Cellular Membrane Localization of Innate Immune Checkpoint Molecule CD47 Is Regulated by Radixin in Human Pancreatic Ductal Adenocarcinoma Cells

In the past decade, immune checkpoint inhibitors have exhibited potent antitumor efficacy against multiple solid malignancies but limited efficacy against pancreatic ductal adenocarcinoma (PDAC). Cluster of differentiation (CD) 47, a member of the immunoglobulin G superfamily, is overexpressed in the surface membrane of PDAC and independently correlates with a worse clinical prognosis. Furthermore, CD47 functions as a dominant macrophage checkpoint, providing a potent “do not eat me” signal to enable cancer cells to evade the innate immune system. Thus, the blockade of CD47 is a promising immunotherapeutic strategy for PDAC. In this study, we determined whether ezrin/radixin/moesin (ERM) family members, which post-translationally modulate the cellular membrane localization of numerous transmembrane proteins by crosslinking with the actin cytoskeleton, contribute to the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC. Immunofluorescence analysis showed that CD47 and ezrin/radixin were highly co-localized in the plasma membrane. Interestingly, gene silencing of radixin but not ezrin dramatically decreased the cell surface expression of CD47 but had little effects on its mRNA level. Furthermore, CD47 and radixin interacted with each other, as determined by a co-immunoprecipitation assay. In conclusion, radixin regulates the cellular membrane localization of CD47 as a scaffold protein in KP-2 cells.


Introduction
Pancreatic ductal adenocarcinoma (PDAC), which accounts for approximately 90% of all pancreatic tumors, is one of the most devastating gastrointestinal malignancies with the highest mortality rate [1]. Most patients are diagnosed at an advanced stage with a metastatic state and respond poorly to current chemotherapy [2][3][4]. In the past decade, immune checkpoint blockade antibodies (Abs) targeting immune checkpoint molecules such as programmed death-1 (PD-1) and PD-ligand 1 (PD-L1) have exhibited potent antitumor efficacy against multiple solid malignancies [5][6][7]. However, immune checkpoint inhibitors have limited efficacy against PDAC [8][9][10]. Therefore, there is an urgent need to develop novel therapeutic strategies against PDAC.
Cluster of differentiation (CD) 47, a member of the immunoglobulin (Ig) G superfamily, is a transmembrane protein widely expressed in most normal and tumor tissues [11][12][13]. CD47 functions as a ligand for signal regulatory protein α (SIRPα) expressed on the cellular surface of innate immune cells including macrophages [14], and behaves as a dominant macrophage checkpoint [15][16][17], thereby providing a potent "do not eat me" signal to the macrophages that enables cancer cells to evade immunosurveillance from the innate immune system [15][16][17]. While lower expression levels of CD47 are observed in normal tissues, almost all cancer cells, including PDAC, overexpress CD47, which is independently correlated with a worse clinical prognosis in a wide variety of malignant tumors [15][16][17][18].
Thus, blocking CD47 signaling is a promising immunotherapeutic strategy for diverse cancer cell types.
The ezrin/radixin/moesin (ERM) family members are the three closely related proteins that post-translationally modulate the cellular membrane localization of numerous cancerrelated transmembrane proteins by crosslinking with the actin cytoskeleton [19][20][21][22]. Our laboratory recently demonstrated that the ERM family post-translationally modulates the cellular surface localization of PD-L1, which belongs to a group of IgG superfamilies similar to CD47, by serving as a scaffold protein in several cancer cell types, including PDAC [23][24][25][26][27]. However, it is unclear whether the ERM family is involved in the cellular membrane localization of CD47 through post-translational modifications in cancer cells. In this study, we determined the role of ERM family members in the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC.

siRNA Treatment
Transfection of siRNAs was conducted as previously described by Kobori et al. [24]. Briefly, the siRNAs targeting human genes of interest and non-targeting control (NC) were transfected into KP-2 cells at a concentration of 5 nM using Lipofectamine RNAiMAX at a volume of 0.20 µL/1.0 × 10 4 cells, followed by continuous cultivation for four days. All the reagents used for siRNA treatment were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

RT-PCR
Total RNA extraction from KP-2 cells and real-time reverse transcription (RT)-polymerase chain reaction (PCR) were performed as previously described by Kobori et al. [24]. A list of the sequences of the gene-specific PCR primers (TaKaRa Bio, Kusatsu, Japan) used is given in Table S1.

