Abstract
A checkpoint protein called the V-domain Ig suppressor of T cell activation (VISTA) is important for controlling immune responses. Immune cells that interact with VISTA have molecules, or receptors, known as VISTA receptors. Immune system activity can be modified by the interaction between VISTA and its receptors. Since targeting VISTA or its receptors may be beneficial in certain conditions, VISTA has been studied in relation to immunotherapy for cancer and autoimmune illnesses. The purpose of this study was to examine the expression levels and interactions between VISTA and its receptors, VSIG3 and PSGL-1, in breast cancer tissues. IHC analysis revealed higher levels of proteins within the VISTA/VSIG3/PSGL-1 axis in cancer tissues than in the reference samples (mastopathies). VISTA was found in breast cancer cells and intratumoral immune cells, with membranous and cytoplasmic staining patterns. VISTA was also linked with pathological grade and VSIG3 and PSGL-1 levels. Furthermore, we discovered that the knockdown of one axis member boosted the expression of the other partners. This highlights the significance of VISTA/VSIG3/PSGL-1 in tumor stroma and microenvironment remodeling. Our findings indicate the importance of the VISTA/VSIG3/PSGL-1 axis in the molecular biology of cancer cells and the immune microenvironment.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Breast cancer is one of the most common types of cancer worldwide. In 2020, an estimated 2.3 million new cases of breast cancer were diagnosed globally [1,2,3]. It is considered to be one of the main causes of cancer morbidity and mortality in women. Early detection, education, and advances in treatment have contributed to improved survival rates and outcomes for breast cancer patients. However, ongoing research and efforts to promote early detection and access to quality care remain important to address this serious health issue
The search for new cancer markers is a crucial element of cancer research, mostly because biomarkers may be identified in the blood, tissues, or other physiological fluids. Therefore, they can aid in the early detection of cancer, even before the appearance of symptoms. Early identification is frequently associated with better treatment results and greater survival rates [5, 10,11,12,13,14,15].
Targeting the VISTA protein, a V-domain Ig suppressor of T cell activation, is becoming increasingly important in cancer research because it has been proven to be an important immunological checkpoint and therapeutic target [16, 17]. A significant number of immune cells express VISTA, which is associated with the control of immunity by controlling the immune response, including inhibition of T cell activation [16,17,18,19,20,21,22]. Recent research findings highlight some of the key aspects of VISTA's involvement in immune regulation and its potential as a target for cancer therapy. The V-set and Immunoglobulin Domain-Containing Protein 3 (VSIG3) and the glycoprotein ligand P-selectin 1 (PSGL-1) bind VISTA, and the signal may be two-way [23,24,25]. At acidic pH, as in the tumor microenvironment (TME), VISTA binds to PSGL-1, but not at physiological pH.
VSIG3 is a member of the Ig superfamily expressed on a variety of non-hematopoietic cells, but its immunosuppressive activity has only recently been described [23,24,25]. The exact mechanism by which VSIG3 signaling suppresses tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocyte (TIL)-mediated responses remains unknown. Human VSIG3 is expressed in tumor cells, including gastric cancer cells and hepatocyte cells [29,30,31,32]. Although VSIG3 knockdown suppresses tumor growth in St-4 gastric cancer cells in vitro, the immunological role of VSIG3 in tumors in vivo has not been demonstrated [33]. Both VSIG3 and PSGL-1 interact with overlapping but distinct regions of VISTA, as shown by structural and mutational analyses.
P-selectin glycoprotein ligand-1 is associated with the immune system and cell adhesion. It is a significant factor in the recruitment and inflammation of leukocytes [34,35,36,37]. PSGL-1, a protein found on activated endothelial cells, is found on the surface of white blood cells (leukocytes) and interacts with P-selectin.
In conditions such as wound healing and tumors, where the pH can be 5.85–6.5, the role of PSGL-1 as an immune checkpoint has been clarified. Therefore, it is plausible that human PSGL-1 is a binding partner of human VISTA in tumors at a low pH [21, 25]. The role of PSGL-1 in antitumor immunity is still unknown, and there are limited data directly addressing its role. Although VISTA and PSGL-1 may function as both ligands and receptors, it remains to be determined whether the VISTA/PSGL-1 interaction occurs exclusively in trans or can also act in cis to inhibit activation [21, 38, 39]. The immunological activity of PSGL-1 is currently being reassessed, making PSGL-1 itself a potential therapeutic target.
VISTA activity causes myeloid and naïve mammalian T cells to become dormant while suppressing T cell activation and cytokine production. It may increase peripheral tolerance by increasing activation-induced T cell apoptosis [21, 22, 41, 42]. VISTA is preferentially upregulated in myeloid-derived suppressor cells (MDSCs) in response to hypoxia and may contribute to the immunosuppressive role of myeloid cells by reducing Toll-like receptor (TLR) signaling, inhibiting cell migration, and inducing myeloid reprogramming toward decreased myeloid cells [25, 43,44,45,46]. Furthermore, there may be a correlation between the overexpression of VISTA and the increased synthesis of the pro-inflammatory cytokines interleukin IL-6, tumor necrosis factor (TNF)-α, and IL-12, as well as increased production of IL-10 and other anti-inflammatory mediators [22, 47,48,49,50].
