Abstract
Diffuse pleural mesothelioma (PM) is a highly aggressive tumour typically associated with short survival. Recently, the effectiveness of first‐line immune checkpoint inhibitors in patients with unresectable PM was reported. CD70–CD27 signalling plays a co‐stimulatory role in promoting T cell expansion and differentiation through the nuclear factor κB (NF‐κB) pathway. Conversely, the PD‐L1 (CD274)–PD‐1 (PDCD1) pathway is crucial for the modulation of immune responses in normal conditions. Nevertheless, pathological activation of both the CD70–CD27 and PD‐L1–PD‐1 pathways by aberrantly expressed CD70 and PD‐L1 participates in the immune evasion of tumour cells. In this study, 171 well‐characterised PMs including epithelioid (n = 144), biphasic (n = 15), and sarcomatoid (n = 12) histotypes were evaluated immunohistochemically for CD70, PD‐L1, and immune cell markers such as CD3, CD4, CD8, CD56, PD‐1, FOXP3, CD68, and CD163. Eight percent (14/171) of mesotheliomas simultaneously expressed CD70 and PD‐L1 on the tumour cell membrane. PMs co‐expressing CD70 and PD‐L1 contained significantly higher numbers of CD8+ (p = 0.0016), FOXP3+ (p = 0.00075), and CD163+ (p = 0.0011) immune cells within their microenvironments. Overall survival was significantly decreased in the cohort of patients with PM co‐expressing CD70 and PD‐L1 (p < 0.0001). In vitro experiments revealed that PD‐L1 and CD70 additively enhanced the motility and invasiveness of PM cells. In contrast, PM cell proliferation was suppressed by PD‐L1. PD‐L1 enhanced mesenchymal phenotypes such as N‐cadherin up‐regulation. Collectively, these findings suggest that CD70 and PD‐L1 both enhance the malignant phenotypes of PM and diminish anti‐tumour immune responses. Based on our observations, combination therapy targeting these signalling pathways might be useful in patients with PM.
Keywords: pleural mesothelioma, immunohistochemistry, CD70, PD‐L1 (CD274), migration, invasion, cellular proliferation, immune evasion
Introduction
Mesothelioma is an aggressive neoplasm arising from mesothelial cells of the pleura, peritoneum, and pericardium [1, 2]. Most cases arise from pleural mesothelial cells, and these lesions are defined as pleural mesothelioma (PM). Despite advances in chemotherapeutic and immunotherapeutic modalities, the prognosis of PM remains poor [3].
CD70 is a type II transmembrane surface protein comprising 193 amino acids with a C‐terminal tumour necrosis factor (TNF) homology domain, making CD70 a member of the TNF superfamily [4, 5]. CD70 expression is tightly regulated on activated T cells, B cells, and mature dendritic cells [6, 7]. CD27, a member of the TNF receptor superfamily (TNFRSF), harbours two complete domains and one incomplete cysteine‐rich domain that are characteristic of TNFRSF [4, 5]. CD27 is a co‐stimulatory immune checkpoint receptor that is constitutively expressed on a broad range of T cells (naïve, αβ, γδ, and memory T cells), natural killer (NK) cells, and B cells. The CD70–CD27 signalling pathway plays a co‐stimulatory role in promoting T cell expansion and differentiation through activation of the nuclear factor κB (NF‐κB) pathway under physiological conditions [7, 8, 9]. Aberrant CD70 expression has been reported to accelerate immune evasion and increase malignant potential in many tumour types [9, 10, 11, 12, 13, 14]. Recently, our group found that CD70 worsens the prognosis of PM by enhancing malignant phenotypes such as PM cell migration and invasion and diminishing anti‐tumour immune responses [15].
PD‐L1 (CD274) has been identified as a cell‐surface glycoprotein belonging to the B7 family [16]. PD‐1 (PDCD1), a physiological receptor for PD‐L1, belongs to the immunoglobulin superfamily, and it is mainly expressed on activated T cells and non‐T lymphocytes such as B and NK cells only upon induction [17, 18]. Physiologically, PD‐L1–PD‐1 signalling is crucial for protecting peripheral tissues from collateral damage during the inflammatory response. This signalling is also crucial for the maintenance of self‐tolerance to avoid autoimmune diseases [19, 20]. When T cells are exposed to chronic antigen stimulation such as that occurring during chronic viral infection or cancer, high PD‐1 expression is induced, leading to T cell exhaustion or anergy [21]. Aberrant PD‐L1 expression has been reported in various tumours including mesothelioma, and in some cases, it is associated with increased tumour aggressiveness and poor clinical outcomes [15, 22, 23, 24, 25].
This study examined the expression status of CD70 and PD‐L1 in PM and analysed their association with clinicopathological parameters as well as the characteristics of tumour‐infiltrating lymphocytes (TILs) and tumour‐infiltrating macrophages (TIMs). Furthermore, in vitro experiments were performed to confirm our observations in clinical specimens and uncover the underlying mechanism.
