# Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells

Doryan Masmoudi, Jérôme Vialaret, Christophe Hirtz, Catherine

Alix-Panabières

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## To cite this version:

Doryan Masmoudi, Jérôme Vialaret, Christophe Hirtz, Catherine Alix-Panabières. Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells. Molecular Oncology, In press, Online ahead of print. 10.1002/1878-0261.13665. hal-04649173

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Submitted on 16 Jul 2024

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REVIEW

Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells

Doryan Masmoudi¹ ID, Jérôme Vialaret², Christophe Hirtz² and Catherine Alix-Panabières¹,³,⁴ ID

1 Laboratory of Rare Circulating Human Cells, University Medical Center of Montpellier, France

2 University of Montpellier, IRMB-PPC, INM, CHU Montpellier, INSERM CNRS, France

3. CREEC/CANECEV, MIVEGEC (CREES), University of Montpellier, CNRS, IRD, France

4 European Liquid Biopsy Society (ELBS), Hamburg, Germany

Keywords

cancer; circulating tumor cells;
glycoproteomics; immunopeptidomics;
proteomics

Correspondence

C. Alix-Panabières, LCCRH, Institut
Universitaire de Recherche Clinique (IURC),
641, avenue du Doyen Gaston Giraud,
34093 Montpellier Cedex 5, France
Fax: +33 (0) 4 11 75 99 33
E-mail: c-panabieres@chu-montpellier.fr

(Received 14 December 2023, revised 19
February 2024, accepted 6 May 2024)

doi:10.1002/1878-0261.13665

Circulating tumor cells (CTCs) are cancer cells that detach from the original site and reach the bloodstream. The most aggressive CTCs survive various immune system attacks and initiate metastasis formation. Importantly, CTCs are not specifically targeted by the current immunotherapies due to the limited knowledge on specific targets. Proteomic profiling can be a powerful tool for understanding some of the immune evasion mechanisms used by cancer cells and particularly CTCs. These mechanisms are generally linked to the expression of specific surface proteins/peptides (i.e. the surfaceome). The study of the peptides that bind to class I molecules of the major histocompatibility complex (MHC-I) and of the various glycoproteins expressed on CTC surface may open a completely new avenue for the discovery of novel mechanisms of immune evasion. In this review, we discuss how immunopeptidomic and glycoproteomic studies of CTCs that interact with immune cells could help to better understand how metastasis-initiator CTCs escape the host immune response. We also describe how immunopeptidomic and glycoproteomic studies are carried out.

1. Introduction

Cancer metastasis is the leading cause of death and
remains a serious challenge despite the many efforts to
improve its diagnosis, therapy and follow-up [1,2]. The
metastatic process is a cascade of events that include
the invasion, intravasation, and dissemination of few
cancer cells in the bloodstream and their subsequent

colonization of distant organs [3]. Once cancer cells
enter the bloodstream, they are called circulating
tumor cells (CTCs) [4]. During the process of intrava-
sation, CTCs leave their protective tumor microenvi-
ronment, become vulnerable to the host immune cells
and are exposed to different environmental insults

