CXCL10-related T lymphocyte infiltration and breast cancer metastasis are regulated by CXCL12

doi:10.21203/rs.3.rs-4686409/v1

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CXCL10-related T lymphocyte infiltration and breast cancer metastasis are regulated by CXCL12

https://doi.org/10.21203/rs.3.rs-4686409/v1

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Background

Breast cancer (BC) is a malignant tumor with the highest incidence rate in women. This work explored the function of CXCL10 and CXCL12 in tumor metastasis.

Methods

CXCL12 and CXCL10 protein expression levels were assessed by IHC in breast cancer tissues. String revealed that CXCL12 interacts with CXCL10 protein molecules. The GEPIA2 database revealed that CXCL12 was negatively correlated with CXCL10. CXCL12. The effects of CXCL12 on invasion and migration were detected by scratch and transwell experiments in breast cancer cells. CD4 + T and CD8 + T cells in the inflammatory microenvironment of breast cancer patients were evaluated with the NGDC database and verified by IHC.

Results

CXCL12 knockdown inhibited migration and invasion and enhanced the expression and secretion of CXCL10 in BC. CXCL10 is responsible for the recruitment of CD4 + and CD8 + T lymphocytes into tumors and enhances antitumor effects. The single-cell data showed that the patients in the CXCL10 + CD4+/CD8 + T-cell group and the CXCL12-CD4+/CD8 + T-cell group had better prognoses.

Conclusions

CXCL12 promoted BC migration and invasion. On the other hand, CXCL12 inhibited the expression and secretion of CXCL10, further inhibiting T lymphocyte infiltration and promoting breast cancer metastasis in the TME.

Breast cancer

CXCL10

CXCL12

T lymphocytes

TME

The International Agency for Research on Cancer (IARC) declared that the incidence of female patients with breast cancer surpassed that with lung cancer as the most commonly diagnosed cancer, with an estimated 2.3 million new cases (11.7%) in 2020[1]. The 5-year relative survival rate of patients with distant metastasis has decreased significantly to 28%[2]. Metastasis is the process by which cancer cells successfully evade immune surveillance[3]. Cancer patients often exhibit weakened immunity in the middle and later stages[4], and T cells are exhausted in tumors[5]. This case provides conditions for cancer cells to spread from the primary tumor to distant organs. Ninety percent of cancer deaths are related to tumor metastasis [6]. The immune system plays an important role in monitoring and killing malignant tumors[7]. With tumor occurrence, the pathological tissue generates an inflammatory microenvironment, which continuously secretes related chemokines to recruit immune cells for aggregation[8]. In addition to macrophages, CD4 + and CD8 + T lymphocytes are common effector cells. The degree of infiltration reflects the body's antitumor immune response status[9].

Chemokines play an important role in immune cell recruitment in the inflammatory tumor microenvironment. More than 44 ligands have been identified in the human genome[10]. C-X-C chemokine receptor type 10 (CXCL10) is a member of the chemokine family[11]. CXCL10 is highly expressed in breast tumors and affects adaptive immunity, angiogenesis, inflammation and hematopoiesis. CXC chemokine receptor-3 (CXCR3) is an interferon-inducible chemokine receptor. As the receptor of CXCL10, CXCR3 has three splice variants, CXCR3-A, CXCR3-B and CXCR3-Alt[12]. Therefore, CXCL10 has a dual function of promoting or inhibiting tumors in tumor cells, depending on the type of CXCR3 receptor[13]. CXCL10 promotes tumor cell proliferation and antiapoptotic effects via CXCR3-A[14]. CXCL10 can directly inhibit tumor cell proliferation via CXCR3-B [15]. CXCR3 plays a crucial role in immunity and inflammation[16]. It is predominantly expressed by a variety of immune cells, including T helper cells, cytotoxic T lymphocytes, dendritic cells, macrophages, natural killer cells and infiltrating lymphocytes[17, 18]. CXCL10 can recruit CXCR3 + T lymphocytes to immune cells, thereby exerting antitumor effects. Tumors with high expression of CXCL10 have a good prognosis [19]. CXCR3 + Tregs can be recruited by CXCL10 in the tumor microenvironment, leading to immunosuppressive and tumor-promoting effects [20]. CXCL10 affects the tumor microenvironment in a paracrine way and plays a role in tumor progression and metastasis[20]. In summary, the promoting or inhibitory effect of CXCL10 on tumor immunity is related to the balance between CXCR3 + T lymphocytes and CXCR3 + Tregs[21]. This work revealed a negative correlation between CXCL12 and CXCL10. The CXCL12/CXCR4 axis is involved in tumor progression, angiogenesis, metastasis, and survival[22]. The CXCL12/CXCR4 signaling pathway has emerged as a potential therapeutic target [23]. In pancreatic cancer, CXCR4/CXCL12 can reduce the ability of T cells to enter the TME [24]. Pancreatic cancer cells evade cancer immune attack[25].