Materials and methods
Tumours and immunohistochemistry
This project was completed under the Office of Human Subject Research Exemption for anonymised specimens. Among 171 diffuse PMs analysed in the present study, 30 cases were included on institutional review board approved protocols at the National Institutes of Health and informed consent was obtained from these patients. One hundred forty‐one additional anonymised PM samples were collected from multiple institutions in Poland without the requirement for patient consent by giving them the opportunity for opt‐out, in accordance with local regulations. Many of the samples were derived from initial biopsy specimens and others from surgical specimen without chemoradiotherapy. Tumours were classified as follows: epithelioid (n = 144), biphasic (n = 15), and sarcomatoid (n = 12) histotypes. Diagnosis was confirmed by using the immunoreactivity for cytokeratin cocktail AE1/AE3, calretinin, WT1, TTF‐1, and CEA. In epithelioid PMs, the nuclear grade (1–3), overall tumour grade (high or low), and histological architecture were defined according to the WHO Classification of Tumours, Thoracic Tumours, 5th Edition [26]. One rectangular tumour sample was used to assemble multi‐tumour blocks containing up to 40 tissue samples. Immunohistochemistry was performed using the Leica Bond‐Max automation (Leica Biosystems, Bannockburn, IL, USA), Ventana BenchMark XT (Roche Diagnostics, Basel, Switzerland), or Ventana BenchMark Ultra (Roche Diagnostics). The dilution of antibodies used in this study is summarised in supplementary material, Table S1. The immunohistochemical data for CD70, PD‐L1, and lymphoid markers including PD‐1 and FOXP3 are cited from our previous reports [15, 25]. CD70 (cell membranous) and PD‐L1 (cell membranous) immunoreactivity was evaluated with a detection cut‐off of 5% under light microscopy for each marker. The numbers of CD3‐, CD4‐, CD8‐, CD56‐, PD‐1‐, and FOXP3‐positive TILs were counted in hot‐spot areas within the tumour microenvironment in high‐power fields (HPFs, ×400) using light microscopy [15, 25]. As for lymphocytes, the numbers of CD68‐ and CD163‐positive TIMs were counted in hot‐spot areas within tumour microenvironment in HPFs.
Fluorescent immunohistochemistry
Antigen retrieval was performed using Histofine deparaffinisation and antigen retrieval buffer pH 9 (Nichirei Biosciences, Tokyo, Japan) according to the manufacturer's protocol. After blocking, primary antibodies were applied at room temperature (RT) for 1 h. The dilution of antibodies is summarised in supplementary material, Table S1. Signals were visualised using secondary antibodies labelled with fluorescein or tetramethylrhodamine applied at a dilution of 1:500 (Molecular Probes®, Thermo Fisher Scientific K. K., Tokyo, Japan).
Cells and plasmids
The MeT‐5A human immortalised mesothelial cell line and six human mesothelioma cell lines, H211, H290, H2052, Y‐MESO‐8A, ACC‐MESO‐1, and ACC‐MESO‐4, were kindly provided by Dr. Yoshitaka Sekido (Aichi Cancer Center Research Institute, Nagoya, Japan). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). The CSII‐CMV‐MCS‐IRES2‐Bsd vector was kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center, Tsukuba, Japan). Stable transfectants were established using CSII‐CMV‐MCS‐IRES2‐Bsd vectors containing the CD70, PD‐L1, or LacZ cDNA sequence followed by IRES2 and the blasticidin or hygromycin B resistance gene. The pLKO.1 vector was procured from Addgene (Cambridge, MA, USA). Short hairpin sequences targeting PD‐L1 and the control sequence were as follows: PD‐L1‐1, 5′‐TCC AGA AAG ATG AGG ATA TTT‐3′; PD‐L1‐2, 5′‐GTT GGA ACG GGA CAG TAT TTA‐3′; Control, 5′‐CCT AAG GTT AAG TCG CCC TCG‐3′.
Fluorescence‐activated cell sorting and immunoblot assays
In fluorescence‐activated cell sorting (FACS) analyses, FITC‐conjugated anti‐CD70 or PE‐conjugated anti‐PD‐L1 antibodies (BioLegend, Inc., San Diego, CA, USA) were applied at a dilution of 1:20 for 1 h on ice to harvest cells. After washing and staining with 7‐AAD (Beckman Coulter, Inc., Brea, CA, USA), the cells were analysed using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Collected data were analysed by FlowJo 7.6.5 software (Tomy Digital Biology Co., Ltd., Taito‐ku, Japan).
Whole‐cell lysates were prepared and subjected to immunoblot analyses using a previously reported procedure [27, 28, 29]. The antibodies used in the immunoblot assays are summarised in supplementary material, Table S2. The signal intensity was measured using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).
