Ezrin and Radixin Differentially Modulate Cell Surface Expression of Programmed Death Ligand-1 in Human Pancreatic Ductal Adenocarcinoma KP-2 Cells

Round 1

Reviewer 1 Report

In the manuscript titled “Ezrin and Radixin Differentially Modulate Cell Surface Expression of Programmed Death Ligand-1 in Human Pancreatic Ductal Adenocarcinoma KP-2 Cells” Kobori et al. by doing Immunoprecipitation assay, western blotting, flow cytometry, confocal laser scanning microscopy, and real-time RT-qPCR attempted to find the expression and subcellular localization of PD-L1 and ERM, to know their role in PDAC using KP-2 cells. The authors discovered that both ezrin and radixin are highly colocalized and interact with PD-L1; ezrin modulates the cell-surface expression of PD-L1 whereas radixin serves as an essential scaffold protein for crosslinking between PD-L1 and the actin cytoskeleton. The study is interesting and important as it could assist in discovering new Immune checkpoint blockade therapies for the treatment of PDAC. I recommend the publication of this work in Immuno in its current form.

Author Response

We would like to thank #Reviewer 1 for the greatest evaluation on our manuscript. Thank you for your time.

Reviewer 2 Report

In the manuscript entitled ''Ezrin and Radixin Differentially Modulate Cell Surface Expression of Programmed Death Ligand-1 in Human Pancreatic Ductal Adenocarcinoma KP-2 Cells'' the authors examine the role of ezrin/radixin/moesin (ERM) family scaffold proteins on the surface localisation of PD-L1 in KP-2 cells, a human PDAC cell line. Since the authors have already published 3 additional papers on the same topic but using different cancer cell lines, I do not find the results of this study novel enough, in order to be published in this journal. Therefore, I reject this manuscript.

Author Response

Response to Reviewer 2 Comments

 

We would like to thank #Reviewer 2 for the meaningful comments on our manuscript. To improve the novelty of this study, we have revised our manuscript as follows with refer to Editor’s suggestions.

 

Modification 1.

We have modified and added following sentence to discuss more detail about the differences in the regulatory mechanisms of PD-L1 expression among three distinct cancer types we have previously reported and to emphasize the differences and the specific character of KP-2 cells when compared to the three distinct cancer cell types in the Discussions.

 

Discussions (Line 383 – 394)

We recently reported that ezrin functions as a scaffold protein for PD-L1, which leads to the cell surface localisation of PD-L1 in human cervical adenocarcinoma HeLa cells and human choriocarcinoma JEG-3 cells, both of which have the highest expression level of ezrin among ERM protein based on the database analysis utilizing DepMap and/or our experimental data [29, 30]. Furthermore, we have found that in human colon adenocarcinoma LS180 cells, ezrin and radixin contributed equally to the cell surface localisation of PD-L1 via physiological interaction and colocalisation with PD-L1, despite extremely low expression level of radixin in LS180 cells [31]. By contrast, another group has shown that moesin interacts with and stabilises PD-L1 in the cell surface membrane by preventing the proteasomal degradation of PD-L1 in human breast cancer adenocarcinoma, MDA-MB-231, though the influence of ezrin and radixin gene suppression remains to be determined [47].

 

Discussions (Line 402 – 408)

Accumulating evidence suggests that radixin principally regulates the surface plasma membrane localization of numerous drug transporters including multidrug resistance protein 2, presumably because radixin is dominantly expressed in the hepatic tissues and cells among ERM proteins [48-53]. Therefore, radixin may primarily contribute to the cell surface localization of PD-L1 in KP-2 cells as a predominant ERM protein, as is the case with ezrin in HeLa, JEG-3, and LS180 cells we have recently reported [29-31].

 

Modification 2.

To confirm that ezrin-mediated transcriptional regulation of PD-L1 might be associated with a decrease in the mRNA expression of proinflammatory cytokines such as interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and interleukin (IL)-6 as described in the discussion section, we have measured the mRNA expression levels of these three proinflammatory cytokines in the ezrin-knockdown cells. The data and related sentences were incorporated into the Materials and Methods, Discussion, and Supplementary Materials.

 

Materials and Methods (Line 112 – 114)

Table 1. Primer sequences used for real-time reverse transcription-polymerase chain reaction analysis.

 

Discussion (Line 416 – 421)

In fact, gene silencing of ezrin decreased the mRNA expression levels of IFN-γ, TNF, and IL-6 in KP-2 cells (Figure S5). Together, these observations raise one possibility that knockdown of ezrin gene may downregulate the mRNA expression levels of pro-inflammatory cytokines, which in turn decrease the mRNA expression level of PD-L1, resulting in a decrease in the cell surface expression of PD-L1 in KP-2 cells.

