# ZEB1 transcription factor induces tumor cell PD-L1 expression in melanoma

Chloé Wirbel, Simon Durand, Félix Boivin, Maud Plaschka, Valentin Benboubker, Maxime Grimont, Laetitia Barbollat-Boutrand, Garance Tondeur, Brigitte Balme, Olivier Harou, et al.

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## To cite this version:

Chloé Wirbel, Simon Durand, Félix Boivin, Maud Plaschka, Valentin Benboubker, et al. ZEB1 transcription factor induces tumor cell PD-L1 expression in melanoma. Cancer Immunology, Immunotherapy, 2025, 74 (4), pp.141. 10.1007/s00262-025-03978-5. hal-05022706

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Distributed under a Creative Commons Attribution 4.0 International License
# ZEB1 transcription factor induces tumor cell PD-L1 expression in melanoma

Chloé Wirbel<fnref n="1" /> · Simon Durand<fnref n="1" /> · Félix Boivin<fnref n="1" /> · Maud Plaschka<fnref n="1" /> · Valentin Benboubker<fnref n="1" /> · Maxime Grimont<fnref n="1" /> · Laetitia Barbollat-Boutrand<fnref n="1" /> · Garance Tondeur<fnref n="2" /> · Brigitte Balme<fnref n="2" /> · Olivier Harou<fnref n="2" /> · Anaïs Eberhardt<fnref n="1" />,<fnref n="2" /> · Stéphane Dalle<fnref n="1" />,<fnref n="2" /> · Jonathan Lopez<fnref n="1" />,<fnref n="3" />,<fnref n="4" /> · Julie Caramel<fnref n="1" />

Received: 29 November 2024 / Accepted: 11 February 2025 / Published online: 8 March 2025
© The Author(s) 2025

## Abstract

Tumor cells can evade antitumor immune response by expressing the PD-L1 ligand, leading to the inhibition of PD-1-expressing T lymphocytes. The mechanisms that regulate PD-L1 expression in cancer cells are imperfectly characterized. The transcription factor ZEB1, a major regulator of phenotype switching in melanoma cells, was shown to promote immune escape in melanoma by repressing T cell infiltration. Using inducible models of phenotype switching and ZEB1 gain/loss-of-function melanoma, we show that ZEB1 binds to the CD274 (PD-L1) promoter, directly enhancing PD-L1 mRNA transcription and its expression at the cell membrane. Furthermore, using single-cell spatial analyses on human primary melanoma samples, we demonstrate the correlation of ZEB1 and PD-L1 expression in tumor cells. Overall, these data identify ZEB1-mediated regulation of PD-L1 tumor expression as a mechanism that could contribute to immune escape in melanoma.

**Keywords** Melanoma · ZEB1 · PD-L1 · Phenotype switching · Pseudo-EMT

## Introduction

Immune checkpoint blockade (ICB) using anti-programmed cell death 1 (anti-PD-1) antibodies has revolutionized the management of metastatic melanoma. Tumor cells can evade antitumor immune response by expressing the PD-L1 ligand, which inhibits PD-1-expressing T lymphocytes (TLs) within the tumor microenvironment. Blocking the PD-1/PD-L1 axis allows the restoration of antitumor immunity, achieving a

clinical benefit in approximately 40% of cases [1]. However, most patients still present primary or acquired resistance to anti-PD-1 therapy. PD-L1 expression has been associated with a significantly lower overall survival [2, 3], and correlates with response to PD-1 blockade [4]. Thereby, defining the mechanisms that regulate PD-L1 expression in melanoma is crucial.

PD-L1 expression in tumor cells is regulated at several levels, including genomic alterations, epigenetic modification, transcriptional regulation, and posttranscriptional and posttranslational modifications [5]. The tumor microenvironment, especially IFN-γ produced by TLs [6], is a crucial regulator of PD-L1 expression. Additionally, TNFα induces PD-L1 expression by activating the nuclear factor κB (NF-κB) pathway in prostate and colon cancer cells [7] or by promoting an epithelial-to-mesenchymal transition in hepatic cancer [8]. Among tumor-intrinsic mechanisms, MYC and oncogenic RAS were shown to regulate its transcription and RNA stability [9], whereas the eIF4F-STAT1 axis controls its translation [10], and CMTM4 and CMTM6 regulate its localization at the cell membrane [11].