Abbreviations

CAFs, cancer-associated fibroblasts; CAR-T, chimeric antigen receptor T cells; CK, cytokeratin; CTCs, circulating tumor cells; CTLs, cytotoxic T lymphocytes; DCs, dendritic cells; DDA, data-dependent acquisition; DIA, data-independent acquisition; ECM, extracellular matrix; EMA, European Medicines Agency; EMT, epithelial-to-mesenchymal transition; EVs, extracellular vesicles; FASL, Fas ligand; FDA, Food and Drugs Administration; FGF, fibroblast growth factor; HLA, human leukocyte antigen; ICIs, immune checkpoint inhibitors; LC, liquid chromatography; MDSCs, myeloid-derived suppressor cells; MHC-I, major histocompatibility complex class I; MMP, matrix metalloproteinase; MS, mass spectrometry; NETs, neutrophil extracellular traps; NK, natural killer; PADI, protein arginine deiminase; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PMN-MDSCs, polymorphonuclear-MDSCs; PTMs, post-translational modifications; SIGLEC, sialic acid-binding immunoglobulin-type lectin; TAA, tumor-associated antigen; TAP, transporter associated with antigen processing; TCR, T cell receptor; TGF, transforming growth factor; Treg, regulatory T cells; TSA, tumor-specific antigen; VEGF, vascular endothelial growth factor.
(e.g. mechanical stress and the pressure exerted by the blood flow) [5]. Therefore, most cancer cells entering the bloodstream do not survive [6]. On the other hand, CTCs that have overcome the host immune system attacks and shear stress constitute a more aggressive subgroup of cells that could reach many distant organs. Liquid biopsy has paved the way to understand CTC biological properties and has led to major discoveries in cancer [7]. This term was coined by Catherine Alix-Panabières and Klaus Pantel in 2010 to describe a blood test used for CTC-based semi-invasive cancer monitoring [8]. Then, liquid biopsy has been extended to other analytes, such as circulating tumor DNA [9], extracellular vesicles (EVs) [10], tumor-educated platelets [11], circulating cell-free RNA [12], and circulating proteins [13]. This assay is an ideal companion tool in the framework of personalized medicine that will be implemented in the next decades [14].

Many efforts have been undertaken to detect CTCs by liquid biopsy in order to use them as prognostic biomarkers and to guide therapy decision-making [13]. As a result, CTCs from different cancer types have been phenotypically characterized to acquire a better knowledge of their function and survival potential. Specifically, it has been established that their phenotype can be different from that of the original tumor and that this confers to CTCs immune escape capabilities [15]. Moreover, due to such phenotypic differences, CTCs may not respond to personalized therapies that target molecules expressed on the primary tumor cell surface. Therefore, studies on CTC immune evasion mechanisms are required. CTCs are frequently characterized with genomic and transcriptomic techniques, but very rarely using proteomic tools. Yet, proteomic profiling of CTCs could bring major insights into the mechanisms of self-recognition by the immune system and into the ligands implicated in immune evasion. This might lead to the identification of new targets for personalized therapies. Immunopeptidomics is gaining interest in the age of mRNA- and peptide-based vaccines as well as chimeric antigen receptor T cells (CAR-T). This entails the investigation of the immunomodulatory peptides (including tumor-specific antigen-derived peptides or neo-peptides) that bind to class 1 molecules of the major histocompatibility complex class 1 (MHC-I) [16]. In the context of cancer, neo-peptides are important for self-immunosurveillance by CD8+ T cells and are central to the quest of new targetable cancer-specific antigens [17]. Recent advances in liquid chromatography (LC) and mass spectrometry (MS) have allowed identifying some MHC-bound peptides that induce immunological

responses [18] and better understanding of how immunological surveillance (i.e. recognition of self vs non-self) works. However, CTC recognition as self by T cells does not fully explain CTC immune evasion mechanisms.

Glycoproteins also are essential interfaces with the immune system and their glycosylation levels and sites regulate these interactions [19]. Aberrant glycosylation is currently considered one of the hallmarks of cancer because it influences cancer aggressiveness and capacity to avoid immunosurveillance [20]. As glycoproteins interact with a wide range of immune cells, such as natural killer (NK) and T cells, they have immunomodulatory properties [21]. Although analyzing glycosylation sites and levels is challenging, glycoproteomics is rapidly developing [22].

In this review, we will describe the role of CTCs in the metastatic cascade and discuss the multiple routes they may take to evade the immune system. In order to identify these mechanisms, we propose the investigation of surface-expressed proteins, the so-called “surfaceome”, and suggest some proteomic-based strategies to analyze them.

The main goal is to identify new therapeutic targets for developing new immunotherapies.

## 2. Circulating tumor cells and the metastatic process

CTCs are cancer cells of epithelial origin found in the blood of patients with cancer. They were first described by Thomas Ashworth who analyzed a blood sample from a patient with metastatic cancer under a microscope in 1869 [23]. He reached the conclusion that CTCs had made their way through the bloodstream from the primary cancer to the vein from which he collected the blood sample. During the metastatic process, these rare cells are released from the initial tumor site. The term metastatic cascade describes a multi-step process: local invasion, intravasation, blood survival, extravasation, and colonization [24,25]. The kinetics of this cascade are influenced by the organotropism of certain cells, the speed of progression of each cancer subtypes, and the nature of the interactions between cancer cells and the host stroma [26,27].