Antibodies against VISTA are being tested in clinical trials for the treatment of numerous malignancies; acid-targeting medicines in the TME can diminish the immunosuppressive action of acid tea and work well with VISTA therapy or checkpoint inhibition. Recent observations have shown that higher VISTA expression is related to better clinical results, whereas PD-L1 expression is associated with worse outcomes in patients with malignant cell carcinoma [17]. VISTA is mostly expressed on TME immune cells in most human malignancies and mouse models, while it has also been found in lung, kidney, and colorectal tumor cells, endometrial tissue, and human ovaries [19, 20, 43, 47, 51,52,53,54,55,56,57,58]. Tumor immunity is impacted by VISTA in both positive and negative ways. In a variety of cancer forms, VISTA functions as an inhibitory immunological checkpoint in addition to potentially serving as a stimulatory immune checkpoint. Since the precise process is yet unknown, it is essential to first determine VISTA's and its partners' potential as cancer diagnostic and predictive tools and to thoroughly define their mechanisms of action. Additionally, upregulation of VISTA in immune cells following immune checkpoint inhibition has been demonstrated in multiple studies [25, 59,60,61,62,63]. In light of this and the significance of VISTA expression on tumor and stromal cells, it seems that VISTA represents a promising target for therapy.
Therefore, this study aimed to evaluate the expression of VISTA and other immune checkpoint molecules in human breast cancer. Understanding the role of immune checkpoint molecules in cancer, including the proteins within the VISTA/VSIG3/PSGL-1 axis, may be crucial for developing effective immunotherapies and prognostic markers. Investigating whether VISTA’s axis expression levels are prognostic in breast cancer patients could provide valuable insights into disease progression and potential therapeutic strategies.
Materials and methods
Patients and clinical samples
The experiments were conducted using archival paraffin blocks of invasive ductal breast carcinomas (n = 284) and fibrocystic breast disease (FBD) mastopathy (n = 27) obtained during surgical resection at the Polish Mother's Memorial Hospital Research Institute in Lodz between 2010 and 2016. Paraffin slices of the malignant tissues were stained with hematoxylin and eosin (H&E) to ensure that immunohistochemistry (IHC) analyses were correct. Table 1 summarizes the clinical data obtained from the hospital archives.
The immunocytochemical expression of VISTA, VSIG3, PSGL-1, CD45, and CD68 was investigated using the ImmunoReactive Score (IRS) developed by Remmele and Stenger, which considers both the intensity of the reaction color (staining) and the proportion of positively stained cells (Table 2).
The final score ranged from 0 to 12. The Mann–Whitney U-test, the ANOVA Kruskal–Wallis test, and the Spearman’s test were used for statistical analysis. When p0.05, differences were considered statistically significant.
Cell lines
Breast carcinoma cell lines MCF-7, T-47D, MDA-MB-231 (obtained from the American Type Culture Collection ATCC, Manassas, VA, USA), SK-BR-3, BT-474 (from the Cell Lines Collection of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Science, Wroclaw, Poland), and MDA-MB-231/BO2 (courtesy of Dr. Philippe Clezardin), as well as ME16C normal breast epithelial cells (American Type Culture Collection ATCC), were used in our study. Breast cancer cell lines were grown in α-MEM supplemented with 10% fetal calf serum (FCS; Invitrogen Carlsbad, CA, USA), antibiotics, and 2 mM l-glutamine (Lonza, Basel, Switzerland). MEGM Bulletkit medium (Lonza, Basel, Switzerland) was used to cultivate Me16C cells. FBS (Sigma) was added to all the media at a final concentration of 10%. The cell lines were grown in 5% CO2 at 37 °C. The aggressiveness potential of each of the investigated cell lines was ranked from the lowest to the highest.
Immunohistochemistry (IHC)
Cancer and non-cancerous tissue samples were fixed in 10% buffered formalin and embedded in paraffin for immunohistochemistry (IHC) assays. Mouse polyclonal antibodies against VISTA (1:500; PA5-113459, Thermo Fisher Scientific), mouse monoclonal antibodies against VSIG3 (1:400, MA5-26624, Thermo Fisher Scientific), rabbit polyclonal antibodies against PSGL-1 (1:500, PA5-140244, Thermo Fisher Scientific), anti-CD45 mouse antibody cocktail (1:200, MA5-13197, Thermo Fisher Scientific), and anti-CD68 mouse monoclonal antibodies (1:200, 14-0688-82, Thermo Fisher Scientific) were used for the immunohistochemical examination of the studied markers. IHC was performed using Autostainer Link 48 (DakoCytomation, Glostrup, Denmark) to provide consistent and repeatable results.
RNA extraction, cDNA synthesis, and real-time PCR reactions
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s protocol. RT-qPCR was performed in 20 l volumes using a 7500 Real-time PCR System with iTaq Universal Probes Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Applied Biosystems also provided the following TaqMan-specific probes used in the experiment: Hs00735289_m1 for C10orf54 (VISTA), Hs00541322_m1 for IGSF11 (VSIG3), Hs00356602_m1 for SELPIG (PSGL-1), and Hs99999903_m1 for ACTB as a reference gene. All reactions were performed in triplicate under the following conditions: polymerase activation at 50 °C for 2 min, initial denaturation at 94 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 15 s, and annealing and elongation at 60 °C for 1 min. The Ct method was used to determine the relative mRNA expression of the markers.