Cellular proliferation, migration, and invasion assays
Cells (5 × 103) were seeded on 12‐well plates. After incubation, cell numbers were measured using CellTiter 96® Aqueous One Solution (Promega, Madison, WI, USA) according to the manufacturer's protocol.
Migration and invasion assays were performed using the Falcon® Permeable Support for 24‐well Plate with 8.0 μm Transparent PET Membrane and Corning® BioCoat™ Matrigel® Invasion Chambers with 8.0 μm PET Membrane (Corning, Corning, NY, USA) according to the manufacturer's procedure. For migration and invasion assays, 4 × 104 cells were used per chamber. Ten percent FBS was used as a chemoattractant. After incubation, the cells were fixed using 100% methanol at RT and stained with Giemsa stain. The number of migrated or invaded cells was counted under a microscope.
Statistical analysis
All statistical analyses were performed using EZR version 1.54 software [30]. The chi‐squared or Fisher's exact test was performed to investigate the statistical correlation between categorical data. To analyse the overall survival of patients with mesothelioma, the log‐rank test was used. For multiple comparisons, a post hoc test was performed. Moreover, Cox proportional hazards regression analysis was performed to analyse the association of survival with factors including age (<65 years versus ≥65 years), sex (male versus female), and tumour histology (epithelioid versus biphasic versus sarcomatoid). The data from immunohistochemical analyses such as PD‐L1 and CD70 expression (double positive versus single positive or negative) and characteristics of TILs or TIMs (high versus low) were also included in the initial model. The cut‐offs for TILs and TIMs were as follows: CD3, 3 (25th percentile); CD4, 28 (the median); CD8, 7 (25th percentile); CD56, 1 (the median); PD‐1, 3 (75th percentile); FOXP3, 19 (75th percentile); CD68, 18 (25th percentile); and CD163, 20 (the median). A backward elimination with a threshold of p = 0.05 was used to select variables in the final model. Cases with missing information were eliminated from the statistical analysis of that parameter. For molecular experiments, an unpaired two‐tailed Student's t‐test or one‐way ANOVA followed by a post hoc test was used.
Results
Expression of CD70, PD‐L1, and immune cell markers in mesothelioma
The results for the diagnostic immunohistochemistry are summarised in supplementary material, Table S3. Representative images for haematoxylin and eosin (H&E) staining and immunohistochemistry for CD70 and PD‐L1 are presented in Figure 1A. Co‐expression of CD70 (green) and PD‐L1 (red) on the cell membrane of PM cells was observed in fluorescent immunohistochemistry (Figure 1B). The clinical, pathological, and immunohistochemical features of the analysed tumours are summarised in Table 1 according to CD70 and PD‐L1 expression on the cell membrane of tumour cells. Thirty‐four (20%) and 54 (32%) of 171 cases showed CD70 and PD‐L1 expression, respectively. Among them, 14 cases (8%) exhibited simultaneous CD70 and PD‐L1 expression on the cell membrane. Biphasic and sarcomatoid PMs tended to show higher positivity for CD70 and PD‐L1 than epithelioid type (p = 0.067). In epithelioid PMs, CD70‐ and PD‐L1‐positive tumours tended to show higher nuclear grades (p = 0.0025) and solid architecture (p = 0.047). PMs co‐expressing CD70 and PD‐L1 contained significantly higher numbers of CD8+ (p = 0.0016), FOXP3+ (p = 0.00075), and CD163+ (p = 0.0011) immune cells within their microenvironment (Table 1 and Figure 2A–C). PMs co‐expressing CD70 and PD‐L1 also tended to contain higher numbers of CD4+ TILs (p = 0.0052) and CD68+ TIMs (p = 0.0051; Table 1).
Figure 1.
A PM case co‐expressing CD70 and PD‐L1. (A) Representative images of H&E staining and immunostaining for CD70 and PD‐L1 in PM cells. Images from independently stained serial sections are presented. (B) Images from a co‐stained section for CD70 (green) and PD‐L1 (red) by fluorescent immunohistochemistry. Mesothelioma cells show membranous signals for CD70 and PD‐L1 simultaneously. Scale bar, 20 μm.
Table 1.