The infiltration of immune cells into solid tumors, their movement within the tumor microenvironment (TME), and interactions with other immune cells are controlled by their directed migration toward gradients of chemokines. Dysregulated chemokine signaling in the TME favors the growth of tumors, exclusion of effector immune cells, and an increase in the abundance of immunosuppressive cells[26]. This work explored the balance between CXCL12 and CXCL10 in the BC microenvironment, as well as their impact on CD4 + T and CD8 + T-cell infiltration. The purpose of this work was to enhance T-cell infiltration and antitumor activity and to provide a basis for clinical treatment.

Cell culture

The MCF-10A cell line (ATCC, CRL-10317) was cultured with MEGM™ Mammary Epithelial Cell Growth Medium and incubated at 37°C in a humidified 5% CO₂ atmosphere. The MDA-MB-231 cell line (ATCC, CRM-HTB-26) was cultured in DMEM (Gibco, 12491015) supplemented with 10% FBS (Gibco, 10091148) and 100 U/ml penicillin/streptomycin (Gibco, 15140122) and incubated at 37°C in a humidified 5% CO₂ atmosphere.

Immunohistochemical assay

Tissue sections were obtained, dewaxed, rehydrated, and incubated with 3% hydrogen peroxide to eliminate endogenous peroxidase activity for 15 min at room temperature. The tissue antigen was retrieved with 0.01 M sodium citrate buffer (pH 6.0). The slides were incubated with the primary antibody at 4°C overnight. Then, the slides were washed three times with PBS. The slides were incubated with the secondary antibody at room temperature for 2 h. Then, the slides were washed three times with PBS. These tissue sections were stained with DAB and imaged by microscopy. The primary antibodies used were anti-CXCL10 (Abcam, ab306587), anti-CXCL12 (Invitrogen, MA5-23759), anti-CD4 (Abcam, ab133616) and anti-CD8 (Abcam, ab4055). The secondary antibody used was goat anti-rabbit IgG H&L (HRP) (Abcam, ab97080).

Western blot analysis

The cells were collected and split to extract plasma and membrane proteins. Proteins were denatured in boiling water for 10 min. Proteins were separated by SDS‒PAGE and transferred to PVDF membranes. The PVDF membranes were blocked in 5% skim milk for 1 hour. Then, the PVDF membranes were incubated with primary antibodies overnight at 4°C. The PVDF membranes were washed three times with TBST and incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Then, the PVDF membranes were washed three times with TBST and imaged with a chemiluminescence instrument.

ELISA

MDA-MB-231 cells (1×10⁴ cells per well) were seeded into 96-well plates. After reaching the indicated time points, the culture supernatants were analyzed with an ELISA kit, including a human CXCL10 ELISA kit (R&D Systems, QK266). The experiment was performed according to the manufacturer’s instructions. The OD values at 450 nm were read with a Multiskan™ FC microplate photometer (Thermo Scientific, 1410101).