SDS-PAGE and western blotting
Cell lines were lysed on ice in Cell Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a cocktail of inhibitors (Sigma, St. Louis, MO, USA), 250 units of Benzonase® (Merck Millipore, Bedford, MA, USA), and 2 mM phenylmethanesulfonyl fluoride (PMSF). Lysates containing 30 µg of total protein were combined with 4 × SDS-PAGE gel loading buffer (200 mM Tris–HCl (pH 6.8), 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol), loaded on 10% acrylamide gels, and separated by SDS-PAGE under reducing conditions before being transferred to PVDF membranes. Following protein transfer, the membranes were incubated for 1 h at room temperature in blocking solution (4% BSA in TBST buffer), followed by overnight incubation at 4 °C with anti-VISTA (1:500), anti-VSIG3 (1:400), and anti-PSGL-1 (1:500) antibodies. The membranes were then washed with TBST buffer and incubated for 1h at room temperature with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies, diluted 1:3000 (709-035-149 and 715035-150, respectively; Jackson ImmunoResearch, Mill Valley, CA, USA), and washed and treated with the Immun-Star HRP Chemiluminescence Kit (Bio-Rad). Rabbit anti-human β-actin monoclonal antibody (#4970; Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000, was used as the internal control. Western blotting results were analyzed using the ChemiDoc MP system (Bio-Rad).
siRNA transfection
Ambion pre-designed siRNAs, including GAPDH siRNA as a positive control and scrambled sequence siRNA as a negative control, were used in the experiments. The specific siRNAs used were as follows: s34467 for C10orf54, s12688 for SELPIG, and s45689 for IGSF11. MDA-MB-231 and T-47D cells were grown in 6-well plates, as previously described. The concentration of siRNA and quantity of transfection reagent were determined experimentally. Cells were trypsinized, centrifuged at 1000 rpm for 5 min at 4 °C, and resuspended in new media before transfection. Ambion's siPORT NeoFX (6µl/well) lipid-based transfection reagent and siRNAs (50 nM final concentration) were individually diluted in OptiMEM and mixed together. After 10 min, the transfection complexes were overlaid with 2 × 105 cells/well. VISTA/VSIG3/PSGL-1 silencing was verified.
Statistical analysis
The Shapiro–Wilk test was performed to assess the normality assumption of the groups investigated. The Wilcoxon signed-rank test was used to examine differences between the LSCC and NMLT groups. In addition, the Spearman correlation test was used to analyze the existing relationships. Prism 8.1.0 (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses. Results were considered statistically significant at p < 0.05.
Results
VISTA/VISG3/PSGL-1 axis is associated with breast tumor features
IHC analysis of the BC and FBD samples revealed that the expression levels of all the studied proteins were significantly elevated in breast cancer cells compared to mastopathy cases (Fig. 1). Membranous expression of VISTA was noticed in 275 (97.01%) IDC samples, compared to 11 (40.74%) in the FBD samples. Moreover, VISTA expression was observed in tumor-infiltrating immune cells (TIICs) in 252 (88.81%) cases of IDC. It is worth mentioning that 27 (9.51%) cases of IDC showed a stromal expression of the VISTA protein in cancer-associated fibroblasts (CAFs), as shown in Fig. 1C and M. VSIG3 showed a membranous expression pattern in 252 (88.81%) cases of IDC and in 36 (12.69%) TIIC areas of IDC cases, compared to 8 (29.63%) cases of FBD. PSGL-1 expression was noticed in 197 (69.40%) cases of IDC and in 207 (73.13%) TIIC regions of IDC cases, compared to 6 (22.22%) cases of FBD.
Using IHC, we determined the membranous localization and expression levels of VISTA, VSIG3, PSGL-1, CD45, and CD68 patterns in 27 cases of FBD and 284 cases of IDC. These values were the following: VISTA (IRS 8.66 ± 2.74 vs. 0.90 ± 1.90, p < 0.0001; Mann–Whitney test), VSIG3 (IRS 5.46 ± 3.39 vs. 0.92 ± 2.03, p < 0.0001; Mann–Whitney test), PSGL-1 (IRS 2.15 ± 2.28 vs. 0.48 ± 1.27, p < 0.0001; Mann–Whitney test), CD45 (IRS 3.07 ± 3.36 vs. 0.02 ± 0.09, p < 0.0001; Mann–Whitney test), and CD68 (IRS 1.92 ± 2.02 vs. 0.09 ± 0.01, p < 0.0001; Mann–Whitney test) (Fig. 2A).
A statistical analysis showed that VISTA shows higher expression levels when comparing the histological grades of IDC (G1 vs. G2, p = 0.02; Mann–Whitney test) and in the TIIC regions of IDC (G1 vs. G2, p = 0.009; G2 vs. G3, p = 0.04; and G1 vs. G3, p = 0.0002; Mann–Whitney test) (Fig. 2B). VSIG3 showed a statistically higher expression in histologically graded G2 cases compared to G1 ones (G1 vs. G2, p = 0.007; Mann–Whitney test) and in G3 vs. G1 cases (G1 vs. G3, p = 0.0087; Mann–Whitney test). The same pattern was noticed when analyzing TIIC regions of IDC samples: (G1 vs. G2, p = 0.043; and G1 vs. G3, p = 0.005; Mann–Whitney test) (Fig. 2.C).
In contrast to VISTA and VSIG3 expression patterns, PSGL-1 showed a decreasing expression trend associated with histopathological grading of IDC (G1 vs. G2, p < 0.0001; G2 vs. G3, p = 0.02; and G1 vs. G3, p < 0.0001; Mann–Whitney test) (Fig. 2D).