Characteristics of PMs classified by CD70 and PD‐L1 expression
| Total No. | Characteristics of PM | |||||
|---|---|---|---|---|---|---|
| CD70+/PD‐L1+ | CD70+/PD‐L1− | CD70−/PD‐L1+ | CD70−/PD‐L1− | |||
| 171 (100%) | 14 (8%) | 20 (12%) | 40 (23%) | 97 (57%) | ||
| [100%] | [100%] | [100%] | [100%] | [100%] | P value | |
| Sex | ||||||
| Male | 97 (100%) | 9 (99%) | 13 (13%) | 18 (19%) | 57 (59%) | 0.15* |
| [69%] | [75%] | [81%] | [53%] | [71%] | ||
| Female | 45 (100%) | 3 (7%) | 3 (7%) | 16 (36%) | 23 (51%) | |
| [31%] | [25%] | [19%] | [47%] | [29%] | ||
| Age, years (mean ± SD) | 59.2 ± 11.9 | 63.5 ± 9.6 | 62.3 ± 12.7 | 60.6 ± 10.8 | 57.8 ± 11.9 | 0.22 † |
| Histology | 0.067* | |||||
| Epithelioid | 144 (100%) | 8 (6%) | 20 (14%) | 35 (24%) | 81 (56%) | |
| [84%] | [57%] | [100%] | [88%] | [84%] | ||
| Biphasic | 15 (100%) | 3 (20%) | 0 (0%) | 3 (20%) | 9 (60%) | |
| [9%] | [21%] | [0%] | [8%] | [9%] | ||
| Sarcomatoid | 12 (100%) | 3 (25%) | 0 (0%) | 2 (17%) | 7 (58%) | |
| [7%] | [21%] | [0%] | [5%] | [7%] | ||
| Nuclear grade | 1.91 ± 0.62 | 2.25 ± 0.46 | 2.05 ± 0.51 | 2.06 ± 0.64 | 1.70 ± 0.60 | 0.0025 † |
| Overall tumour grade | 0.17* | |||||
| High | 38 (100%) | 3 (8%) | 6 (16%) | 13 (34%) | 16 (42%) | |
| [26%] | [38%] | [30%] | [37%] | [20%] | ||
| Low | 106 (100%) | 5 (5%) | 14 (13%) | 22 (21%) | 65 (61%) | |
| [74%] | [63%] | [70%] | [63%] | [80%] | ||
| Histological architecture | 0.047* | |||||
| Tubulopapillary | 81 (100%) | 2 (2%) | 13 (16%) | 20 (25%) | 46 (57%) | |
| [56%] | [25%] | [65%] | [57%] | [57%] | ||
| Solid | 53 (100%) | 6 (11%) | 7 (13%) | 15 (28%) | 25 (47%) | |
| [37%] | [75%] | [35%] | [43%] | [31%] | ||
| Others | 10 (100%) | 0 (0%) | 0 (0%) | 0 (0%) | 10 (100%) | |
| [7%] | [0%] | [0%] | [0%] | [12%] | ||
| CD3+ TILs (/HPF) | 8.5 (2–24) | 20.5 (13.5–30.75) | 14 (4–22) | 11 (3–40) | 5 (2–16.75) | 0.013 ‡ |
| CD4+ TILs (/HPF) | 25 (13–48.5) | 46.5 (31.75–67.75) | 19 (11–34) | 40 (16–78) | 22 (11–35.75) | 0.0052 ‡ |
| CD8+ TILs (/HPF) | 15 (7–32.75) | 27.5 (10.75–41.75) | 14.5 (6.75–32.75) | 20 (9.75–65) | 11 (6–24) | 0.0016 ‡ |
| CD56+ TILs (/HPF) | 1 (0–3) | 1 (0.25–1.75) | 0 (0–1) | 1 (0–4.75) | 1 (0–5) | 0.11 ‡ |
| PD‐1+ TILs (/HPF) | 1 (0–2) | 2 (0.25–5.75) | 1 (0–2) | 1 (0–2.5) | 0 (0–2) | 0.13 ‡ |
| FOXP3+ TILs (/HPF) | 4 (1–12) | 22.5 (10.75–41.75) | 6 (2.75–10.25) | 6.5 (2–13.5) | 3 (1–9) | 0.00075 ‡ |
| CD68+ TIMs (/HPF) | 20 (9.5–31.25) | 40 (29–67.75) | 22 (14–27.25) | 19 (8–36.75) | 18 (8–29) | 0.0051 ‡ |
| CD163+ TIMs (/HPF) | 20.5(8–56.25) | 81.5 (24.75–169) | 20 (3.75–38) | 33.5 (12–75.5) | 17.5 (7–39.75) | 0.0011 ‡ |
P values were calculated by Fisher's exact test.
One‐way ANOVA was used to calculate the P values. Data are presented as the mean ± SD.
The Kruskal–Wallis test was used for analyses. Numbers in curved and square brackets give row and column percentages, respectively. Data for TILs and TIMs are presented as the median (25th–75th percentile). The Bonferroni‐corrected P value for significance was 0.0036 (0.05/14). Others contain trabecular, adenomatoid, small cell, and signet‐ring like features.
Figure 2.
