuppression and enhancing antitumor effects. Insights into the interaction between cancer and the immune system have helped in maximizing the benefits of immunotherapy. Notably, immunotherapy has achieved significantly stronger antitumor responses than monotherapy in several patients. Therefore, several immunotherapy-based treatment strategies have been proposed. The principle of immunotherapy is multifaceted. Immune checkpoint dysregulation is an important event during malignant transformation. This process allows tumor cells to resist immune responses, reduces the activation of T cells, prevents tumor surveillance, and enhances tumor survival. In this context, the clinical application of immune checkpoint inhibitors blocks immune checkpoint-related pathways, thus reactivating immune cells. Hence, treatment with immune checkpoint inhibitors disrupts immune resistance in tumor cells, strengthens the activity of T cells against cancer cells, and boosts the immune response [5]. Research has shown that both tumor-infiltrating lymphocytes (TILs) and checkpoint molecules can serve as indicators for the effectiveness of immune checkpoint inhibitors in BC [6]. Higher TIL levels can lead to improved immunotherapy outcomes, and the number of TILs is positively correlated with progression-free survival [7].
Interestingly, TNBC is the most immunogenic type of breast malignancy because it exhibits higher levels of TILs than other BC subtypes. The inherent heterogeneity of TNBC has important implications for drug development, clinical diagnosis, and treatment in this type of cancer. According to Oura et al. [8], the tumor immune microenvironment (TIME) plays a key role in TNBC metastasis. Identifying TIME biomarkers can help elucidate the causes of tumor heterogeneity in TNBC, enabling the development of targeted treatment strategies.
In this study, differentially expressed genes (DEGs) between primary and
metastatic BC lesions were identified, with a focus on genes related to the TIME.
After identifying the hub gene CD8A, immune cell infiltration (ICI)
analysis was performed. The results showed that CD8A expression was
closely related to the infiltration of CD8
All gene expression data were obtained from the Gene Expression Omnibus (GEO) database. RNA-seq expression data from 31 patients with primary BC and 17 patients with metastatic BC were obtained from the GSE102818 dataset. Additionally, the scRNA-seq data of 1534 cells from six TNBC samples (GSE118389), as well as the scRNA-seq data of patient-derived BC xenograft tumors and matched lung macrometastases (GSE202695), were acquired.
We used the “Limma” package and GSE102818 data to identify the genes differentially expressed between primary and metastatic BC lesions. DEGs were identified by comparing gene expression between the 31 primary BC samples and 17 metastatic BC samples.
To explore the functional relationships and interactions among the identified DEGs, we constructed a PPI network using the STRING database. This network analysis helped us understand the potential roles of these genes in tumor-related biological processes.
We conducted gene enrichment analysis on the identified DEGs using R software (version 4.3.2, The R Foundation, https://www.r-project.org/). The analysis was performed using the “clusterProfiler” package, “org.Hs.eg.db” package, and “enrichplot” package. Accordingly, we explored the biological functions and pathways associated with the identified DEGs.
We obtained MSI and TMB scores from The Cancer Genome Atlas (TCGA). Spearman’s method was employed to analyze the relationship of MSI and TMB with the expression of specific genes.
We used the cBioPortal platform and BC data from TCGA for gene mutation analysis and explored the genetic alterations in the genes of interest to better understand their potential roles in BC.
To investigate the correlation between ICI and CD8A expression, we utilized the TIMER platform (https://cistrome.shinyapps.io/timer/). Pearson correlation coefficients were calculated to explore the role of CD8A in the modulation of immune responses.
We applied the “SingleR” package to analyze the scRNA-seq datasets GSE118389 and GSE202695. This analysis allowed us to determine the specific expression patterns of CD8A within the TIME, as previously described [9].
Immunohistochemical staining was performed to examine primary and metastatic BC tissue. Tissue sections were dewaxed, and antigen retrieval was performed in citrate buffer (pH 6.0) at 95 °C for 20 min. Subsequently, the sections were treated with 3% peroxidase for 20 min and then use endogenous peroxidase to blocked for 30 min. Finally, the sections were incubated with the anti-CD8A antibody (ab237709, Abcam) overnight at 4 °C. The following day, the sections were washed and incubated with HRP-Conjugated Streptavidin and biotinylated goat anti-mouse IgG (H+L). DAB staining was performed, followed by hematoxylin counterstaining. The protein expression levels were assessed by five pathologists.
All data were subjected to statistical analysis using R software (version 4.0.3, https://www.r-project.org/). Statistical tests were performed, as appropriate, for the specific analyses conducted. A p value of 0.05 or below indicated statistical significance.
To understand the differences in gene expression between primary BC and metastatic lesions, we analyzed differential gene expression using data from 31 primary BC samples and 17 metastatic BC lesions (obtained from the GSE102818 dataset). The analysis yielded 63 DEGs (Fig. 1A,B). Subsequently, we attempted to identify the potential biological functions of these DEGs. Gene enrichment analyses—including both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses—revealed the association of DEGs with various tumor-related processes. These included the humoral immune response, lymphocyte differentiation, G protein-coupled receptor binding, and chemokine activity (Fig. 1C,D).