 

Supplementary Materials (Line 64 – 71)

RNA interference of ezrin reduces the mRNA expression levels of proinflammatory cytokines in KP-2 cells

 

Figure S5. RNA interference of ezrin reduces the mRNA expression levels of proinflammatory cytokines in KP-2 cells. Cells were treated with the transfection medium (Untreated), transfection reagent (Lipofectamine), nontargeting control (NC) siRNA, and specific siRNA for ezrin at the concentration of 5 nM, and then cultured for 4 days. Gene expression levels of interferon (IFN)-ɤ, tumor necrosis factor (TNF), and interleukin (IL)-6 mRNA normalized with β-actin in cells treated with each siRNA relative to that in cells treated with the transfection reagent alone as determined by real-time reverse transcription-polymerase chain reaction. n = 3, *p < 0.05 vs. Lipofectamine. All data were expressed as the mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s test.

 

 

Reply Point 3.

In order to state the advantage that we used KP-2 cells in this study more clearly, we have incorporated and modified data and sentence in the Results section.

 

Results (Line 239 – 244)

First, we examined the gene expression profiles of PD-L1 and ERM in a variety of human pancreatic cell lines registered in the public database of the Cancer Cell Line Encyclopedia (CCLE) [32] and the Cancer Dependency Map (DepMap) portal data explorer [33, 34]. The database analysis showed that the relative mRNA expression levels of PD-L1, ezrin, and radixin in KP-2 cells were moderate to higher than those in other cells. By contrast, KP-2 cells had considerably lower levels of moesin (Figure 1a).

 

Results (Line 252 - 254)

Note that the expression pattern of ERM in KP-2 cells is quite similar to that in human clinical PDAC tissues [37-40].

 

Legend for Figure 1 (Line 257 – 260)

(a) Relative gene expression patterns of PD-L1 and each ERM in various types of human pancreas cell lines evaluated by utilizing the Cancer Dependency Map (DepMap), Broad (2021): DepMap 21Q4 Public. Scatter plots show the expression levels (log2 (TPM+1)) of four genes of interest in each human pancreas cell line.

Reviewer 3 Report

The authors presented how radixin and ezrin differentially modulate the cell surface localisation of PD-L1 in KP-2 cells, highlighting a potential therapeutic target to improve the current Immune checkpoint blockade therapy in pancreatic ductal adenocarcinoma.

I have only a comments on fluorescence: please insert a negative staining control for all fluorescence images.

Author Response

Response to Reviewer 3 Comments

 

We would like to thank #Reviewer 3 for the greatest evaluation and valuable suggestions on our manuscript. We have carefully read your comments and suggestions and have made the corrections in the revised version of manuscript. Detailed responses to your comments are listed below, and we highlighted all changes with word track changes in the file labeled ‘Revised Manuscript with Track Changes’. We hope this revised manuscript would be satisfactory for publication in Immuno.

 

Comment 1.  The authors presented how radixin and ezrin differentially modulate the cell surface localisation of PD-L1 in KP-2 cells, highlighting a potential therapeutic target to improve the current Immune checkpoint blockade therapy in pancreatic ductal adenocarcinoma.

I have only a comments on fluorescence: please insert a negative staining control for all fluorescence images.

 

Reply Comments.

We would like to appreciate #Reviewer 3’s valuable suggestion. According to #Reviewer 3’s comment, we have added following data and sentence in the Materials and Methods and Results sections in addition to Supplementary Materials.

 

Materials and Methods (Line 141 – 144)

For negative control staining, cells were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) antibody ReadyProbes (R37116; Thermo Fisher Scientific) at a dilution of approximately 1:12.5 in blocking buffer without incubation with primary antibodies against PD-L1 and each ERM.

 

Materials and Methods (Line 162 – 167)

For negative control staining, cells were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) antibody ReadyProbes (R37116; Thermo Fisher Scientific) or an Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) antibody ReadyProbes (R37117; Thermo Fisher Scientific) both at a dilution of approximately 1:12.5 in blocking buffer without incubation with primary antibodies against PD-L1 and each ERM.

 

Results (Line 271 – 274)

None of fluorescence signals were observed in cells incubated with goat anti-rabbit IgG (H+L) secondary antibodies conjugated with an Alexa Fluor 488 or an Alexa Fluor 594 without first antibodies against PD-L1 or each ERM (Figure S3).

 

Supplementary Materials (Line 23 – 30)

Figure S3. Negative fluorescence staining for KP-2 cells in confocal laser scanning microscopy analysis. (a) Negative fluorescence staining in Figure 2. Left; Fluorescence image of goat anti-rabbit IgG (H+L) secondary antibody conjugated with an Alexa Fluor 488 without primary antibodies against PD-L1 or each ERM. Middle; Counterstaining with the plasma membrane by using phalloidin conjugated to the tetramethylrhodamine (TRITC), which binds F-actin with high selectivity and affinity. Right; Phase-contrast image. Scale bars: 50 μm. (b) Negative fluorescence staining in Figure 3. Left and middle; Fluorescence images of goat anti-rabbit IgG (H+L) secondary antibodies conjugated with an Alexa Fluor 488 or an Alexa Fluor 594, respectively, without primary antibodies against PD-L1 or each ERM. Right; Phase-contrast image. Scale bars: 50 μm. All images were captured by confocal laser scanning microscopy.