Immunoblotting
Preparation of whole cell lysates from KP-2 cells and Western blotting were carried out as previously described by Kobori et al. [24]. The source and dilution ratio of all the primary and secondary Abs used and the original immunoblots are shown in Table S2 and Figure S1, respectively.

Double Immunofluorescence Staining and Confocal Laser Scanning Microscopy
Double immunofluorescence staining and confocal laser scanning microscopy (CLSM) analysis were performed as previously described [23,24], with some modifications. The source and dilution ration of all the primary and secondary Abs used are listed in Table S2. To quantify the colocalization ratio of CD47 with ezrin and radixin, the coefficients of Pearson's correlation and Mander's overlap were calculated from the two-or three-dimensional reconstructed images using the NIS-Elements Ar Analysis software (Nikon Instrument, Tokyo, Japan). Figure S2 shows negative control staining in KP-2 cells using each respective secondary Ab conjugated with Alexa Fluor 488 or 594 without primary Abs.

Co-Immunoprecipitation
Co-immunoprecipitation assays were conducted as previously described by Kobori et al. [23,24], with some modifications. The source and dilution ratio of all the  Table S2 and Figure S1, respectively.

Flow Cytometry
The cell surface expression levels of CD47 in KP-2 cells were measured using flow cytometry as previously described [24,25], with some modifications, using allophycocyanin (APC)-conjugated anti-CD47 Ab (323124; Bio Legend, San Diego, CA, USA) and an EC800 Flow Cytometry Analyzer and EC800 software (Sony Imaging Products and Solutions, Tokyo, Japan). Gene expression of CD47 and ezrin/radixin in tumor tissues derived from patients with PDAC and normal tissues was assessed using the GEPIA database [29]. The database is a freely available interactive web server used for analyzing the RNA-sequencing (seq) expression data of 9736 tumors and 8587 normal tissues based on data from The Cancer Genome Atlas (TCGA) [30] and the Genotype-Tissue Expression (GTEx) project [31,32].

Analysis for Gene-Level Correlation with Survival Rate of Patients with PDAC Using UALCAN
The relationship between the prognosis of patients with PDAC and the relative mRNA expression of CD47 and ezrin/radixin levels in the tumor tissues was assessed with UALCAN, an interactive web source. This allows the assessment of the relationship between gene expression levels of interest and survival probability for patients with over 20,000 protein-coding genes in 33 different tumor types using RNA-seq data obtained from the TCGA project [33,34].

Statistical Analysis
Data are presented as the mean ± standard error of the mean (SEM) or the boxand-whisker plots with boxes indicating the median as well as the values of 25% and 75% quartiles and whiskers representing the range of values with jitter plots. Statistical comparisons between two and multiple groups were performed with a one-way analysis of variance (ANOVA) using GEPIA and ANOVA followed by Dunnett's test using Prism version 3 (GraphPad Software, La Jolla, CA, USA), respectively. Correlation analysis was performed with Pearson's correlation coefficient. Differences were considered statistically significant at p < 0.05.

Gene and Protein Expression of CD47 and the ERM Family in KP-2 Cells
We checked the mRNA expressions of CD47 and ERM family members in 50 human pancreatic adenocarcinoma cell lines registered in the public databases of the Cancer Cell Line Encyclopedia (CCLE) and Cancer Dependency Map (DepMap) portal [35][36][37]. Database analysis indicated that the mRNA expressions of CD47, ezrin, and radixin were detected in KP-2 cells, while that of moesin was located near the limit of detection (Figure 1a), which is consistent with real-time RT-PCR data ( Figure S3) and our previous observations [24]. Similarly, Western blot analysis revealed the presence of CD47, ezrin, and radixin proteins in KP-2 cells. In contrast, moesin was undetected in KP-2 cells, whereas it was detected in HeLa cells (Figure 1b), in which moesin is normally expressed at the mRNA and protein levels [25,38,39]. Because several studies on clinical human PDAC tissues and freshly isolated primary human PDAC revealed the presence of ezrin and radixin, but not moesin, at the mRNA and protein levels [40][41][42][43], we selected KP-2 cells in which the expression profiles of the ERM family are similar to those found in human PDAC tissues and freshly isolated primary human PDAC.
it was detected in HeLa cells (Figure 1b), in which moesin is normally expressed at the mRNA and protein levels [25,38,39]. Because several studies on clinical human PDAC tissues and freshly isolated primary human PDAC revealed the presence of ezrin and radixin, but not moesin, at the mRNA and protein levels [40][41][42][43], we selected KP-2 cells in which the expression profiles of the ERM family are similar to those found in human PDAC tissues and freshly isolated primary human PDAC.