Characteristics of TILs and overall survival of patients with PM according to CD70 and PD‐L1 expression. (A) Numbers of CD8+ immune cells within the tumour microenvironment. The post hoc test revealed significant differences between CD70−/PD‐L1− cases and CD70−/PD‐L1+ (p = 0.024) or CD70+/PD‐L1+ cases (p = 0.010). (B) Numbers of FOXP3+ immune cells within the tumour microenvironment. The post hoc test revealed significant differences between CD70+/PD‐L1+ cases and CD70−/PD‐L1− (p = 0.013), CD70+/PD‐L1− (p = 0.038), or CD70−/PD‐L1+ cases (p = 0.029). (C) Numbers of CD163+ macrophages within the tumour microenvironment. The post hoc test revealed significant differences between CD70+/PD‐L1+ cases and CD70−/PD‐L1− (p = 0.004), or CD70+/PD‐L1− cases (p = 0.015). (D) Kaplan–Meier curves for patients with mesothelioma with or without CD70 and PD‐L1 expression. The post hoc test revealed significant differences between CD70+/PD‐L1+ and CD70−/PD‐L1− (p < 0.0001) or CD70+/PD‐L1− cases (p = 0.038).
Survival analyses of patients with mesothelioma
The characteristics of the 63 patients with mesothelioma analysed for survival are summarised in Table 2. Patients were followed up for up to 120 months. The log‐rank test revealed worse clinical outcome in patients with CD70‐ (p = 0.0043) and PD‐L1‐positive tumour (p = 0.00042). Patients with PM containing higher numbers of CD3+ (p < 0.0001), CD4+ (p = 0.0028), CD8+ (p = 0.00076), FOXP3+ (p < 0.0001), and CD163+ immune cells (p = 0.026) showed worse clinical outcome. In epithelioid PMs, higher nuclear grade (p = 0.054) and solid architecture (p = 0.063) tended to associate with worse clinical outcome (Table 2). Survival was significantly shorter in patients with PM expressing CD70 and PD‐L1 on the cell membrane (1.5 months; 95% confidence interval [CI] = 0–11.5; p < 0.0001; Figure 2D). Multivariate Cox proportional hazards regression analysis identified CD70 and PD‐L1 expression on mesothelioma cells (hazard ratio [HR] = 9.35; 95% CI = 3.02–28.94; p < 0.001), higher FOXP3+ TIL accumulation (HR = 2.27; 95% CI = 1.05–4.91; p = 0.038), and CD3+ TIL accumulation (HR = 7.80; 95% CI = 3.20–19.04; p < 0.001) as potential independent risk factors. In contrast, higher PD‐1+ TIL accumulation (HR = 0.41; 95% CI = 0.20–0.86; p = 0.018) and CD56+ TIL accumulation (HR = 0.45; 95% CI = 0.24–0.82; p = 0.0091) were identified as potential independent favourable factors in patients with mesothelioma (Table 3). Tumour histology and the numbers of TIMs had limited impacts on the survival of PM patients in our models.
Table 2.
Characteristics of the 63 patients analysed for survival
| Characteristics | P value | |
|---|---|---|
| Sex, no. (%) | ||
| Male | 47 (75) | 0.21 |
| Female | 16 (25) | |
| Age, years (mean ± SD) | 61.0 ± 10.6 | 0.59 |
| Histology, no. (%) | ||
| Epithelioid | 53 (84) | 0.35 |
| Biphasic | 7 (11) | |
| Sarcomatoid | 3 (5) | |
| Nuclear grade, mean ± SD | 2.0 ± 0.62 | 0.054 |
| Overall tumour grade, no. (%) | ||
| High | 16 (30) | 0.27 |
| Low | 37 (70) | |
| Histological architecture, no. (%) | ||
| Tubulopapillary | 30 (57) | 0.063 |
| Solid | 21 (40) | |
| Others | 2 (4) | |
| Tumour positive for membranous CD70, no. (%) | 20 (32) | 0.0043 |
| Tumour positive for membranous PD‐L1, no. (%) | 15 (24) | 0.00042 |
| CD3+ TILs (/HPF), median (25th–75th percentiles) | 11 (3–29.75) | <0.0001 |
| CD4+ TILs (/HPF), median (25th–75th percentiles) | 28 (9–61.75) | 0.0028 |
| CD8+ TILs (/HPF), median (25th–75th percentiles) | 16 (7–34) | 0.00076 |
| CD56+ TILs (/HPF), median (25th–75th percentiles) | 1 (0–6) | 0.27 |
| PD‐1+ TILs (/HPF), median (25th–75th percentiles) | 1 (0–3) | 0.069 |
| FOXP3+ TILs (/HPF), median (25th–75th percentiles) | 6 (2–19) | <0.0001 |
| CD68+ TIMs (/HPF), median (25th–75th percentiles) | 26 (18–45) | 0.10 |
| CD163+ TIMs (/HPF), median (25th–75th percentiles) | 20 (7–67) | 0.026 |
P values were calculated by log‐rank test.
Table 3.