In melanoma, a pseudo-epithelial-to-mesenchymal transition (pseudo-EMT) allows the reversible transition from a proliferative/melanocytic state to an invasive cell state

<figure><img src="image_3.png" /></figure>

<footnote>Julie Caramel
julie.caramel@lyon.unicancer.fr</footnote>

<footnote n="1">¹ Cancer Research Center of Lyon, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, “Cancer Cell Plasticity in Melanoma” Team, Lyon, France</footnote>

<footnote n="2">² Dermatology Unit, Hospices Civils de Lyon, CH Lyon Sud, 165 Chemin du Grand Revoyet, 69495 Pierre Bénite Cedex, France</footnote>

<footnote n="3">³ Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France</footnote>

<footnote n="4">⁴ Biochemistry and Molecular Biology Unit, Hospices Civils de Lyon, CH Lyon Sud, 165 Chemin du Grand Revoyet, 69495 Pierre Bénite Cedex, France</footnote>
with neural crest-like stem cell (NCSC) features [12–14]. Melanoma cell phenotype switching is mainly controlled by the microphthalmia transcription factor (MITF) [12, 15]. In addition, we previously showed that the EMT-inducing transcription factor, zinc finger E-box binding 1 (ZEB1), negatively regulates MITF-dependent proliferative/melanocytic programs and promotes AP-1-driven invasive and stem-like programs [16, 17]. ZEB1 expression is associated with a significant decrease in the expression of differentiation antigens, including MLANA, tyrosinase and MC1R, while simultaneously enhancing nerve growth factor receptor (NGFR) expression, a NCSC marker [16–18]. Melanoma cell plasticity promotes tumor progression, distant migration, resistance to targeted therapy, and more recently escape from the immune system [19, 20]. Indeed, the NGFRhigh NCSC state was associated with immune exclusion and resistance to ICB [21, 22], and we recently demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8+ T cell infiltration by repressing the expression of the T cells attracting chemokine CXCL10, thus promoting resistance to anti-PD-1 treatment [23]. In lung and colorectal carcinomas, ZEB1 regulates PD-L1 tumor expression [24–26], suggesting that ZEB1-mediated phenotype switch in melanoma might regulate the expression of immune checkpoint ligands promoting immune escape.

Herein, using inducible models of phenotype switching and ZEB1 gain/loss of function, we investigated the role of ZEB1 in the regulation of tumor cell PD-L1 expression, and further analyzed their concomitant expression using single-cell spatial analyses on human primary melanoma samples. Our results demonstrate that ZEB1 binds to the CD274 (PD-L1) promoter, directly enhances PD-L1 mRNA transcription and its expression at the cell membrane.

# Materials and methods

## Culture cell lines

A375 human melanoma cell line was purchased from ATCC and was cultured in DMEM complemented with 10% FBS (Cambrex) and 100 U/ml penicillin-streptomycin (Invitrogen). Previously described patient-derived short-term cultures, GLO and C-09.10, established from BRAFV600 metastatic melanomas [16], were grown in RPMI complemented with respectively 20 and 7% FBS and 100 U/ml penicillin-streptomycin. TNFα (100ng/mL) and TGFβ (10ng/mL) (PeproTech) were replaced in the culture medium every 3 days. Generation of ZEB1-overexpressing C-09.10 cells using retroviral infection and HA-Zeb1 in a pBabe-puro vector was described previously [18]. Generation of Zeb1 knocked-out A375 cells was also described previously [16].

## Immunoblot analyses

Cells were washed twice with phosphate-buffered saline (PBS) containing CaCl₂ and then lysed in a 100 mM NaCl, 1% NP40, 0.1% SDS, 50 mM Tris pH 8.0 RIPA buffer supplemented with a complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitors (Sigma-Aldrich). Loading was controlled using anti-GAPDH or anti-actin. Horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibodies (Glostrup) were used as secondary antibodies. Western blot detections were conducted using the Luminol reagent (Santa Cruz). Western blot digital imaging was performed with ChemiDoc™ MP Imager (Bio-Rad). Primary antibodies used: anti-ZEB1 (1/500, Sigma #HPA027524, rabbit); anti-NGFR (1/1000, Cell Signaling #8238S, rabbit); anti-MITF (1/500, Millipore #MAB3747, mouse); and anti-GAPDH (1/20000, Sigma-Aldrich, Cat# ABS16, rabbit).