Genes differentially expressed between primary and metastatic breast cancer (BC) lesions. (A) Volcano plot showing the distribution of differentially expressed genes (DEGs). (B) Heatmap denoting the DEGs (primary vs. metastatic BC lesions). (C,D) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene enrichment analysis of the DEGs.
After the DEGs were identified, we visualized their interactions using a PPI network (Fig. 2). In-depth analysis revealed that CD8A was a key hub gene in this network and had the highest number of nodes (i.e., 74), which highlighted its centrality within this network (Fig. 3A). Furthermore, we validated the expression of CD8A using TCGA data. Interestingly, our analysis indicated that there was no significant difference in CD8A expression levels between BC and normal tissues (Fig. 3B). Moreover, in our pan-cancer analysis, CD8A expression was not significantly correlated with TMB or MSI (Fig. 3C,D).
Protein–protein interaction network of differentially expressed genes.
CD8A is an important hub gene. (A) Node analysis of
the differentially expressed gene–protein network. (B) Expression levels of
CD8A in 33 types of tumors. (C) Association of CD8A expression
with tumor mutational burden in 33 types of tumors. (D) Association of
CD8A expression with microsatellite instability in 33 types of tumors.
*p
To further understand the role of CD8A in BC, we explored the mutations in this gene using the cBioPortal platform. The overall mutation rate of CD8A was 0.6% (Fig. 4A), and a detailed examination revealed various mutation types (Fig. 4B). Moreover, several genes showed co-alterations in the presence of CD8A mutations, including DNAH6, MAT2A, and PTCD3 (Fig. 4C). Notably, K92N was the primary type of CD8A mutation (Fig. 4D). Interestingly, CD8A mutations were correlated with copy number alterations such as deep deletions, high amplifications, and arm-level gains in BC macrophages (Fig. 4E).
Mutation status of CD8A in breast cancer (BC). (A)
Oncoprint of CD8A in BC. (B) Types of CD8A mutations in BC. (C)
Frequency of gene alterations in the presence and absence of CD8A
mutations. (D) Site of CD8A mutations in BC. (E) Association of
CD8A mutations in BC with copy number alterations in immune cells.
*p
We explored the correlations between CD8A expression and ICI.
CD8A expression exhibited a strong correlation with PD-L1 (CD274, R =
0.581) and PD1 (PDCD1, R = 0.841) expression (Fig. 5A,B). Additionally, we
analyzed the association between CD8A expression and the infiltration of
various immune cells. CD8A expression exhibited a negative correlation
with tumor purity. Moreover, it exhibited a
positive correlation with the infiltration of B cells, CD8
Correlation between CD8A expression and immune cell infiltration (ICI). (A) Correlation between CD8A and PD-L1 (CD274) expression. (B) Correlation between CD8A and PD1 (PDCD1) expression. (C) Correlation between CD8A expression and ICI. (D) Survival analysis based on CD8A expression and ICI in BC patients.
Using scRNA-seq data, we identified CD8A-expressing immune cells to
understand the distribution of CD8A expression within the TIME of BC.
Our findings showed that CD8A was predominantly expressed in CD8
Immune cell distribution of CD8A in primary breast cancer (BC) lesions. (A) tSNE plot illustrating the scRNA-seq data obtained from immune cells in the BC microenvironment. (B,C) Cellular distribution of CD8A expression in the BC immune microenvironment. (D) Cellular distribution of the top 10 differentially expressed genes in the BC immune microenvironment.
Immune cell distribution of CD8A in lung metastases of breast cancer (BC). (A) tSNE plot illustrating the scRNA-seq data obtained from immune cells in BC lung metastases. (B,C) Cellular distribution of CD8A in the the Tumor IMmune Estimation Resource (TIME) of BC lung metastases. (D) Cellular distribution of DEGs in the TIME of BC lung metastases.
Finally, we validated our findings using clinical BC tissue samples. The results were consistent with our previous analyses, revealing high CD8A expression levels in the TIME of primary BC foci (Fig. 8A) and a significant down-regulation of CD8A expression in the TIME of BC lung metastases (Fig. 8B). These results confirmed the significance of CD8A in BC progression.
Immunohistochemical staining for CD8A in clinical breast cancer and lung metastasis samples. Black box, cancerous tissue; red box, adjacent normal tissue; blue box, immune cells. High CD8A expression levels in the TIME of primary BC foci (A) ,down-regulation of CD8A expression in the TIME of BC lung metastases (B).
TNBC, which accounts for 15% to 20% of all BC cases, is a highly aggressive breast malignancy. Pathologically invasive ductal carcinoma is the most common subtype of TNBC [10]. Clinically, TNBC is often accompanied by lung and brain metastasis, which typically occurs within the first 3 years after the initial diagnosis. TNBC is prone to recurrence even after neoadjuvant and adjuvant treatments, including chemotherapy, and it has a lower overall survival rate than other types of BC [11].