Following cell-to-cell adhesion loss and extracellular matrix (ECM) breakdown, cancer cells start to undergo epithelial-to-mesenchymal transition (EMT) and develop a transient and partial mesenchymal phenotype that allows them to acquire resistance to apoptotic signals [25]. Such cells can initiate intravasation. This stage entails interactions with the endothelial cells that form the blood vessels. These interactions are orchestrated by
EMT regulators. For example, ZEB1 (an E-cadherin repressor) silencing in prostate cancer cells results in the decrease of N-cadherin expression (normally upregulated in EMT) and the reduction of transendothelial migration [28]. Furthermore, when overexpressed, the transcription factor TWIST1 (an EMT inducer) can influence vascular mimicry whereby aggressive tumor cells can mimic the embryonic vasculogenic network by interacting with endothelial cells [29].

When they reach the bloodstream, cancer cells are exposed to a strong difference in pressure and few of them will survive. Indeed, although aggressive tumors can release thousands of cancer cells into the bloodstream, only few of them will colonize to a new site [30]. The few studies that tried to estimate the half-life of breast CTCs in blood reported values ranging from 30 min to 2.4 h [31,32]. It has been predicted that just one CTC in 10 000 can initiate metastasis [33]. However, CTCs may aggregate and form clusters to increase their chances of survival. Homotypic clusters are composed only of CTCs. Conversely, in heterotypic clusters, CTCs interact with different blood cell types, such as neutrophils and platelets [32,34]. By traveling in the bloodstream in a cluster, CTC resistance to the immune system attacks is enhanced via paracrine signaling or direct contacts [35], as well as their protection against anoikis [36]. Therefore, CTC clusters have a stronger invasive potential than CTCs on their own [37]. However, CTC clusters have been mainly observed in blood samples from patients with metastatic cancer. Only one report mentioned their detection in patients with early-stage breast cancer [38]. Lastly, during their dissemination, single (up to 20 µm in diameter) and clustered CTCs are stopped due to the reduction in the blood vessel size (3–7 µm), resulting very rarely in extravasation, a crucial stage in the metastatic cascade [39].

An important feature of the metastatic process is that cancer cells from specific tumor types appear to preferentially invade specific organs. For instance, CTCs from breast cancer have a strong affinity for lung, liver, bone, and brain. This organ selectivity is the logical consequence of the “seed and soil” theory introduced by Stephen Paget in 1889 [40]. Paget hypothesized that like a seed that will only develop in fertile soil, a metastatic cancer cell or cancer stem cell will only extravasate if the microenvironment is suitable. Recently, Welch and Hurst proposed a third concept, called “climate”, that refers to how each individual’s overall health and genetic inheritance might influence metastasis formation [41].

To reach a new site, cancer cells must first adhere to a new barrier of endothelial cells in a process called

extravasation. Selectins are a family of receptors that govern cancer cell interaction with endothelial cells. E-selectin, a cytokine-inducible endothelial cell adhesion receptor, has been implicated as a mediator of carcinoma cell interaction with endothelial cells and of metastasis to the liver [42]. Adherence to endothelial cells is influenced by the cancer cell ability to bind to selectins. This is governed by the glycosylation level of proteins at the cancer cell surface. Indeed, abnormal protein glycosylation is one of the hallmarks of cancer. Sialyl Lewis a and Sialyl Lewis x are carbohydrates that play a role in cancer cell adherence to vascular endothelium through selectins [43,44]. Thus, accumulation of carbohydrate ligands on the cancer cell surface is strongly linked to the occurrence of hematogenous metastases. Selectin expression on the surface of endothelial cells is influenced not only by cancer cells, but also by specific cellular components and the secretion of specific compounds. For instance, myeloid-derived suppressor cells (MDSC; a type of immune cells) can increase E-selectin expression on the surface of endothelial cells through interleukin (IL)-1 secretion, contributing to the establishment of the pre-metastatic niche [45]. Moreover, activated platelets can secrete chemotactic factors, such as CXCL5/7, leading to the recruitment of granulocytes, an essential component of the future metastatic niche [46].