The Spearman correlation test revealed a high positive correlation between VISTAIDC and VISTATIIC (r = 0.51, p < 0.0001), VISTAIDC and VSIG3IDC (r = 0.53, p < 0.0001), VISTAIDC and VSIG3TIIC (r = 0.33, p < 0.0001), VISTAIDC and CD45 (r = 0.49, p < 0.0001), VISTAIDC and CD68 (r = 0.46, p < 0.0001), VISTATIIC and VSIG3IDC (r = 0.66, p < 0.0001), VISTATIIC and VSIG3TIIC (r = 0.43, p < 0.0001), VISTATIIC and CD45 (r = 0.31, p < 0.0001), VSIG3IDC and VSIG3TIIC (r = 0.61, p < 0.0001), VSIG3IDC and CD45 (r = 0.33, p < 0.0001), VSIG3IDC and CD68 (r = 0.29, p < 0.0001), PSGL-1IDC and PSGL-1TIIC (r = 0.36, p < 0.0001), PSGL-1IDC and CD45 (r = 0.32, p < 0.0001), and PSGL-1IDC and CD68 (r = 0.27, p < 0.0001), as showed in Fig. 3. Additionally, a statistical analysis showed a positive correlation between ER status and VISTAIDC (r = 0.23, p = 0.0071).
We used Kaplan–Meier to analyze the effect of the VISTA/VSIG3/PSGL-1 axis on the survival time of patients with IDC. We observed that higher overall VISTA expression, both in IDC and TIIC areas, had a positive impact on overall time compared to the groups with lower VISTA expression (VISTAIDC p = 0.0035, HR = 0.33, 95% CI = 0.1622–0.6997; VISTATIIC p = 0.0033, HR = 0.35, 95% CI = 0.1702–0.7014) (Fig. 4A, B).
A similar situation was noticed for VSIG3 expression in IDC samples, whereas elevated VSIG3 expression levels correlated with a better overall survival time for IDC patients (p = 0.0705, HR = 0.46, 95% CI 0.1992–0.9373) (Fig. 4C).
Moreover, CD45-positive regions of tumor-infiltrating lymphocytes (TILs) were identified in 125 (96.89%) samples, and 128 (99.22%) cases showed CD68-positive regions of tumor-associated macrophages (TAMs). The expression levels of CD45 and CD68, ranging from low to high, were observed in only 16 (12.40%) and 12 (09.30%) BC cases, respectively. Moreover, the Spearman correlation test revealed a strong positive correlation between hormone receptor status and the tumor features of BC cells and the CD45- and CD68-positive regions. CD45 correlations with CD68 (r = 0.84, p < 0.0001), ER (r = 0.40, p < 0.0001), PR (r = 0.34, p < 0.0001), HER2 (r = 0.34, p < 0.0001), and pN (r = 0.29, p < 0.001), but not pT (r = 0.10), were noticed. Similarly, CD68 correlated with ER (r = 0.33, p0.001), PR (r = 0.33, p0.001), HER2 (r = 0.30, p0.001), and pN (r = 0.27, p0.001), but not with pT (r = 0.08).
VISTA, VISG3, and PSGL-1 expression patterns vary among breast cancer cells
To evaluate the functional role of the VIST/VSIG3/PSGL-1 receptor pathway, its expression levels were examined in different human breast cancer cell lines, including MCF-7, BT-474, SK-BR-3, T-47D, MDA-MB-231, MDA-MB-231/BO2, and Me16C, as normal epithelial cell lines, as shown in Fig. 5. The increased aggressiveness of cancer cells leads to a significant increase in the expression levels of the studied transcripts and their protein products. These results indicate that the most elevated gene expression levels were present in the most aggressive cell lines, T-47D (C10orf54 p < 0.01, IGSF11 p < 0.0001, SELPLG p < 0.0001) and MDA-MB-231 (C10orf54 p < 0.0001, IGSF11 p < 0.0001, SELPLG p < 0.0001), compared to Me16C cells (Fig. 5).
Therefore, the T-47D and MDA-MB-231 cell lines were selected as optimal cell models for in vitro siRNA knockdown experiments. Protein level analysis using the WB technique may support the statement that the elevated expression of the VISTA, VSIG3, and PSGL-1 receptors increases with the aggressiveness of breast cancer cells. VSIG3 and PSGL-1 were visibly overexpressed in the T-47D (p < 0.0001, p < 0.0001) and MDA-MB-231 (p < 0.0001, p < 0.0001) cell lines compared to the Me16C epithelial cell line (Fig. 5E–F). A similar trend in the rising expression levels of VISTA protein was observed in the most aggressive breast cancer cell lines (Fig. 5D).
siRNA-related knockdown shows crosstalk in VISTA/VSIG3/PSGL-1 axis
To evaluate the effect of siRNA-mediated silencing on the genes of the VISTA/VSIG3/PSGL-1 axis, specific siRNAs were transfected into the MDA-MB-231 and T-47D breast cancer cell lines for 24 h and 48 h. The efficacy of this transfection was measured using RT-qPCR and Western blotting. As shown in Fig. 6, we observed a statistically significant downregulation of all the silenced genes during the 24 h and 48 h time periods.