## RT-qPCR

Total RNA was isolated using RNeasy Kit (QIAGEN) and reverse transcribed using a high cDNA capacity reverse transcription kit following the manufacturer's instructions (Fisher Scientific). Quantitative PCR was done using the Azure Cielo (Azure Biosystem). Human GAPDH was used for normalization. The following primers were used:

ZEB1_Forward GAGGTAAAGCGTTTATAGCCTCTAT CA, and ZEB1_Reverse AGGGCACACCAGAAGCCAG, CD274_Forward AGGGAGAATGATGGATGTGAAA and CD274_Reverse TGCTGGATTACGTCTCCTCC.

## Flow cytometry analyses

To analyze the expression of cell surface markers, about 500.000 cells per condition were incubated with anti-PD-L1/CD274 (PE, 1/100, 29e.2a3 from BioLegend) and anti-NGFR/CD271 (Alexa Fluor 647, 1/100, from BD Pharmingen) antibodies for 1h in the dark before being counted on a BD LSRFortessa™ flow cytometer (BD Biosciences-IN) on the flow cytometry core facility (CYLE, CRCL, Lyon). DAPI was used to evaluate cell viability. Data were analyzed using the FlowJo 10.8.1 software.

## Chromatin immunoprecipitation

The ChIP assay was carried out according to the protocol from the iDeal ChIP-seq kit for transcription factors (Diagenode, Denville, NJ, USA) as previously described [16]. Briefly, cells from one 15-cm dish were cross-linked with 1% formaldehyde at RT for 10 min and quenched in 125 mM glycine for 5 min. The cross-linked chromatin was
isolated and sonicated to generate DNA fragments aver-
aging 200–500 bp in length by Bioruptor plus sonication
device (Diagenode). Chromatin fragments were immuno-
precipitated with antibodies directed against ZEB1 (1 µg,
Genetex, GTX105278, RRID:AB_11162905), or IgG (1
µg, Bio-Rad, PRABP01, RRID:AB_321631) as negative
control. qPCR primers on promoters used for quantifica-
tion: CD274_Forward CTCGCTGGGCACTTTAGGAC and
Reverse TACTGCCCCCTAGACCATCG; MITF_Forward
CTGAAGATCCCAGCGGGTTG and Reverse GAGGT-
GACTCCAAGCGAACT.

ZEB1 ChIP-seq data generated in A375 and GLO cells
[16] are publicly available on the Gene Expression Omnibus
(GEO) database under accession number GSE246672.

Human tumor samples

Retrospective melanoma tumor samples were obtained
through the Biological Resource Centre of the Hospital
Lyon Sud, Hospices Civils de Lyon. Human tumor sam-
ples were used with the patient's written informed consent.
This study was approved by the local ethics committee
(Comité Scientifique et Éthique des Hospices Civils de
LYON CSE-HCL-IRB 00013204, approval n° 22_680) and
the French data protection authority (Commission nationale
de l'informatique et des libertés, approval n° 22_5680) for
the collection of associated clinical data. A cohort of n =
22 cutaneous primary melanoma patient lesions was used
for multi-immunofluorescence analyses. Associated clinical
data are listed in Supplementary Table 1. Four tumor lesions
were excluded because of ripped tissue. Eighteen samples
were included in the final analysis.

Seven-color immunofluorescence multiplex
analyses and immunohistochemical staining

A 3-µm tissue sections were cut from formalin-fixed
paraffin-embedded human melanoma specimens. The sec-
tions underwent immunofluorescence staining using the
OPAL™ technology (Akoya Biosciences) on a Leica Bond
RX, on the Research pathology platform East (ANAPATH
RECHERCHE, CRCL, Lyon). DAPI was used for nuclei
detection. Sections were digitized with a Vectra Polaris
scanner (Perkin Elmer, USA). An autofluorescence treat-
ment of images was carried out using the Inform software
(PerkinElmer). Quantification of the number and percentage
of the staining was carried out using the Inform software.
Antibodies used were as followed: anti-SOX10 (OPAL-
480, Santa Cruz #sc-365692, 1/1000, 30 min incubation,
mouse); 1/40, 30 min incubation, rabbit); anti-ZEB1 (OPAL-
570, Sigma #HPA027524, 1/100, 30 min incubation, rabbit);
anti-PD-L1 (OPAL-620, Diagomics #1-PR292, 1/100, 60
min incubation, rabbit); anti-CD68 (OPAL-690, Abcam