Cox proportional hazards regression analysis of patients with mesothelioma
| Hazard ratio | 95% CI | |||
|---|---|---|---|---|
| Min | Max | P value | ||
| PD‐1+ TILs (/HPF) | ||||
| ≥3 | 0.41 | 0.20 | 0.86 | 0.018 |
| CD56+ TILs (/HPF) | ||||
| ≥1 | 0.45 | 0.24 | 0.82 | 0.0091 |
| FOXP3+ TILs (/HPF) | ||||
| ≥19 | 2.27 | 1.05 | 4.91 | 0.038 |
| CD3+ TILs (/HPF) | ||||
| ≥3 | 7.80 | 3.20 | 19.04 | <0.001 |
| Tumour CD70 and PD‐L1 | ||||
| Double positive | 9.35 | 3.02 | 28.94 | <0.001 |
Cox proportional hazards regression analysis was performed to analyse the association of survival with other factors. The initial model included age, sex, tumour histology, and data for immunohistochemical staining for CD70 and PD‐L1 (double positive versus others), CD3, CD4, CD8, CD56, CD68, CD163, PD‐1, and FOXP3 expression in tumours, TILs or TIMs. A backward elimination with a threshold of p = 0.05 was used to select variables in the final model.
CD70 and PD‐L1 expression in human PM cell lines
Among the mesothelial and PM cells analysed, ACC‐MESO‐1 cells uniquely expressed CD70. MeT‐5A, H211, H2052, and H290 cells expressed PD‐L1. Unfortunately, no PM cell line expressed endogenous CD70 and PD‐L1 simultaneously. Expression of CD27 and PD‐1, the canonical receptors for CD70 and PD‐L1, respectively, was undetectable in PM cells (Figure 3A). ACC‐MESO‐1 and MeT‐5A cells expressed CD70 and PD‐L1, respectively, on the cell membrane (Figure 3B).
Figure 3.
Immunoblotting and FACS of PM cells. (A) Immunoblots showing CD70 and PD‐L1 expression in PM cells. Note that CD27 and PD‐1, the canonical receptors for CD70 and PD‐L1, respectively, were undetectable in PM cells. As a positive control, CD27‐ or PD‐1‐transfected Jurkat cells were used. (B) FACS analyses revealed the CD70+ (left) or PD‐L1+ (right) fractions in ACC‐MESO‐1 and MeT‐5A cells, respectively.
PD‐L1 depletion increased the proliferation and suppressed the migration of PM cells
Recently, our group revealed that CD70 enhanced the migration and invasion of PM cells via MET–ERK activation without expression of its canonical receptor CD27 in vitro [15]. In the present study, we hypothesised that PD‐L1 plays the same roles as CD70 because CD70 and PD‐L1 co‐expression resulted in the worst prognosis in patients with PM (Figure 2D and Table 3). To assess this hypothesis, additional molecular experiments were performed. First, PD‐L1 was depleted in MeT‐5A and H2052 cells via stable transfection of pLKO.1 vectors targeting PD‐L1 (Figure 4A). PD‐L1 depletion significantly enhanced the proliferation of mesothelial and mesothelioma cells (Figure 4B). In contrast, the migration of these cells was significantly suppressed (Figure 4C,D).
Figure 4.
PD‐L1 knockdown increased the proliferation and decreased the migration of mesothelial and mesothelioma cells. (A) Immunoblots presenting the reduced expression of PD‐L1 in MeT‐5A or H2052 cells following PD‐L1 depletion. (B) Results of cellular proliferation assays revealing the enhanced proliferation of PD‐L1‐depleted cells. Assays were performed in triplicate. Data are presented as the mean ± SD. *p < 0.05; **p < 0.01. (C) Results of migration assays illustrating that PD‐L1 depletion reduced the migration of MeT‐5A and H2052 cells. Assays were performed in triplicate. Data are presented as the mean ± SD. **p < 0.01. (D) Representative images of migration assays for MeT‐5A cells. Scale bar = 100 μm.
CD70 and PD‐L1 cooperatively increase the migration and invasion of PM cells
To examine the function of co‐expressed CD70 and PD‐L1 in PM cells, Y‐MESO‐8A and ACC‐MESO‐4 cells were stably transfected for CD70 and/or PD‐L1 as well as LacZ for their controls, because endogenous CD70 and PD‐L1 were undetectable in these cells (Figures 3A and 5A).
Figure 5.
CD70 and PD‐L1 additively increased the migration and invasion of PM cells. (A) Immunoblots of Y‐MESO‐8A and ACC‐MESO‐4 cells co‐transfected with CD70 and PD‐L1. (B) Results of cellular proliferation assays illustrating that PD‐L1 slightly suppressed the proliferation of PM cells. The effects of CD70 expression on PM cell proliferation were different in PM cells. Assays were performed in triplicate. Data are presented as the mean ± SD. *p < 0.05; **p < 0.01. (C) Results of the migration and invasion assays illustrating that exogenous CD70 and PD‐L1 expression significantly increased the migration and invasion of Y‐MESO‐8A and ACC‐MESO‐4 cells. Assays were performed in triplicate. Data are presented as the mean ± SD. *p < 0.05; **p < 0.01. (D) Representative images of migration and invasion assays in ACC‐MESO‐4 cells. Scale bar = 100 μm.