The downregulation of the VISTA gene in MDA-MB-231 cells resulted in overexpression of VSIG-3 after 24 h and 48 h (p < 0.001 and p < 0.0001, respectively) and a higher expression of PSGL-1 after 48 h (p < 0.0001) compared to the control cells. At the same time, in T-47D cells, overexpression of PSGL-1 after 24 h (p < 0.0001) and VSIG3 (p < 0.0001) after 48 h was observed. However, overexpression of VSIG3 after 24 h (p < 0.0001) and PSGL-1 after 48 h (p < 0.0001) was not observed. The downregulation of VSIG3 resulted in the overexpression of VISTA and PSGL-1 after 24 h in MDA-MB-231 cells. Similarly, in T-47D cells, we observed overexpression of VISTA (p < 0.1) and PSGL-1 (p < 0.0001) mRNAs after 24 h and overexpression of only VISTA (p < 0.0001) after 48 h. siRNA specific for the PSGL-1 transcript resulted in a significant reduction in the expression levels of VISTA (p < 0.01) and overexpression of VSIG3 (p < 0.0001) after 24 h. After 48 h, overexpression of both VISTA (p < 0.0001) and VSIG3 (p < 0.0001) was observed.
Figure 7 illustrates that mRNA expression levels correlated with protein abundance in the MDA-MB-231 cell line after 48 h of gene knockdown. We observed statistically significant overexpression of PSGL-1 (p < 0.001) protein after VISTA knockdown, significant downregulation of VISTA (p < 0.0001) and PSGL-1 (p < 0.0001) expression after VSIG3 silencing, and a decrease in VSIG3 (p < 0.0001) after PSGL-1 inhibition.
Discussion
The VISTA, VSIG-3, and PSGL-1 proteins are responsible for the regulation of the immune system and immune cells, as has been well documented. A recent study found that the binding interactions between VISTA and PSGL-1 are pH-dependent, and VISTA was identified as a binding partner for VSIG3 (also known as IGSF11) in two independent protein interaction studies [21, 23, 64]. To date, VISTA, VSIG-3, and PSGL-1 have not shown any well-established direct interactions or known relationships in the context of cancer. In recent years, the direct binding capacity of these proteins has been demonstrated in mouse antibody models using an SG7 inhibitor [65]. To our knowledge, this is the first study demonstrating the significance of the VISTA/VSIG3/PSGL-1 axis in human cancer cells. According to our findings, VISTA/VSIG3/SPGL-1 proteins are expressed in breast cancer cells as well as in lymphocytes (CD45 +) and macrophages (CD68 +), both of which are immune cells that infiltrate tumors. An increase in breast cancer cell aggressiveness correlates with an increase in the levels of proteins within the VISTA/VSIG3/PSGL-1 axis, as we have observed. These receptor levels are simultaneously overexpressed both in the most aggressive types of BC and on the surfaces of immune system cells. Notably, stromal expression of the VISTA receptor was observed in nearly 10% of the studied IDC cases. The importance of this discovery has yet to be described because of the lack of sufficient data [66,67,68]. Nevertheless, further studies are needed to determine the role of the VISTA/VSIG3/PSGL-1 axis in the tumor microenvironment and BC progression. The specificity of the IHC reaction shows that only very adjacent stromal cells are VISTA-positive, which may implicate their role in crosstalk between cancer cells, tumor-infiltrating immune cells (TAMs and TILs), and the extracellular matrix.
Results from both our own and previously published research on human breast cancer indicate that poorly differentiated BC cells have higher rates of VISTA expression. Since poorly differentiated tumors frequently contain cancer stem cells, it is reasonable to report that high VISTA expression in patients with high proportions of cancer stem cells likely serves as a mechanism of immune evasion and resistance to immunotherapy. Cancer stem cells are self-renewing cells with a high potential for tumorigenicity that reside in specific tumor microenvironment niches. Within this microenvironment, cancer stem cells are thought to be one of the main causes of immunosuppression. Few studies have been conducted in this field. Nevertheless, one important molecular explanation for the detrimental effect of immune checkpoint on cancer immunotherapy is the increase in the percentage of cancer stem cells and their interaction with cells inside the tumor’s immune microenvironment and inflammatory infiltrate. Immune checkpoint protein targeting may be viewed as an innovative tactic that enhances standard immune checkpoint therapy in reviving the antitumor side of tumor immunity, decreasing tumor recurrence, and producing long-lasting effects.
These findings highlight the significance of tumor stroma and microenvironment remodeling in cancer progression. The effective manipulation of all stromal components, for example, the extracellular matrix, fibroblasts, endothelial cells, and immune system cells, remains at the root of successful cancer evolution. Many components, such as innate and adaptive immune cells, play many functions during cancer progression and can either promote or suppress tumor formation, depending on local and systemic factors. Our findings strongly support the assertion that the expression of VISTA/VSIG3/PSGL-1 in both cancerous and immune cells may play a key role in the interactions between BC cells and their microenvironment. Changes in the expression levels of these receptor genes following siRNA knockdown further illustrate how easily and freely cancer cells may modify their gene expression processes to help them survive in their niche. This could be due to the fact that VISTA is a co-inhibitory molecule that lowers T-cell-mediated immunity while promoting immune escape. Our findings support the relevance of the VISTA/VSIG3/PSGL-1 axis as a biological target for immunotherapy and a predictive biomarker in breast cancer. Interrelationships and linkages between VISTA/VSIG3/PSGL-1 were observed at the RNA and protein levels at the same time.