#EPR20545, 1/8000, 30 min incubation, rabbit). The cutoff
value to define tumor cells based on SOX10 signal intensity
was set up at 2. Cutoff values to define positivity were 3 for
ZEB1, 1.3 for PD-L1 and 4 for CD68.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 8
software (GraphPad Software, Inc., San Diego, USA). Data
are presented as mean ± SEM. To determine significant dif-
ferences between two groups, parametric data were analyzed
using Student's t-test. All statistical tests were two-tailed. The
p-values obtained were considered significant when < 0.05.

Melanoma public datasets analysis

ZEB1 ChIP-seq data were retrieved from the Gene Expres-
sion Omnibus (GEO) database under accession number
GSE246672. The analysis was performed as previously
described in Durand et al. [16].

Single-cell RNA-seq data of melanoma cell lines
from Wouters et al. [27] were downloaded from GEO
(GSE134432). Single-cell RNA-seq data were analyzed and
visualized using Seurat (4.3.0).

Results

Phenotype switching toward ZEB1<sup>high</sup>/NGFR<sup>high</sup>
invasive/neural crest-like state is associated
with increased PD-L1 expression at the cell
membrane

Besides ZEB1 overexpression, TNFα combined or not with
TGFβ treatment is a potent inducer of phenotype switch-
ing toward invasive/neural crest-like states in melanocytic
patient-derived short-term cultures, as assessed by increased
ZEB1 and NGFR expression, and decreased MITF expres-
sion (Fig. 1A, Supp. Figure 1A). Flow cytometry analyses
of NGFR membrane expression further showed that TNFα
± TGFβ treatment significantly increases the proportion of
NGFR<sup>high</sup> cells in C-09.10 following 7 or 14 days of treat-
ment, to similar levels compared to ZEB1-overexpressing
cells (Fig. 1B, Supp. Figure 1B). We therefore used these
models of phenotype switching toward ZEB1<sup>high</sup>/NGFR<sup>high</sup>
invasive/stem-like states to investigate the expression of PD-
L1 at the cell membrane by flow cytometry. We observed a
significant increase in PD-L1 membrane expression in both
GLO and C-09.10 cell lines upon TNFα ± TGFβ treatment
and in ZEB1-overexpressing C-09.10 cells (Fig. 1C). Inter-
estingly, cells expressing PD-L1 concomitantly expressed
NGFR, as evidenced by the significant increase in double
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 1** Phenotype switching toward ZEB1<sup>high</sup>/NGFR<sup>high</sup> dedifferentiated state is associated with increased PD-L1 expression at the cell membrane. C-09.10 cells were treated for 7 (D7) and 14 days (D14) with TNFα and TGFβ, and GLO cells were treated with TNFα for 7 (D7) and 14 days (D14). A Western blot analyses of ZEB1, NGFR, and MITF expression. GAPDH was used as loading control. B NGFR expression measured by flow cytometry (mean ± SEM) in C-09.10 and GLO cells (n = 3), in C-09.10 control (CT) or overexpressing ZEB1</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

(ZEB1) (n = 5). C PD-L1 expression measured by flow cytometry (left), the percentage of the PD-L1+ population is indicated for each condition (right) (mean ± SEM) (n = 3). D NGFR and PD-L1 expression measured by flow cytometry (left), the percentage of the NGFR+/PD-L1+ population is indicated (mean ± SEM) (n = 3). P values were determined by a two-tailed unpaired Student's t-test (B-D). Differences were considered statistically significant at \*P ≤ 0.05, \*\*P < 0.01, \*\*\*P < 0.001, and \*\*\*\*P < 0.0001
positive PD-L1<sup>high</sup>/NGFR<sup>high</sup> cells in the C-09.10 cellular models (Fig. 1D). Moreover, analysis of single-cell RNA-seq public datasets of melanoma cell lines with well-characterized phenotypes [27] showed that both PD-L1 and ZEB1 display an increased expression in mesenchymal-like melanoma cells (Supp. Figure 1C).