Regarding cellular proliferation, PD‐L1 slightly suppressed the proliferation of PM cells. The effects of CD70 expression on PM cell proliferation were different in PM cells (Figure 5B). Conversely, compared with the findings in the LacZ/LacZ‐transfected controls, CD70 and PD‐L1 increased the migration and invasion of Y‐MESO‐8A and ACC‐MESO‐4 cells. In addition, the effects of CD70 and PD‐L1 on the migration and invasion of PM cells were additive (Figure 5C,D).
PD‐L1 enhanced the mesenchymal characteristics of PM cells
To uncover the mechanism by which PD‐L1 accelerates the migration and invasion of PM cells, we performed immunoblot analyses for epithelial–mesenchymal transition (EMT)‐related genes. Immunoblotting revealed N‐cadherin and/or VIM up‐regulation in Y‐MESO‐8A and ACC‐MESO‐4 cells transfected with PD‐L1 (Figure 6). E‐cadherin expression was undetectable in all of PM cell lines (supplementary material, Figure S1). Meanwhile, Snail1/2 up‐regulation was detected in Y‐MESO‐8ALacZ/PD‐L1 cells.
Figure 6.
PD‐L1 enhanced the mesenchymal phenotype of PM cells. Immunoblots presenting N‐cadherin up‐regulation in Y‐MESO‐8A and ACC‐MESO‐4 cells transfected with PD‐L1. Note that Snail1/2 up‐regulation was detected in Y‐MESO‐8ALacZ/PD‐L1 cells. E‐cadherin expression was undetectable.
Discussion
In the present study, 171 diffuse PM tissues were immunohistochemically evaluated for the expression of CD70, PD‐L1, PD‐1, FOXP3, and other immune cell markers to assess their effects on clinicopathological parameters and survival. Co‐expression of CD70 and PD‐L1 was identified as a potential independent risk factor for the survival of patients with mesothelioma (HR = 9.35, p < 0.001). PMs co‐expressing CD70 and PD‐L1 contained significantly higher number of CD8+ (p = 0.0016), FOXP3+ (p = 0.00075), and CD163 (p= 0.0011) immune cells within their microenvironments. For the mechanistic aspects of our observation, both immune evasion and enhanced motility and invasiveness attributable to the PD‐L1‐induced mesenchymal phenotypes were considered.
PD‐L1 was expressed in 32% (54/171) of PM tumours in our cohort. Several reports identified a significant association between PD‐L1 expression and non‐epithelioid histology in mesothelioma [22, 23]. In our cohort, biphasic (40%, [6/15]) and sarcomatoid (42%, [5/12]) mesotheliomas displayed slightly higher rates of PD‐L1 positivity than epithelioid mesothelioma (30%, [43/144]). Sarcomatoid mesothelioma has often been considered to exhibit enhanced invasiveness because of EMT [31]. While E‐cadherin down‐regulation was not detected because E‐cadherin expression was undetectable in PM cells, our observations, namely the enhanced invasiveness and decreased proliferation of PD‐L1‐induced PM cells with increased N‐cadherin and VIM expression, are partly in line with this notion [32]. It has been reported that EMT‐induced tumour cells and cancer stem cells share some gene expression and phenotypic features [33]. Recently, our group immunohistochemically identified the co‐expression of PD‐L1 and ALCAM (CD166), a stemness gene, in PM [25] and colorectal cancer [34, 35]. These observations indicate that PD‐L1‐positive tumour cells harbour cancer stem cell properties. This point should be elucidated in the near future.
CD70 has been reported to regulate malignant potential in haematological and solid malignancies [9, 10, 11, 12, 13, 14]. Furthermore, CD70 expression on tumour cells has been suggested to contribute to immune evasion through (1) T cell apoptosis [11, 36, 37, 38], (2) regulatory T cell (Treg) expansion [9, 39, 40], and (3) T cell exhaustion [41]. In fact, our group recently revealed that CD70 enhanced the migration and invasion of PM cells via MET–ERK signalling activation independent of the canonical CD70–CD27 axis [15]. Furthermore, in syngeneic mouse models, significantly higher numbers of FOXP3+ (p < 0.05), PD‐1+ (p < 0.05), and HAVCR2+ TILs (p < 0.05) were found in the microenvironment of CD70‐positive PMs, indicating Treg induction and an exhausted TIL phenotype [15]. In the present study, CD70‐ and PD‐L1‐positive PMs contained significantly higher numbers of Tregs (Table 1 and Figure 2B). This observation indicated that the PD‐L1–PD‐1 axis can induce Tregs within the tumour microenvironment in cooperation with CD70. Although the mechanism has not fully elucidated, the Tregs induced by tumour‐expressed CD70 and PD‐L1 might be an optional biomarker for future clinical decision‐making.