Q-PCR assay

Total RNA was extracted with TRIzol solution (Invitrogen, 15596026). Reverse transcription of cDNA was performed with HiScript QRT SuperMix for qPCR (+ gDNA wiper) according to the manufacturer’s instructions (Vazyme, R123-01). qPCR was performed in triplicate with AceQ qPCR SYBR Green Mastermix on an ABI VII7 ABI/viia7 real-time fluorescence quantitative PCR instrument. The following qPCR primers were used: CXCL10, 5′-AACCTCCAGTCTCAGCACCATGAA-3′ (forward) and 5′-AGGTACAGCGTAA GGTTCTAGAGAG-3′ (reverse); CXCL12, 5′-TGCCAGAGCCAACGTCAAG-3′ (forward) and 5′-CAGCCGGGCTACAATCTGAA-3′ (reverse); and GAPDH, 5’-GAGTCAA CGGATTTGGTCGT-3’ (forward) and 5’-TTGATTTTGGAGGGATCTCG-3’ (reverse).

Woundhealing assay

MDA-MB-231 cells were seeded in a 24-well plate and grown to 90% confluence to form a confluent monolayer. A wound was formed by dragging the tip of the pipette. Cell migration images were captured at 0 and 24 h by microscopy, and the cell pixel areas were calculated at 0 and 24 h.

Migration area = 24 h-cell pixel area − 0 h-cell pixel area.

Transwell assay

MDA-MB-231 cells were seeded into the transwell upper chamber at 5000 cells/well and cultured for 24 h. The upper chamber was washed with PBS. The migrating cells were fixed with methanol for 30 min, and 0.1% crystal violet was added for 20 min. Finally, the cells were viewed under a microscope, photographed, and counted.

Statistical analysis

R 3.4.3 and GraphPad Prism 8.01 (GraphPad Software, La Jolla, CA, USA) were used for our statistical analyses. Categorical variables were evaluated using Fisher's exact test or the χ2 test. For continuous variables, paired-samples Student's t test was utilized, and for multiple groups of continuous variables, one-way ANOVA was used. Genes linked to overall survival were identified by calculating hazard ratios (HRs) with 95% confidence intervals (CIs) based on univariate and multivariate Cox regression models, which were used to measure survival. For statistical significance, a cutoff of P < 0.05 was utilized.

Expression level of CXCL10 in breast cancer

Analysis of the CanerSEA database revealed that CXCL10 is associated with inflammation in the tumor microenvironment (TME) (Fig. 1A). The TIMER2.0 database revealed that CXCL10 was upregulated in breast cancer. Compared with LumA and LumB, CXCL10 was more highly expressed in basal-like and Her2 breast cancer (Fig. 1B). The volcano plots showed that CXCL10 expression was upregulated (Fig. 1C). Analysis of the TIMER2.0 database revealed that patients with high expression of CXCL10 had a better survival prognosis (Fig. 1D). The immunohistochemistry results confirmed that CXCL10 was upregulated in breast cancer tissues (Fig. 1E). CXCL10 expression increased as malignant progression progressed (Fig. 1F).

The expression level of CXCL10 in different cell types

Then, the single-cell GSE148673 dataset was analyzed with CellMarker 2.0, and 16 major cell types were identified (Fig. 2A). The information exchange relationships between 16 types of cells were analyzed with NGDC (Fig. 2B). Here, we focused on the interactions between breast cancer cells and 15 other types of cells (Fig. 2C). CXCL10 was highly expressed in macrophages, breast cancer cells and monocytes and expressed at low levels in different types of CD4 + and CD8 + T cells (Fig. 2D-E).