Colocalization of CD47 with Ezrin and Radixin in KP-2 Cells
The counterstaining of CD47 with F-actin, a typical plasma membrane marker, implies that in KP-2 cells, CD47 was principally localized in the cellular membrane (Figure

Colocalization of CD47 with Ezrin and Radixin in KP-2 Cells
The counterstaining of CD47 with F-actin, a typical plasma membrane marker, implies that in KP-2 cells, CD47 was principally localized in the cellular membrane ( Figure S4). Double immunofluorescence staining showed that in KP-2 cells, CD47 was highly colocalized with ezrin (the coefficient of Pearson's correlation and Mander's overlap were 0.93 and 0.94, respectively) and radixin (the coefficient of Pearson's correlation and Mander's overlap were both 0.94) in the plasma membrane ( Figure 2). On the other hand, negative control staining showed no fluorescence signal ( Figure S2). S4). Double immunofluorescence staining showed that in KP-2 cells, CD47 was highly colocalized with ezrin (the coefficient of Pearson's correlation and Mander's overlap were 0.93 and 0.94, respectively) and radixin (the coefficient of Pearson's correlation and Mander's overlap were both 0.94) in the plasma membrane ( Figure 2). On the other hand, negative control staining showed no fluorescence signal ( Figure S2).

Knockdown Activity of Small Interfering (si) RNAs against Ezrin and Radixin in KP-2 Cells
We checked the inhibitory potency of small interfering (si) RNAs against ezrin and radixin on each target at mRNA and protein levels in KP-2 cells. Exposure of KP-2 cells to siRNAs against ezrin and radixin markedly diminished both the mRNA and protein expression levels of their respective targets (Figure 3), with little influence on cell viability ( Figure S5).

Knockdown Activity of Small Interfering (si) RNAs against Ezrin and Radixin in KP-2 Cells
We checked the inhibitory potency of small interfering (si) RNAs against ezrin and radixin on each target at mRNA and protein levels in KP-2 cells. Exposure of KP-2 cells to siRNAs against ezrin and radixin markedly diminished both the mRNA and protein expression levels of their respective targets (Figure 3), with little influence on cell viability ( Figure S5).

siRNA-Mediated Knockdown of Radixin Decreased Cell Surface Expression Levels of CD47 in KP-2 Cells
We determined whether ezrin and radixin gene silencing affected the mRNA and cell surface membrane expression of CD47 in KP-2 cells. siRNA against ezrin had little influence on the mRNA and cell surface membrane expression of CD47 in KP-2 cells (Figure 4). How-ever, radixin gene silencing significantly decreased the cell surface membrane expression of CD47 with little impact on its mRNA expression (Figure 4).

siRNA-Mediated Knockdown of Radixin Decreased Cell Surface Expression Levels of CD47 in KP-2 Cells
We determined whether ezrin and radixin gene silencing affected the mRNA and cell surface membrane expression of CD47 in KP-2 cells. siRNA against ezrin had little influence on the mRNA and cell surface membrane expression of CD47 in KP-2 cells ( Figure  4). However, radixin gene silencing significantly decreased the cell surface membrane expression of CD47 with little impact on its mRNA expression (Figure 4).

Molecular Interactions between CD47 and Radixin in KP-2 Cells
We investigated whether CD47 interacts with radixin in KP-2 cells using co-immunoprecipitation experiments. Radixin protein was detected in the whole-cell lysates (input) of KP-2 cells and immunoprecipitates with an anti-CD47 Ab but not with an isotype-matched control IgG Ab (Figure 5a). Similarly, reverse immunoprecipitation with an anti-radixin Ab detected the protein expression of CD47 but not with an isotype-matched control IgG Ab ( Figure S6), suggesting an intrinsic protein-protein interaction between CD47 and radixin in KP-2 cells (Figure 5b).