Macrophages have been reported to play critical roles in not only development but also immune evasion of PM [2, 42, 43]. Infiltration of mesothelial cells with asbestos fibres causes the release of inflammatory cytokines, which attract immune stimulatory macrophages. Persistent cytokine release by activated macrophages and mesothelial cells accelerates malignant transformation of the mesothelial cells. After malignant transformation, mesothelioma cells attract immune‐suppressing cells, such as myeloid‐derived suppressor cells, Tregs, and tumour‐associated macrophages with M2‐like phenotype and CD163 expression [2, 42, 43]. In the present study, PMs co‐expressing CD70 and PD‐L1 contained significantly higher number of CD163+ macrophages within their microenvironments (p = 0.0011). Not only the inhibition of Tregs but also targeting macrophages might be potential therapeutics in PM patients.
Monoclonal antibodies (mAbs) blocking immune checkpoint pathways such as the CTLA‐4 and PD‐1 axes have been introduced for cancer therapy, and significant anti‐cancer effects have been observed [44, 45, 46]. Recently, first‐line immune checkpoint inhibitors against CTLA‐4 and PD‐1 were reported to provide better outcomes than systemic chemotherapy in patients with unresectable PM [3]. Regarding CD70, CD70–CD27 axis‐targeting mAbs such as ARGX‐110 (a blocking antibody causing antibody‐dependent cellular cytotoxicity) and CDX1127 (varlilumab, an agonistic antibody specific for CD27) have been developed and introduced into clinical trials [7, 47, 48, 49]. Recently, it was reported that an agonistic anti‐CD27 antibody can synergise with a blocking antibody against PD‐L1 to restore the function of CD8+ effector T cells [50]. Based on the PD‐L1 [25] and CD70 [15] expression patterns and the worst clinical outcomes of patients with PMs co‐expressing CD70 and PD‐L1, combination therapies targeting the PD‐L1–PD‐1 and CD70–CD27 axes should be considered.
The limitations of this study included the small number of patients and the lack of patient clinical information including the history of asbestos exposure, smoking, and treatment modalities. Tumour staging was also difficult because many of the samples analysed in the present study were small biopsy specimens, which are non‐informative for this purpose. Furthermore, intra‐tumoral and marker heterogeneity should be considered when analysing tissue arrays containing small specimens. Studies of these clinical parameters featuring a larger cohort, a longer follow‐up period, and large tumour samples might be needed to optimise prognostication models and identify additional characteristics of PM. In the present study, serial sections of tumour tissue array were used in the cohort analyses. Three‐colour fluorescent immunohistochemical staining was only performed in a representative case. Application of the multi‐colour immunohistochemical staining to the cohort analyses might be useful for the further characterisation of tumours, TILs, and TIMs within tumour microenvironment.
In conclusion, the present study demonstrated that the expression of CD70 and PD‐L1 on tumour cells was associated with shorter survival in patients with PM. The cohort and molecular studies indicated that CD70 and PD‐L1 worsened the prognosis of patients with PM by enhancing invasiveness and immune evasion. CD70, PD‐L1, and the other immune cell markers identified in this study could be useful for prognosis and treatment planning, including immune‐modulating therapy, in patients with PM. Combination therapies targeting the CD70–CD27 and PD‐L1–PD‐1 axes might be useful therapies for PM.
Author contributions statement
SI conceived, designed, and supervised the overall study. PC, RL, JR, JS, PW, KO, WB, DSS, RH, JL and MM collected clinical samples and analysed clinical data. SI, JL and MM performed histological analyses, tissue processing, and immunohistochemical staining. SI, AU, MK, ANS and ST performed molecular experiments. SI performed statistical analyses, made the figures and tables, and wrote the manuscript. ST provided the facilities. All authors read and provided final approval of the submitted manuscript.
Supporting information
Figure S1. E‐cadherin expression in PM cells
Table S1. Antibodies and conditions for immunohistochemistry
Table S2. Antibodies and dilutions for immunoblot analyses
Table S3. Results for the diagnostic immunohistochemistry in PM
Acknowledgements
This work was supported by a Grant‐in‐Aid for Scientific Research (C) (to SI, 20K07410) from Japan Society for the Promotion of Science. We thank Taeko Yamauchi and Koji Kato (Nagoya City University) for their assistance with immunohistochemical staining.
No conflicts of interest were declared.
Data availability statement
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1. E‐cadherin expression in PM cells
Table S1. Antibodies and conditions for immunohistochemistry
Table S2. Antibodies and dilutions for immunoblot analyses
Table S3. Results for the diagnostic immunohistochemistry in PM
Data Availability Statement
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.