The interaction between CXCL10 and CXCL12

The String database revealed that CXCL10 strongly interacts with CCL13, CXCL6, PF4V1, and CXCL12 (Fig. 3A). CXCL10 expression was positively correlated with that of CCL13 (R = 0.19) (Fig. 3B) but not with that of CXCL6 or PF4V1 (Fig. 3C-D). In addition, it showed a negative correlation with CXCL12 (R=-0.12) (Fig. 3E). Furthermore, the GEPIA2 results showed that the four interacting proteins were expressed at low levels in breast cancer tissue, but CXCL12 was significantly differentially expressed (Fig. 3F-I). Western blot analysis revealed that CXCL10 was highly expressed in breast cancer cells, while CXCL12 was expressed at low levels in breast cancer cells (Fig. 3E).

The expression level of CXCL12 in different cell types

Analysis of the TIMER2.0 database revealed that CXCL12 is expressed at low levels in different types of breast cancer (Fig. 4A). Bioinformatics analysis confirmed that CXCL12 expression was downregulated in breast cancer (Fig. 4B). Analysis of the GEPIA2 database revealed that CXCL12 expression decreased with the progression of breast cancer (Fig. 4C). On the survival prognosis side, patients with high CXCL12 expression had a poorer survival prognosis, while patients with low CXCL12 expression had a better survival prognosis (Fig. 4D). The IHC results showed that CXCL12 was downregulated in breast cancer. Analysis of the NGDC database revealed that CXCL12 was expressed at low levels in breast cancer cells, CD4 + T cells, and CD8 + T cells and was highly expressed in fibroblasts and macrophages (Fig. 4F-G).

Effects of CXCL12 on TNBC cell migration and invasion

To explore the role of CXCL12 in breast cancer. In this study, a CXCL12 knockdown vector, which has an effective knockdown efficiency, was constructed (Fig. 5A). With CXCL12 knockdown, the migration and invasion ability of MDA-MB-231 cells were reduced (Fig. 5B-E), but the expression and secretion of CXCL10 were increased (Fig. 5F-G).

CD4 + and CD8 + T-cell-related immune infiltration is affected by CXCL10 and CXCL12 in terms of survival prognosis

Analysis of the Cancer SEA database revealed that low expression of CXCL12 and high expression of CXCL10 are closely related to inflammation (Fig. 6A). CXCL12 is highly expressed in CD4 + EPICs, CD4 + nonregulatory T cells (QUANTISEQ), CD4 + memory resting-CIBERSORT T cells and CD4 + memory resting-CIBERSORT-ABS T cells and has low expression in other CD4 + T-cell types. CXCL10 is weakly expressed in CD4 + T cells (nonregulatory), QUANTISEQ T cells, CD4 + native CIBERSORT T cells, CD4 + native CIBERSORT-ABS T cells, CD4 + memory resting CIBERSORT T cells, and CD4 + memory resting CIBERSORT-ABS T cells but is highly expressed in other CD4 + T-cell types. CXCL12 is expressed at low levels in CD8 + T cells. CXCL10 was highly expressed in CD8 + T cells (Fig. 6B). On the survival prognosis side, patients with low CXCL10 expression (CXCL10-) and low CD8 + T cells (CD8 + T) had the worst prognosis. Patients with high CXCL10 expression (CXCL10+) and CD8 + T status had a better prognosis, while patients with CXCL10 + and high CD8 + T-cell status (CD8 + T) had the best prognosis. Similarly, patients with CXCL10- and low CD4 + T cells (CD4 + T cells) had the worst prognosis. Patients with CXCL10- and high CD4 + T cells (CD4 + T) have a favorable prognosis. Patients with CXCL10 + and CD4 + T cells had a good prognosis, while those with CXCL10 + and CD4 + T cells had a better prognosis. The prognosis of patients with high CXCL12 expression (CXCL12+) and CD8 + T status was the worst, especially in the first 120 months. Patients with CXCL12 + and CD8 + T cells have a worse prognosis. Patients with low CXCL12 expression (CXCL12-) and CD8 + T status had a worse prognosis, while patients with CXCL12- and CD8 + T status had a relatively good prognosis. Patients with CXCL12 + and CD4 + T status had the worst prognosis, while patients with CXCL12 + and CD4 + T status had a poor prognosis. Patients with CXCL12- and CD4 + T cells had a poor prognosis, while patients with CXCL12- and CD4 + T cells had a worse prognosis (Fig. 6C-F). Furthermore, CD4 + T cells and CD8 + T cells in the tissue were detected by immunohistochemistry. The results showed significant enrichment of CD4 + T cells and CD8 + T cells in the tumor tissue (Fig. 6G).