Gene Expression Analysis of CD47 and Ezrin/Radixin in Clinical PDAC Tissue
We assessed CD47 and ezrin/radixin gene expression levels in clinical tissue samples derived from PDAC tumors and normal tissues. The relative mRNA expression levels of CD47 and ezrin in PDAC tumors were significantly higher than those in normal tissues. Additionally, the relative mRNA expression levels of radixin in PDAC tumors were moderately higher than in normal tissues ( Figure 6).

Molecular Interactions between CD47 and Radixin in KP-2 Cells
We investigated whether CD47 interacts with radixin in KP-2 cells using co-immunoprecipitation experiments. Radixin protein was detected in the whole-cell lysates (input) of KP-2 cells and immunoprecipitates with an anti-CD47 Ab but not with an isotypematched control IgG Ab (Figure 5a). Similarly, reverse immunoprecipitation with an antiradixin Ab detected the protein expression of CD47 but not with an isotype-matched control IgG Ab ( Figure S6), suggesting an intrinsic protein-protein interaction between CD47 and radixin in KP-2 cells (Figure 5b).

Correlation Analysis of CD47 and Ezrin/Radixin Gene Expression Levels in Tumor Tissues with Survival Probability in Patients with PDAC
Finally, we assessed the relationship between the gene expression levels of CD47 and ezrin/radixin in tumor tissues derived from patients with PDAC and their survival probabilities based on the TCGA database using UALCAN, a comprehensive and interactive web resource for in-depth analysis of cancer OMICS data [33,34]. The survival probability in PDAC patients with higher expression of CD47 and radixin was not significant but substantially lower (p = 0.093 and p = 0.071, respectively) than in those with low/medium expression of CD47 and radixin, respectively (Figure 7a-c). To further clarify the relationship between CD47 and ezrin/radixin mRNA expressions in clinical PDAC tissues, we employed a gene correlation analysis of CD47 with ezrin and radixin in PDAC. Gene correlation analysis revealed a significant positive correlation between CD47 and radixin (Pearson's correlation coefficient: 0.43) (Figure 7d). In contrast, there was no correlation between CD47 and ezrin.

Gene Expression Analysis of CD47 and Ezrin/Radixin in Clinical PDAC Tissue.
We assessed CD47 and ezrin/radixin gene expression levels in clinical tissue samples derived from PDAC tumors and normal tissues. The relative mRNA expression levels of CD47 and ezrin in PDAC tumors were significantly higher than those in normal tissues. Additionally, the relative mRNA expression levels of radixin in PDAC tumors were moderately higher than in normal tissues ( Figure 6).

Correlation Analysis of CD47 and Ezrin/Radixin Gene Expression Levels in Tumor Tissues with Survival Probability in Patients with PDAC
Finally, we assessed the relationship between the gene expression levels of CD47 and ezrin/radixin in tumor tissues derived from patients with PDAC and their survival proba-

Discussion
In this study, we confirmed that KP-2 cells derived from human PDAC had sufficient expression of CD47, ezrin, and radixin at both the mRNA and protein levels, while displaying a lack of moesin (Figure 1; Figure S3). Previous studies have found that CD47 is highly expressed in clinical PDAC samples and numerous human PDAC cell lines [18,[44][45][46][47]. Furthermore, several studies on clinical human PDAC tissues and freshly isolated primary human PDAC have revealed the presence of ezrin and radixin, but not moesin, at the mRNA and protein levels [40][41][42][43], implying that KP-2 cells have expression profiles similar to those of the ERM family found in patients with PDAC. Furthermore, the confocal laser scanning microscopy (CLSM) analysis provided evidence for the first time that CD47 was highly colocalized with ezrin and radixin in the surface membrane of KP-2 cells (Figure 2). Altogether, KP-2 cells have a expression profile of CD47 and ERM analogous to that found in human clinical PDAC tissues and primary human PDAC, offering an optimal experimental model for examining the involvement of the ERM family in the cellular membrane localization of CD47 in human PDAC.
CD47 is a transmembrane protein commonly overexpressed in the surface membrane of multiple cancer cells to protect them from phagocytosis upon direct interaction with SIRPα expressed on the macrophage surface membrane [48]. A growing body of evidence has demonstrated that CD47 expression is endogenously modulated by many cellular