VISTA is an immunological checkpoint protein, and its overexpression, together with increased levels of the VSIG3 and PSGL-1 receptors in the tumor microenvironment, may be involved in the modulation of immune responses to cancer. There is growing evidence of the importance of the immunosuppressive role of VISTA in cancer progression [20, 41, 57, 62, 63, 69,70,71,72,73]. The presence of VISTA on the surface of cancer cells can not only modulate immune cells but also potentially play a much broader role in cancer biology. Targeting multiple immune checkpoints may be a turning point in cancer therapy. Therefore, in this study, we investigated the crosstalk between VISTA and its receptors, VSIG3 and PSGL-1. Our research revealed a strong correlation between VISTA expression and PSGL-1 and VSIG-3 in IDC samples, indicating that this axis functions in concert with immune cells to promote the advancement of BC. In cases of breast cancer, inhibiting the VISTA pathway could improve antitumor immunity. When combined with other treatments, such as chemotherapy and radiotherapy, inhibition of the VISTA pathway could effectively limit tumor growth and reduce inflammation, which is helpful in the management of autoimmune disorders.
Nevertheless, further investigation of the role of VISTA in breast cancer and its potential as a therapeutic target may provide useful insights into the development of novel treatment options for this disease.
Conclusions
The current study has demonstrated that the expression patterns of the VISTA/VSIG3/PSGL-1 axis were increased in infiltrating breast cancer cells and immune cells. To the best of our knowledge, this is the first study to show direct crosstalk between these immunological receptors, resulting in greater adaptation of tumor cells to their microenvironment. The knockdown of one of the receptors by siRNA resulted in an increase in other investigated genes, indicating the relevance of the VISTA/VSIG3/PSGL-1 axis in breast cancer progression. Despite our important findings, there remains a significant amount of work yet to be accomplished in this field to demonstrate the entire molecular basis of tumor microenvironmental mechanisms, as well as the role of VISTA receptors and their partners in immune regulation and cell interactions in human malignancies.
Data availability
All data associated with this study are present in the manuscript. Materials used in this study are available from the corresponding author upon reasonable request.
References
Giaquinto AN et al (2022) Breast cancer statistics, 2022. CA Cancer J Clin 72(6):524–541
Huang Y et al (2023) Global trends in BRCA-related breast cancer research from 2013 to 2022: a scientometric analysis. Front Oncol 13:1197168
Luftner D et al (2023) Update breast cancer 2022 part 6—advanced-stage breast cancer. Geburtshilfe Frauenheilkd 83(3):299–309
Arzanova E, Mayrovitz HN (2022) The epidemiology of breast cancer. HN Mayrovitz (eds) In Breast Cancer. Brisbane (AU).
Go YM et al (2023) Exposome epidemiology for suspect environmental chemical exposures during pregnancy linked to subsequent breast cancer diagnosis. Environ Int 178:108112
Goel N et al (2023) Translational epidemiology: genetic ancestry in breast cancer: what is the role of genetic ancestry and socioeconomic status in triple-negative breast cancer? Adv Surg 57(1):1–14
Karim AM et al (2023) Triple-negative breast cancer: epidemiology, molecular mechanisms, and modern vaccine-based treatment strategies. Biochem Pharmacol 212:115545
Muller V et al (2023) Epidemiology, clinical outcomes, and unmet needs of patients with human epidermal growth factor receptor 2-positive breast cancer and brain metastases: a systematic literature review. Cancer Treat Rev 115:102527
Qian X et al (2023) Epidemiology and clinicopathologic features of breast cancer in China and the United States. Transl Cancer Res 12(7):1826–1835
(2022) Liquid biopsy prototype assay for breast cancer. Cancer 128(22):3905.
Duque G et al (2022) Cancer biomarkers in liquid biopsy for early detection of breast cancer: a systematic review. Clin Med Insights Oncol 16:11795549221134832
Holler A et al (2023) Diagnostic and prognostic biomarkers of luminal breast cancer: where are we now? Breast Cancer (Dove Med Press) 15:525–540
Sivakumar S et al (2022) Tissue and liquid biopsy profiling reveal convergent tumor evolution and therapy evasion in breast cancer. Nat Commun 13(1):7495
Nejatipour Z et al (2023) Looking for biomarkers in interferon response pathway to predict response to oncolytic HSV-1 in breast cancer: an ex vivo study. Cancer Biomark.
Neves Rebello Alves L et al (2023) Biomarkers in breast cancer: an old story with a new end. Genes (Basel) 14(7).