**ZEB1 is recruited on the CD274 promoter and induces its transcription**

We then investigated how ZEB1 may promote PD-L1 expression upon melanoma cells dedifferentiation. In our recent ZEB1 chromatin immunoprecipitation sequencing (ChIP-Seq) dataset [16], we observed that ZEB1 is bound to the transcription start site (TSS) of the *CD274* (*PD-L1*) promoter in ZEB1<sup>high</sup> A375 cells, as well as in differentiated GLO cells upon TNFα-induced phenotype switching (Fig. 2A). We next validated by ChIP-QPCR the binding of ZEB1 on the *PD-L1* promoter in A375 cells (Fig. 2B) and in GLO cells treated with TNFα compared to the control condition (Fig. 2C). Enrichment levels were similar to those on the *MITF* promoter, which served as a positive control.

Our results therefore suggest that ZEB1 might promote *PD-L1* transcriptional activation by binding to its promoter. Consistently, increased PD-L1 mRNA expression was observed upon TNFα treatment in both C-09.10 and GLO cells (Fig. 2D). To confirm a specific role of ZEB1 in this induction, we quantified the expression of PD-L1 mRNA in ZEB1 gain/loss-of-function models (Fig. 2E). In C-09.10 cells overexpressing ZEB1 (C-09.10 ZEB1), we observed a trend toward an increased PD-L1 mRNA expression (Fig. 2F). Accordingly, in A375 cells knocked-out for ZEB1 (A375 AZ1) [16], PD-L1 mRNA expression exhibited a 40% decrease, indicating that ZEB1 participates in PD-L1 expression regulation (Fig. 2F). Altogether, our results demonstrate that ZEB1 binds to *PD-L1* promoter and directly induces its transcription, leading to a significant increase in PD-L1 expression at the cell membrane.

**ZEB1 is quantitatively and spatially associated with PD-L1 in human primary melanoma samples**

Next, we wanted to further study the correlation between ZEB1 and PD-L1 tumor-intrinsic expression in human samples. Bulk RNA-seq analyses on tumor samples are severely biased since PD-L1 is also expressed by immune cells (notably macrophages) and ZEB1 by stromal and immune cells [23], thus precluding robust analysis of cell type-specific expression. We therefore designed a multiplex immunofluorescence panel to precisely investigate tumor cell-specific correlation between ZEB1 and PD-L1 expression in human primary melanoma samples, using SOX10 to distinguish the tumor cells from the stromal tissue, as well as

CD68 to identify macrophages. We reconstructed the whole-
slide images using R.

In human tumors, PD-L1 is not only expressed by tumor
cells, but also by infiltrating immune cells (mainly CD68+
macrophages and DC) [28]. We therefore excluded PD-L1+
CD68+ cells from the analysis to focus on PD-L1 expres-
sion in the tumor cells compartment. The analysis of PD-L1
expression in SOX10+ melanoma cells showed a frequent
expression at the periphery of tumor nodules or closely asso-
ciated with CD68+ cells infiltrating the tumor (Fig. 3A and
B). Importantly, we observed that PD-L1 expression was
mainly found in tumor cells expressing a high level of ZEB1.
Fifteen percent of ZEB1 positive cells in all tumors were
positive for PD-L1 (min 1.62%, max 44.3%, mean 15.0%),
compared to 4.21% in ZEB1 negative cells (min. 0.35%, max
15.6%, mean 4.21%) (p = 0.0030, Fig. 3C). By segregating
tumors in ZEB1^high and ZEB1^low tumors, we further showed
that PD-L1 tumor cell expression was significantly higher in
ZEB1^high compared to ZEB1^low tumors (Fig. 3D). Moreover,
we observed a significant correlation between ZEB1 expres-
sion and PD-L1 expression in tumor cells (r² = 0.4368, p
= 0.002823, Fig. 3E). Altogether, these results confirm that
ZEB1 is quantitatively and spatially associated with PD-L1
expression in human primary melanoma samples.