Discussion
In this study, we confirmed that KP-2 cells derived from human PDAC had sufficient expression of CD47, ezrin, and radixin at both the mRNA and protein levels, while displaying a lack of moesin (Figure 1; Figure S3). Previous studies have found that CD47 is highly expressed in clinical PDAC samples and numerous human PDAC cell lines [18,[44][45][46][47]. Furthermore, several studies on clinical human PDAC tissues and freshly isolated primary human PDAC have revealed the presence of ezrin and radixin, but not moesin, at the mRNA and protein levels [40][41][42][43], implying that KP-2 cells have expression profiles similar to those of the ERM family found in patients with PDAC. Furthermore, the confocal laser scanning microscopy (CLSM) analysis provided evidence for the first time that CD47 was highly colocalized with ezrin and radixin in the surface membrane of KP-2 cells (Figure 2). Altogether, KP-2 cells have a expression profile of CD47 and ERM analogous to that found in human clinical PDAC tissues and primary human PDAC, offering an optimal experimental model for examining the involvement of the ERM family in the cellular membrane localization of CD47 in human PDAC.
CD47 is a transmembrane protein commonly overexpressed in the surface membrane of multiple cancer cells to protect them from phagocytosis upon direct interaction with SIRPα expressed on the macrophage surface membrane [48]. A growing body of evidence has demonstrated that CD47 expression is endogenously modulated by many cellular events, including transcriptional, translational, and post-translational modifications such as glycosylation and ubiquitination [48,49]. However, few studies have focused on the post-translational modification process of CD47, although it can affect its cellular membrane localization and functionality [49]. Members of the ERM family have recently emerged as key regulators of numerous cancer-related transmembrane proteins involved in cellular surface membrane localization and functional activity through post-translational modifications [19,26,50]. Importantly, we have demonstrated that the ERM family posttranslationally regulate the cellular membrane localization of PD-L1, whose structure is similar to that of CD47, through molecular interactions, presumably by working as scaffold proteins in several cancer cell types, including PDAC [23][24][25][26][27]51]. Our recent study identified radixin as the predominant scaffold protein that modulates the cellular membrane localization of PD-L1 in KP-2 cells [24]. In this study, RNA interference-mediated knockdown of radixin but not ezrin significantly decreased the cell surface expression levels of CD47 without changes in its mRNA level in KP-2 cells (Figure 4). Furthermore, co-immunoprecipitation experiments revealed intrinsic molecular interactions between radixin and CD47 ( Figure 5). These findings imply that in KP-2 cells, radixin may primarily contribute to the cellular membrane localization of CD47 as a predominant scaffold protein through protein-protein interactions ( Figure 5). These data are further supported by the lower survival probabilities of patients with PDAC with higher expression of CD47 and radixin in clinical PDAC tissues, where a positive correlation between CD47 and radixin was observed (Figures 6 and 7). Differences in the ERM proteins responsible for the cellular membrane localization of CD47 may be, at least in part, due to the divergent expression profiles of ERM depending on cancer cell type. In addition, evidence indicates that radixin plays a key role in the plasma membrane localization of certain drug transporters, including multidrug resistance protein 2 (MRP-2), possibly because radixin is predominant among ERM proteins in hepatic tissues and cells [52][53][54][55][56][57]. Hence, radixin may principally regulate the cellular membrane localization of CD47 in KP-2 cells as a dominant scaffold protein, as is the case with MRP-2 in hepatocytes. However, this issue has yet to be elucidated and should be addressed in future studies.
In summary, we observed cellular membrane co-localization and protein-protein interactions between CD47 and radixin in KP-2 cells. Furthermore, radixin may be the predominant scaffold protein responsible for the cellular membrane localization of CD47 in KP-2 cells, possibly through post-translational modifications. Thus, modulating CD47 expressions in the cellular membrane of PDAC by therapeutic agents targeting radixin such as siRNA may be an attractive immunotherapeutic strategy independent of the PD-1/PD-L1 axis. This novel macrophage checkpoint blockade therapy through its scaffold protein might offer possible therapeutic application for PDAC patients, the majority of which does not benefit from the current immune checkpoint blockade therapies.