Ait Boujmia OK (2021) V-domain Ig suppressor of T cell activation (VISTA) inhibition is a new approach to cancer therapy: a bibliometric study. Naunyn Schmiedebergs Arch Pharmacol 394(6):1057–1065
Cao X et al (2020) VISTA expression on immune cells correlates with favorable prognosis in patients with triple-negative breast cancer. Front Oncol 10:583966
Blando J et al (2019) Comparison of immune infiltrates in melanoma and pancreatic cancer highlights VISTA as a potential target in pancreatic cancer. Proc Natl Acad Sci USA 116(5):1692–1697
Hou Z et al (2021) Prognostic significance and therapeutic potential of the immune checkpoint VISTA in pancreatic cancer. J Cancer Res Clin Oncol 147(2):517–531
Jlassi A et al (2023) VISTA+/CD8+ status correlates with favorable prognosis in Epithelial ovarian cancer. PLoS ONE 18(3):e0278849
Johnston RJ et al (2019) VISTA is an acidic pH-selective ligand for PSGL-1. Nature 574(7779):565–570
Kuklinski LF et al (2018) VISTA expression on tumor-infiltrating inflammatory cells in primary cutaneous melanoma correlates with poor disease-specific survival. Cancer Immunol Immunother 67(7):1113–1121
Wang J et al (2019) VSIG-3 as a ligand of VISTA inhibits human T-cell function. Immunology 156(1):74–85
Xie X et al (2021) Structural basis of VSIG3: the ligand for VISTA. Front Immunol 12:625808
Yuan L et al (2021) VISTA: a mediator of quiescence and a promising target in cancer immunotherapy. Trends Immunol 42(3):209–227
Kim H et al (2020) CD44 can compensate for IgSF11 deficiency by associating with the scaffold protein PSD-95 during osteoclast differentiation. Int J Mol Sci 21(7).
Kim H et al (2020) IgSF11 regulates osteoclast differentiation through association with the scaffold protein PSD-95. Bone Res 8:5
Tang XY et al (2022) IGSF11 and VISTA: a pair of promising immune checkpoints in tumor immunotherapy. Biomark Res 10(1):49
Eom DS et al (2012) Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11. PLoS Genet 8(8):e1002899
Ghouzlani A et al (2020) The promising IgSF11 immune checkpoint is highly expressed in advanced human gliomas and associates to poor prognosis. Front Oncol 10:608609
Hayano Y et al (2021) IgSF11 homophilic adhesion proteins promote layer-specific synaptic assembly of the cortical interneuron subtype. Sci Adv 7(29).
Jang S et al (2016) Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity. Nat Neurosci 19(1):84–93
Watanabe T et al (2005) Identification of immunoglobulin superfamily 11 (IGSF11) as a novel target for cancer immunotherapy of gastrointestinal and hepatocellular carcinomas. Cancer Sci 96(8):498–506
(2020) Expression of Concern: inflammatory molecule, PSGL-1, deficiency activates macrophages to promote colorectal cancer growth through NFkappaB signaling. Mol Cancer Res 18(6):939.
(2020) Correction: Inflammatory molecule, PSGL-1, deficiency activates macrophages to promote colorectal cancer growth through NFkappaB signaling. Mol Cancer Res 18(6):938.
DeRogatis JM et al (2021) PSGL-1 immune checkpoint inhibition for CD4(+) T cell cancer immunotherapy. Front Immunol 12:636238
Handa K et al (1995) P-selectin-dependent adhesion of human cancer-cells - requirement for coexpression of a psgl-1-like core protein and the glycosylation process for sialosyl-le(x) or sialosyl-le(a). Int J Oncol 6(4):773–781
Kappelmayer J, Nagy B Jr (2017) The interaction of selectins and PSGL-1 as a key component in thrombus formation and cancer progression. Biomed Res Int 2017:6138145
Li J et al (2022) Retraction: inflammatory molecule, PSGL-1, deficiency activates macrophages to promote colorectal cancer growth through NFkappaB signaling. Mol Cancer Res 20(7):1178
Li J et al (2017) Inflammatory molecule, PSGL-1, deficiency activates macrophages to promote colorectal cancer growth through NFkappaB signaling. Mol Cancer Res 15(4):467–477
Li N et al (2023) Chemotherapy induces immune checkpoint VISTA expression in tumor cells via HIF-2alpha. Biochem Pharmacol 210:115492
Ma S et al (2022) The expression of VISTA on CD4+ T cells associate with poor prognosis and immune status in non-small cell lung cancer patients. Bosn J Basic Med Sci 22(5):707–715
Pan J et al (2021) Inhibition of lung tumorigenesis by a small molecule CA170 targeting the immune checkpoint protein VISTA. Commun Biol 4(1):906
Pilones KA et al (2020) Converging focal radiation and immunotherapy in a preclinical model of triple negative breast cancer: contribution of VISTA blockade. Oncoimmunology 9(1):1830524
Thakkar D et al (2022) Rationally targeted anti-VISTA antibody that blockades the C-C' loop region can reverse VISTA immune suppression and remodel the immune microenvironment to potently inhibit tumor growth in an Fc independent manner. J Immunother Cancer 10(2).