Fibroblasts also play a role in remodeling the ECM and in establishing the pre-metastatic niche. Their differentiation into cancer-associated fibroblasts (CAFs) and the release of matrix metalloproteinases (MMPs) allow them to prepare the environment for cancer cell invasion [47]. Tumor-associated macrophages also exert an important role in ECM remodeling by secreting MMP-2 and MMP-9 [48]. Moreover, cancer cells can produce EVs to convey molecular information that may influence the function of distant NK cells, T cells, and antigen-presenting cells to prepare an immunosuppressive environment in the future metastatic niche [49]. EVs display specific organotropism depending on their integrin expression profile [50]. However, even when CTCs find the optimal conditions for extravasation and the environment is favorable, a new tumor will not form automatically. Indeed, cancer cells (particularly breast cancer cells) can remain dormant for an extended period of time (i.e. tumor dormancy) [31]. Two different hypotheses have been proposed to explain this phenomenon. Dormant cancer cells may withdraw completely from the cell cycle (i.e. cancer cell dormancy) [51] or they continue to proliferate, but their number is counterbalanced by the number of cells that die (micro-metastatic model) [51]. In the cancer cell dormancy model, cells can enter in a prolonged
state of mitotic arrest in the G₀-₁ phase [52]. In the micro-metastatic model, cells proliferate but the tumor does not grow in size. This control is intimately related to the process of neovascularization. Indeed, increasing the concentration of angiogenic agents (e.g. FGF or VEGF) promotes the tumor growth capacity by 'waking up' dormant cells [53]. When the microenvironment becomes favorable, cancer cells will leave dormancy and initiate a new metastasis. For instance, in mice, tobacco-induced inflammation leads to the formation of neutrophil extracellular traps (NETs) that cleave ECM laminins. This cleavage generates a new epitope that activates the integrin signaling cascade in cancer cells, converting dormant cells into aggressive metastatic cells [54].

### 3. CTC interactions with blood cells during their journey

Following the detailed description of the various steps of the metastatic cascade, this new section briefly describes the interactions between CTCs and the surrounding cells in the blood compartment. The most aggressive CTCs can interact with cells in the blood compartment, such as platelets, immune cells, and CAFs. These interactions help CTCs to evade the immune system, and also facilitate adhesion, intravasation and invasion. To achieve this, CTCs skillfully use the blood cell plasticity to their advantage.

Cancer progression is strongly influenced by recruiting and activating platelets [55]. These anucleated cells, derived from the maturation of megakaryocytes in the bone marrow, are involved in thrombosis and hemostasis that occur also during inflammation, one of the hallmarks of cancer. TGF-β secretion by platelets and direct contact with cancer cells activate the TGF-β/SMAD and NF-kB pathways [56]. These pathways are involved in the cancer cell invasive capacities and initiate their EMT. Platelets can also form clusters with CTCs in which CTCs are protected against the shear stress of the bloodstream [57], against NK cell-mediated cytolysis [58] and against the immune system attacks. Platelets also facilitate CTC extravasation. Indeed, ATP secretion by tumor-educated platelets activates the P2Y₂ receptor, thus opening the transendothelial barrier and allowing CTC extravasation [59]. Taken together, these data demonstrate that platelets support CTCs on their journey.

CTCs interact with other cell types in the blood compartment. Neutrophils are an extremely abundant circulating white cell type and are also implicated in cancer development. Indeed, increased neutrophil number has been associated with poor prognosis. The

association of CTCs with neutrophils in the bloodstream enhances their metastatic capacity by driving the cell cycle [60]. Furthermore, activated neutrophils can release DNA-histone complexes and NETs that are involved in cancer progression [61]. NETs can sequester CTCs by binding to β1 integrins (expressed on the cancer cell surface), thereby promoting their dissemination [62]. Spiegel et al. [63] showed that neutrophils play a role in immune evasion by inhibiting NK cell function and also promote CTC extravasation by secreting IL-1β and MMPs. Although the underlying mechanisms remain poorly understood, CTC interaction with neutrophils facilitates cancer cell dissemination.