CXCL10, an important molecule that regulates immune cell activity, plays a crucial role in the tumor immune microenvironment[27]. CXCL10 participates in the regulation of the inflammatory microenvironment [28]in tumor tissue. CXCL10 was upregulated in four types of breast cancer tissues: basal, Her2E, LumA and LumB. At the same time, the immunohistochemical results showed that CXCL10 was highly expressed in breast cancer tissues. CXCL10 expression was correlated with the survival rate and TNM classification of tumor patients[29]. Patients with high expression of CXCL10 had a better survival prognosis, while patients with low expression had a poor survival prognosis. It has been concluded that the immune system recruits T lymphocytes by secreting CXCL10 and enhances antitumor ability in the process of malignant progression of breast cancer[30]. When the immune system is disrupted, CXCL10 secretion is reduced, and the number of immune cells is decreased in the late stage. Breast cancer metastasizes more often in the late stage[31]. These data indicate that CXCL10 could be utilized as a biomarker to predict the prognosis of patients with breast cancer.

To describe the tumor microenvironment of breast cancer more clearly, single-cell data from the GSE148673 dataset were analyzed with CellMarker 2.0[32]. The 16 major cell types are displayed. The crosstalk between breast cancer cells and 15 other types of cells was analyzed by NGDC[33]. The results showed that various types of cells interact with each other in the TME in a very complex relationship. The expression of CXCL10 in different types of cells was further analyzed. CXCL10 is highly expressed in macrophages, breast cancer cells and monocytes[34] and expressed at low levels in different types of CD4 + and CD8 + T cells[35]. CXCL10 is secreted by breast cancer tissues, recruits CXCL10 CD4 + and CD8 + T cells into tumors via CXCR3 receptors, and enhances antitumor effects[29]. GEPIA2 revealed that CXCL10 was negatively correlated with CCL12. The western blot results also showed that CXCL10 was overexpressed in MDA-MB-231 cells, while CXCL12 was expressed at low levels. Analysis of the TIMER2.0 database revealed that CXCL12 was expressed at low levels in different types of breast cancer. CXCL12 expression decreases with the clinical progression of breast cancer[36]. Patients with high CXCL12 expression had poorer survival, while those with low CXCL12 expression had better prognosis[37]. IHC showed that CXCL12 expression was downregulated in breast cancer. These data revealed that the incidence of breast cancer was negatively correlated with tumor progression. The single-cell data showed that CXCL12 was expressed at low levels in breast cancer cells, CD4 + T cells and CD8 + T cells but was highly expressed in fibroblasts and macrophages. CXCL12 levels in tumor tissues are associated with fibroblasts and macrophages[38]. The data showed that CXCL12 expression was downregulated in breast cancer, but CXCL12 expression was still high, as shown in Fig. 4A. Therefore, a CXCL12 knockdown vector was constructed to explore the function of CXCL12 in breast cancer. With CXCL12 knockdown in MDA-MB-231 cells, migration and invasion were reduced[39], while CXCL10 expression and secretion were increased. These data showed that low levels of CXCL12 can inhibit the metastasis of breast cancer and enhance the chemotaxis of T cells by promoting the secretion of CXCL10. Analysis of the Cancer SEA database revealed that low expression of CXCL12 and high expression of CXCL10 were closely related to inflammation. Among most T-cell types, CXCL12 is expressed at low levels, and CXCL10 is highly expressed. Patients in the CXCL10 low gene expression + low T-cell CD8+/CD4 + group had the worst clinical prognosis, and patients in the CXCL10 high gene expression + high T-cell CD8+/CD4 + group had a better clinical prognosis. The data revealed that high levels of CXCL10 and T lymphocytes play a role in antitumor effects[30]. Patients in the CXCL12 high gene expression + low T-cell CD8+/CD4 + group had a poor clinical prognosis, and patients in the CXCL12 low gene expression + high T-cell CD8+/CD4 + group had a good clinical prognosis. The data revealed that low levels of CXCL12 were beneficial for exerting antitumor effects. Furthermore, CD4 + T cells and CD8 + T cells in the tissue were detected by immunohistochemistry. The results showed that CD4 + T cells and CD8 + T cells were significantly enriched in tumor tissue. These data proved that invasion and metastasis were effectively inhibited by CXCL12 knockdown in breast cancer and that the expression and secretion of CXCL10 were increased. CXCL10 promoted CD4 + and CD8 + T lymphocyte infiltration, further enhanced antitumor activity, and improved patient survival and prognosis.