Yum JI, Hong YK (2021) Terminating cancer by blocking VISTA as a novel immunotherapy: hasta la vista, baby. Front Oncol 11:658488
Deng J et al (2019) Hypoxia-induced VISTA promotes the suppressive function of myeloid-derived suppressor cells in the tumor microenvironment. Cancer Immunol Res 7(7):1079–1090
Hosseinkhani N et al (2021) The role of V-domain Ig suppressor of T cell activation (VISTA) in cancer therapy: lessons learned and the road ahead. Front Immunol 12:676181
Lines JL et al (2014) VISTA is a novel broad-spectrum negative checkpoint regulator for cancer immunotherapy. Cancer Immunol Res 2(6):510–517
Mo J et al (2023) Targeting STAT3-VISTA axis to suppress tumor aggression and burden in acute myeloid leukemia. J Hematol Oncol 16(1):15
Akdogan O et al (2022) Effect of neoadjuvant therapy on tumor tissue PD-L1 and VISTA expression levels in non-small-cell lung cancer. Immunotherapy 14(14):1121–1131
Boger C et al (2017) The novel negative checkpoint regulator VISTA is expressed in gastric carcinoma and associated with PD-L1/PD-1: a future perspective for a combined gastric cancer therapy? Oncoimmunology 6(4):e1293215
Christenson RH et al (2011) Analytical performance and clinical concordance of the cancer biomarkers CA 15–3, CA 19–9, CA 125 II, Carcinoembryonic Antigen, and Alpha-Fetoprotein on the Dimension Vista(R) System. Clin Biochem 44(13):1128–1136
Li L et al (2021) Expression and clinicopathological significance of Foxp3 and VISTA in cervical cancer. Am J Transl Res 13(9):10428–10438
Li W et al (2023) Immune inactivation by VISTA predicts clinical outcome and therapeutic benefit in muscle-invasive bladder cancer. BMC Cancer 23(1):661
Mortezaee K, Majidpoor J, Najafi S (2022) VISTA immune regulatory effects in bypassing cancer immunotherapy: updated. Life Sci 310:121083
Rezouki I et al (2023) High VISTA expression is linked to a potent epithelial-mesenchymal transition and is positively correlated with PD1 in breast cancer. Front Oncol 13:1154631
Tagliamento M et al (2021) VISTA: A promising target for cancer immunotherapy? Immunotargets Ther 10:185–200
Huang X et al (2020) VISTA: an immune regulatory protein checking tumor and immune cells in cancer immunotherapy. J Hematol Oncol 13(1):83
Tagliamento M, Bironzo P, Novello S (2020) New emerging targets in cancer immunotherapy: the role of VISTA. ESMO Open 4(Suppl 3):e000683
Long L et al (2022) Different clinical significance of novel B7 family checkpoints VISTA and HHLA2 in human lung adenocarcinoma. Immunotherapy 14(6):419–431
Sener GY et al (2023) Comparison of PD-L1 and VISTA expression status in primary and recurrent/refractory tissue after (chemo)radiotherapy in head and neck cancer. Strahlenther Onkol 199(8):761–772
Zapala L et al (2023) Immune checkpoint receptor VISTA on immune cells is associated with expression of T-cell exhaustion marker TOX and worse prognosis in renal cell carcinoma with venous tumor thrombus. J Cancer Res Clin Oncol 149(7):4131–4139
Yang W et al (2017) Construction of a versatile expression library for all human single-pass transmembrane proteins for receptor pairings by high throughput screening. J Biotechnol 260:18–30
Mehta N et al (2020) An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA. Sci Rep 10(1):15171
Joshi RS et al (2021) The role of cancer-associated fibroblasts in tumor progression. Cancers (Basel) 13(6).
Kharaishvili G et al (2014) The role of cancer-associated fibroblasts, solid stress and other microenvironmental factors in tumor progression and therapy resistance. Cancer Cell Int 14:41
Rosenbaum SR et al (2020) FOXD3 regulates VISTA expression in melanoma. Cell Rep 30(2):510–524
Rezagholizadeh F et al (2024) Unraveling the potential of CD8, CD68, and VISTA as diagnostic and prognostic markers in patients with pancreatic ductal adenocarcinoma. Front Immunol 15:1283364
Iadonato S et al (2024) Corrigendum: a highly potent anti-VISTA antibody KVA12123 - a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors. Front Immunol 15:1365240
Hosseinkhani N et al (2024) Dual silencing of tumor-intrinsic VISTA and CTLA-4 stimulates T-cell mediated immune responses and inhibits MCF7 breast cancer development. Gene 896:148043
Digomann D et al (2023) VISTA ligation reduces antitumor T-cell activity in pancreatic cancer. Cancers (Basel) 15(8).
Shekari N et al (2023) VISTA and its ligands: the next generation of promising therapeutic targets in immunotherapy. Cancer Cell Int 23(1):265
Acknowledgements
The authors would like to thank Agnieszka Baranska, Elżbieta Polejko, and Bozena Przygocka for their technical support with histological slide preparation. We would like to express our sincere gratitude to Francisco Jose Vare Alvarez for his exceptional editorial work.
Funding
This research was funded by the Wroclaw Medical University (SUBK.A352.22.017) under the title "Modulation of the immune response by immunoglobulin suppressor T-cell activation domain V (VISTA) and its partners PSGL-1 and VSIG3 as potential diagnostic and predictive biomarkers in invasive ductal breast carcinoma.” Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Bioethics Committee of Wroclaw Medical University (No. 411/2023N).
Author information
Authors and Affiliations
Contributions
M.O. contributed to conceptualization, writing—original draft, methodology, visualization, formal analysis, supervision, resources, funding acquisition; M.O., B.S., and H.R. contributed to data curation; M.O., M.M., and A.P. contributed to investigation; M.O., A.P., and M.M contributed to writing—review and editing. All authors have read and agreed to the published version of the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Informed consent
Informed consent for histopathological examination with further research using the collected tumor material was obtained from all subjects involved in this study.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Olbromski, M., Mrozowska, M., Piotrowska, A. et al. The VISTA/VSIG3/PSGL-1 axis: crosstalk between immune effector cells and cancer cells in invasive ductal breast carcinoma. Cancer Immunol Immunother 73, 136 (2024). https://doi.org/10.1007/s00262-024-03701-w
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00262-024-03701-w