The present study demonstrates that CD8A plays a critical role in the progression of TNBC. CD8A, which is primarily expressed in cytotoxic T lymphocytes and natural killer cells, is a key mediator of the immune system’s defense against cancer cells. In this study, we discovered that CD8A is significantly up-regulated in metastatic TNBC. This highlights the role of CD8A in the advanced stages of TNBC, particularly during its spread to distant sites.
In this study, we re-mined high-throughput data on tumor characteristics from existing public databases. We analyzed and integrated these data to obtain novel insights regarding TNBC [12]. Previously, a genomic study of 158 TNBC patients (TCGA dataset) revealed that some TNBC tumors only exhibited a few genomic changes, while others contained hundreds of somatic mutations and showed the simultaneous activation of multiple signaling pathways. This suggests that a single drug or therapeutic agent would likely be ineffective against TNBC [13]. Further detailed RNA-seq showed that about 36% of mutations in TNBC were expressed, and the gene mutations were most commonly concentrated in TP53 (80%), followed by PIK3CA and PTEN.
The increased expression of CD8A in metastatic TNBC is not merely a
correlative finding. Instead, it has profound prognostic implications. Our
analysis revealed that higher CD8A expression levels are associated with
more favorable survival outcomes. This finding suggests CD8A could serve
as a valuable prognostic marker in TNBC. Hence, patients with elevated
CD8A expression may have a better prognosis, potentially due to the
heightened immune response against cancer cells in the metastatic
microenvironment. The presence of CD8A-expressing CD8
Immune evasion is a well-established strategy that allows tumors to survive and
spread in the body. In this context, the role of CD8A in recognizing and
eliminating cancer cells is quite important. Antigen-presenting cells present
cancer antigens to tumor-infiltrating CD8
Subgroup analysis can reveal the relationship between the number/proportion of
different subtypes of TIL and the prognosis of cancer patients. Hence, different
subgroups of TILs have different prognostic values for cancer. Of all the TIL
subgroups, tumor-infiltrating CD8
Consistent with these results, we found that CD8A is specifically expressed in CD8+ T cells within the TIME of primary BC. Moreover, high CD8A expression leads to better patient outcomes in BC, and CD8A expression is significantly related to PD1 expression, PD-L1 expression, and ICI. Subsequently, we explored the relationship between CD8A expression and genetic alterations in infiltrating immune cells, especially deep deletions. Examinations of a patient-derived xenograft model using scRNA-seq technology revealed that the temporal and spatial evolution of tumor genomes largely conforms to Darwinian evolutionary laws during in vivo BC progression. That is, the change in genome copy number is relatively stable even during the malignant transformation of BC cells. However, somatic mutations exhibit great heterogeneity during tumor development, and this acquired heterogeneity is even more pronounced in TNBC [24, 25].
In this study, we also analyzed the distribution of CD8A expression
using scRNA-seq data from a patient-derived TNBC xenograft tumor and matched lung
metastases model. The findings showed that CD8A expression is
down-regulated in BC lung metastases. These results suggest that high
CD8A expression in tumor-infiltrating CD8
The strength of this study lies in the combined transcriptome and scRNA-seq analysis of TNBC metastasis-associated biomarkers. Nevertheless, this study also has certain shortcomings. First, it was difficult to find a comparable BC metastasis transcriptome dataset for validation. Second, we only verified protein expression using clinical samples of primary and secondary BC lesions and did not establish an independent BC metastasis model to further clarify the role of CD8A in BC metastasis. Further experiments to clarify the mechanisms underlying the role of CD8A during BC metastasis are warranted.
Here, we comprehensively analyzed transcriptome and scRNA-seq datasets to identify the key genes involved in TNBC. Our results indicated that CD8A expression was correlated with BC metastasis, and a high level of CD8A expression was associated with improved survival outcomes in TNBC patients. Hence, CD8A might be a potential prognostic marker in TNBC and could also play a role in BC metastasis.
TNBC, triple-negative breast cancer; BC, breast cancer; DEGs, differentially expressed genes; scRNA-seq, single-cell RNA sequencing; TIME, tumor immune microenvironment; TILs, tumor-infiltrating lymphocytes; GEO, Gene Expression Omnibus; PPI, protein–protein interaction; TMB, tumor mutational burden; MSI, microsatellite instability; ICI, immune cell infiltration.
The corresponding author will provide the datasets upon reasonable request.
JC and ST drafted the manuscript and revised it critically and finally approved the version to be published; JC, ST, TL, and HF designed the study and analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
The study’s protocol was approved by the Affiliated Hospital of Nanjing University Medical School (protocol No. 2021220374).
Not applicable.
This research received no external funding.
The authors declare no conflict of interest.
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