Tumor-associated macrophages are implicated in the primary tumor development, and also in CTC dissemination and extravasation. A study showed that when CTCs from small cell lung cancer were co-cultured with peripheral blood mononuclear cells, they induced the differentiation of monocytes into macrophages to enhance angiogenesis, invasion, and immunosuppression [64]. Moreover, recent results suggest that the interaction between macrophages and CTCs promotes epithelial-to-mesenchymal plasticity and confers greater resistance to shear stress [65]. CTCs may fuse with macrophages to increase their chances of survival. The presence of these macrophage-CTC fusions in the blood is a poor prognostic factor [66,67]. When injected in mice, they can spread and colonize in distant sites [68]. The use of leukocyte and CTC markers to identify these macrophage-CTC fusion events may be useful for predicting the prognosis of patients with ovarian cancer. A better understanding of the interactions between macrophages and CTCs would help to clarify the metastatic cascade.

MDSCs are a population of immature myeloid cells with immunosuppressive properties toward T lymphocytes, dendritic cells (DC) and NK cells. It has been hypothesized that clusters of CTCs with MDSCs form a defensive shield to avoid immune surveillance and to facilitate dissemination [69]. Sprouse et al. [70] demonstrated higher CTC dissemination in mice after co-injection of CTCs (isolated from patients with melanoma or metastatic breast cancer) and polymorphonuclear-MDSCs (PMN-MDSCs) compared with injection of CTCs alone or PMN-MDSCs alone. Co-culture of CTCs with PMN-MDSCs induced the pro-tumor differentiation of PMN-MDSCs via paracrine Nodal signaling, leading to increased production of reactive oxygen species [70] that can inhibit T cell differentiation and activation [71]. Indirectly, IL-8 produced by cancer cells chemotactically attracts MDSCs and induces NET formation by MDSCs [72].
CAFs play an important role in tumor microenvi-
ronment remodeling and angiogenesis, but few data
are available on their interactions with CTCs. CAF
depletion in mice reduced the number of metastases
because CTCs that travel with CAFs in the blood-
stream display increased viability and colonization
potential [73]. Moreover, through intercellular contacts
and soluble factors, activated CAFs protect CTCs
from shear stress [74]. Many evidences suggest that
CAFs mediate CTC cluster formation [75].

All these studies show that CTCs do not travel
alone in the bloodstream and that they adroitly sub-
vert the functions of blood cells to increase their
capacity for dissemination [76]. Nevertheless, little is
known about the nature of these interactions.

4. How can CTCs evade immune cell attacks?

The immune system is a complex and dynamic net-
work of different cell types that can discriminate
between self and non-self. Cancer cells are not totally
considered non-self and they sometimes escape the
immune system. Moreover, immune cells can display
both pro-tumor and anti-tumor activities. In the fol-
lowing section, we will discuss and propose molecular
mechanisms of immune evasion by CTCs (Fig. 1;
Table 1).

Self-recognition is an essential step in immune sur-
veillance. Each cell is probed via the crosstalk between
MHC-I molecules expressed on its surface and the
T cell receptors (TCR) on CD8⁺ T cells. The presence
of a tumor-associated antigen (TAA) bound to MHC-
I molecules is essential for the initiation of the immune
response. CTCs exploit different mechanisms to escape
this surveillance.

First, cancer cells can downregulate MHC-I expres-
sion at their surface, thus hindering surveillance by
cytotoxic T lymphocytes (CTLs) [77,78]. It has also
been reported that MHC-I expression is frequently
downregulated in micro-metastatic carcinoma cells
from bone marrow aspirates [79]. However, MHC-I
downregulation promotes NK cell cytotoxic function
[80]. To avoid activation of CTLs and NK cells, CTCs
must express MHC-I but without TAA presentation.
For instance, in *in vitro* experiments, platelets can con-
fer to cancer cells in which MHC-I is downregulated a
non-suspicious MHC-I phenotype, known as “pseudo-
normal”, to avoid CTL and NK cell activation [81]. In
addition, expression of cytokeratin (CK) 8 and its het-
erodimeric partners CK-18 and CK-19 by cancer cells
can inhibit MHC-I interaction with TCR on CTLs by
binding to their activation domain [82]. This

mechanism has been demonstrated using carcinoma
cell lines derived from a subclavicular lymph node
metastasis. CTCs also express CKs at their surface.
Moreover, platelet aggregation to CTCs may promote
loss of expression of MIC-A and MIC-B in CTCs
(two ligands of the NKG2D receptor that is expressed
on NK cells) [83].