Discussion

The expression of ligands of inhibitory immune checkpoints
by tumor cells is a major mechanism of escape from immune
surveillance. Tumor cell plasticity is also increasingly recog-
nized as a tumor-intrinsic mechanism of immune escape [20].
Previous experiments in syngeneic mouse models demon-
strated that ZEB1, a major regulator of melanoma plasticity
in melanoma, driving cells toward a dedifferentiated state,
promotes immune escape and tumor growth in a CD8+ TLs-
dependent manner [23]. Here, we demonstrated that ZEB1
directly binds to the CD274 promoter, induces PD-L1 mRNA
transcription, and increases its expression at the cell mem-
brane. This work provides additional mechanistic insight into
how ZEB1 may promote immune escape by directly increas-
ing tumor cell PD-L1 expression potentially inhibiting PD-1+
CD8+ TLs.

A link between EMT and PD-L1 expression was previ-
ously reported in epithelial tumors [25, 29]. In their study,
Mak et al. observed that a pan-cancer EMT signature corre-
lated with high expression of multiple immune checkpoints,
including PD-L1. In NSCLC, miR-200 is a negative regula-
tor of both ZEB1 and PD-L1, and ZEB1 represses miR-200
expression leading to an indirect increase of PD-L1 [24]. In
melanoma cells, in which ZEB1 expression is not regulated
by miR-200 [17], we rather demonstrate by ChIP assay that
ZEB1 directly binds to the CD274 promoter, to efficiently
<figure><img src="image_10.png" /><figcaption>**Fig. 2** ZEB1 is recruited on the CD274 promoter and induces its transcription. A Geneviewer captures of ZEB1 chromatin immunoprecipitation sequencing in untreated (GLO NT, green), or TNFα-treated GLO cells for 14 days (GLO TNF D14, red) and in A375 (blue). B-C ZEB1 ChIP-qPCR on CD274 promoter in A375 cells (AS3 control) (n = 2) (B) and in GLO NT and GLO TNF D14 (n = 4) (C). MITF promoter was used as a positive control. D PD-L1 mRNA relative expression measured by qPCR (mean ± SEM) in C-09.10 untreated or treated 7 and 14 days with TNFα and TGFβ (respectively D7 and D14) (n = 5), in GLO untreated and treated 7 and 14 days with TNFα (respectively</figcaption></figure>

D7 and D14) (n = 5). E Western blot analyses of ZEB1 expression in C-09.10 cells overexpressing ZEB1 and in A375 control (AS3) or knocked-out for ZEB1 (AZ1). GAPDH was used as loading control. F PD-L1 mRNA relative expression measured by qPCR (mean ± SEM) in C-09.10 control (CT) or overexpressing ZEB1 (n = 5) and in A375 control (AS3) and A375 knocked-out for ZEB1 (AZ1) (n = 5). P values were determined by a two-tailed unpaired Student's t-test (B-D, F). Differences were considered statistically significant at \*P ≤ 0.05, \*\*P < 0.01, \*\*\*P < 0.001, and \*\*\*\*P < 0.0001
<figure><img src="image_11.png" /><figcaption>**Fig. 3** Quantitative and spatial correlation of ZEB1 and PD-L1 expression in human primary melanoma samples. A Reconstitution of a ZEB1 high, a ZEB1 intermediary (ZEB1inter), and a ZEB1 low tumor according to the intensity of ZEB1 in tumor cells (3–5: blue; 5–7: orange; > 7: red); the positivity for PD-L1 alone (yellow), or ZEB1 and PD-L1 (orange) and CD68 (magenta) is indicated. For ZEB1, PD-L1, and ZEB1 and PD-L1 markers, only tumor cells with a SOX10 intensity superior to 2 were analyzed. B Representative picture of multi-immunofluorescence OPAL staining for SOX10 (white), ZEB1 (red), PD-L1 (yellow), CD68 (magenta) visualized on Phenochart software (scale bar = 20µm). C Percentage of PD-L1 positive tumor cells</figcaption></figure>