In conclusion, CXCL10 can be produced by antigen-presenting cells (dendritic cells or macrophages) and by tumor cells. The continuous expression of CXCL10 could recruit more CD4 + T cells and CD8 + T cells into the TME to kill tumor cells. The chemokine CXCL12 is expressed in tumors and is correlated with poor patient survival. CXCL12 is involved in driving migration and invasion in BC and inhibits CXCL10 expression and secretion in the TME, further inhibiting T lymphocyte infiltration and promoting breast cancer metastasis (Fig. 7). As described above, CXCL12 intervention combined with CXCL10 overexpression may provide a rationale for achieving better therapeutic strategies for BC patients.

Acknowledgments

The authors gratefully acknowledge the funds of the Key Project of Anhui Provincial Department of Education (No. KJ2019A0341). Key natural science project of Bengbu Medical University (No.2023byzd067). We acknowledge the TCGA, GEPIA2, CanerSEA, CellMarker 2.0, NGDC and TIMER2.0 databases for free use.

Ethical Statements:

not applicable

Author contributions

Conceptualization, X.L. (Xianfu Liu) and X.Z. (Xiaojing Zhang); methodology, X.L. (Xianfu Liu) and Y.C. (Yansong Chen); software, X.L. (Xianfu Liu) and J.T. (Jingwei Tang); validation, X.L. (Xianfu Liu), J.T. (Jingwei Tang) and H.Z. (Hao Zhang); formal analysis, X.L. (Xianfu Liu); investigation, X.L. (Xianfu Liu); resources, X.L. (Xianfu Liu); data curation, X.L. (Xianfu Liu); writing—original draft preparation, X.L. (Xianfu Liu) and X.Z. (Xiaojing Zhang); writing—review and editing, X.Z. (Xiaojing Zhang); project administration, X.L. (Xianfu Liu) and X.Z. (Xiaojing Zhang); funding acquisition, X.L. (Xianfu Liu). All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

The authors have no conflicts of interest to declare.

Ethics approval

This cell line-based study was exempt from ethics approval.

Informed consent

All authors contributed significantly to the work and are in agreement with the content of the manuscript. All the authors gave their consent to the publication of this manuscript.

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CXCL10-related T lymphocyte infiltration and breast cancer metastasis are regulated by CXCL12

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Cell culture

Immunohistochemical assay

Western blot analysis

ELISA

Q-PCR assay

Woundhealing assay

Transwell assay

Statistical analysis

Results

Expression level of CXCL10 in breast cancer

The expression level of CXCL10 in different cell types

The interaction between CXCL10 and CXCL12

The expression level of CXCL12 in different cell types

Effects of CXCL12 on TNBC cell migration and invasion

Discussion

Declarations

References

Additional Declarations

Status:

Version 1