(SOX10 > 2, PD-L1 > 1.3) in ZEB1 positive cells (ZEB1 > 3) and ZEB1 negative cells (right). D ZEB1 intensity (left) measured by multiplex immunofluorescence (mean ± SEM) in ZEB1<sup>high</sup> tumors (n = 6, ZEB1<sup>+</sup> cells > 10%) and in ZEB1<sup>low</sup> tumors (n = 12). PD-L1 intensity (right) measured by multiplex immunofluorescence (mean ± SEM) in ZEB1<sup>high</sup> (n = 6) and ZEB1<sup>low</sup> (n = 12) tumors. E Spearman correlation between ZEB1 intensity and PD-L1 intensity in all tumors. ZEB1<sup>high</sup> tumors are shown in red and ZEB1<sup>low</sup> tumors in green. P values were determined by a two-tailed unpaired Student's t-test (C, E). Differences were considered statistically significant at \*P ≤ 0.05, \*\*P < 0.01, \*\*\*P < 0.001, and \*\*\*\*P < 0.0001
induce PD-L1 transcription. Accordingly, PD-L1 mRNA expression was upregulated upon ZEB1-overexpression. Altogether, we demonstrate that ZEB1 is a direct positive regulator of PD-L1 expression in melanoma tumor cells.

Besides tumor-intrinsic/cell autonomous regulation, PD-L1 tumor expression might be further increased in the immune tumor microenvironment by the presence of TNFα ± TGFβ, favoring ZEB1 expression and phenotype switching. Interestingly, our ChIP experiments showed a significant increase in ZEB1 recruitment on the CD274 gene promoter upon TNFα ± TGFβ treatment, suggesting that the integration of external stimuli might amplify tumor-intrinsic regulation of PD-L1 expression. TNFα expression is associated with resistance to immunotherapy. Consistently, TNF blockade increases the CD8+ TLs tumor infiltration and decreases PD-L1 expression in melanoma mouse models [30]. This is consistent with the recently proposed model from the Kumar-Jolly laboratory, suggesting that intra-tumor heterogeneity in PD-L1 expression is controlled by a cooperation between IFNγ signaling and tumor-intrinsic TF activity [31].

Although PD-L1 was found positively associated with response to immunotherapy in melanoma patients, its expression alone is not a sufficient predictive marker. In addition, there is currently no standardized methodology to measure PD-L1 expression, and its evaluation can differ between assays, mainly due to the high variability of immunohistochemistry analyses across distinct centers [32]. In the present study, we used multiplex immunofluorescence to precisely investigate the spatial localization of PD-L1+ tumor cells and their co-localization with ZEB1 expression, as a surrogate of dedifferentiated melanoma cells, and observed a preferential association between PD-L1 and ZEB1 expression in primary tumor samples from advanced melanoma patients. Overall, our results identify ZEB1-mediated regulation of PD-L1 tumor expression as a mechanism that could contribute to immune escape in melanoma lesions. It will be interesting to further investigate in the future whether ZEB1/PD-L1 melanoma cell heterogeneity associates with immunotherapy response in patients.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00262-025-03978-5.

**Acknowledgements** We would like to thank the Biological Resource Centre of the Hospital Lyon Sud, Hospices Civils de Lyon, the Research pathology platform East (ANAPATH RECHERCHE) and the flow cytometry core facility (CYLE).

**Author contributions** CW performed and analyzed most experiments, prepared figures, and wrote the manuscript. SD performed experiments and statistical analyses, prepared figures, and wrote the manuscript. FB, MP, VB, and LB performed and analyzed experiments. MG performed multi-IF staining. GT, BB, and OH performed pathological examination. AE and SDa provided human samples and clinical data and wrote

the manuscript. JL co-supervised the project and wrote the manuscript. JC conceived and supervised the project and wrote the manuscript.

**Funding** This work was funded by the Ligue Nationale contre le Cancer (Comité de l'Ain), the Lyon Integrated Research Institute in Cancer (SIRIC LYriCAN INCa-DGOS-Inserm_12563 and Lyrican + INCa-DGOS-INSERM-ITMO cancer_18003), the Institut Convergence PLAsCAN (ANR-17-CONV-0002), the ERiCAN program of Fondation MSD-Avenir (Reference DS-2018-0015), the Institut National contre le Cancer (INCA-DGOS PRTK_2017-022), the Société Française de Dermatologie (SFD), and the association Melarnaud and Vaincre le Mélanome. MelBase is sponsored by the French National Cancer Institute (INCa). CW was supported by a fellowship from Agence Régionale de Santé Auvergne Rhône Alpes and SD by a fellowship from the Association pour la Recherche contre le Cancer (ARC).

**Data availability** No datasets were generated or analyzed during the current study.

## Declarations

**Conflict of interest** The authors declare that there are no competing interests.

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