Second, CTCs express some glycoproteins and
receptors on their surface. The glycoprotein CD47
binds to its receptor signal-regulatory protein α
(SIRPα) that is expressed on the surface of DCs and
macrophages and inhibits phagocytosis [84]. Genomic
analyses in patients with colorectal cancer have shown
that CTCs overexpress CD47, thus inducing a “don’t
eat me” signal [85]. CD47-expressing CTCs have also
been found in patients with metastatic non-small cell
lung cancer and are associated with poor progression-
free survival [86]. CD47 expression on the CTC surface
has also been reported in metastatic head and neck
squamous cell carcinoma [87] and metastatic breast
cancer [88]. Moreover, sometimes, CD47 expression on
CTCs and on the original tumor can show a lack of
concordance [88].

Third, to avoid lysis induced by CTLs, CTCs may
express ligands, such as FASL [89]. In primary breast
cancer, a flow cytometry study showed that CTC-
positive patients have more FAS-positive T cells [89].
Binding of FASL to the FAS transmembrane receptor
expressed on the surface of CD8⁺ T cells initiates apo-
ptosis [90]. Moreover, autocrine secretion of soluble
FASL may constitute an additional mechanism of
escape from T-cell surveillance [91]. Although this
mechanism is poorly understood and not explicitly
proven for CTCs, there is some evidence of the FAS/
FASL pathway implication in the immune escape
by CTCs.

CTCs can also keep CD8⁺ T cells in an inactive
state by expressing immune checkpoint proteins.
Much current research is focusing on these interac-
tions, and programmed cell death protein 1 (PD-1)
and its ligand (PD-L1) perfectly illustrate the phe-
nomenon. The interaction between PD-1 and PD-L1
maintains immune tolerance by reducing cytokine
production and suppressing T-cell proliferation [92].
CTCs from various cancer types can express PD-L1
[93–95] and this is generally considered a poor prog-
nostic marker [93]. Moreover, PD-L1 expression het-
erogeneity in CTCs, compared to the primary tumor,
may confer an advantage regarding immune evasion
[15]. This non-exhaustive review of possible immune
evasion mechanisms in CTCs illustrates the variety of
mechanisms involved, but also our limited
knowledge.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Immune evasion mechanisms of circulating tumor cells (CTCs). The schematic highlights the most important known mechanisms of immune evasion by CTCs. Through epigenetic and genetic mechanisms, the major histocompatibility complex I (MHC-I) is downregulated in CTCs and this allows escaping identification by CD8⁺ T cells. Cytokeratin 8, 18 and 19 inhibit the interaction with the TCR expressed on CD8⁺ T cells. Platelets can confer a pseudonormal phenotype to the MHC-I of CTCs, thus avoiding recognition by natural killer (NK) cells. Platelets also promote the downregulation of MIC A and MIC B, ligands of the NKG2D activating receptor. In addition, in CTCs, secretion of MIC A and MIC B is induced, promoting the protection against NK cell cytotoxic effects. Metalloproteases secreted by CTCs cleave the FAS receptor ligand (FASL), thus inhibiting the action of CD8⁺ T cells. FASL binding to the FAS receptor on CD8⁺ T cells inhibits CTC apoptosis. Expression of the immune checkpoint protein PD-L1 maintains CD8⁺ T cells in an inactive state. Expression of CD47 on CTC surface sends a "don't eat me" signal by binding to the SIRPα receptor on macrophages. Created with BioRender.com. CTCs, circulating tumor cells; MHC, major histocompatibility complex; NK, natural killer; TCR, T-cell receptor.</figcaption></figure>

**5. Immunotherapy in patients with cancer**

Today, immunotherapy is considered a therapeutic
revolution in cancer treatment. However, patient

response to treatment is variable and the underlying
reasons are still elusive. Several classes of immunother-
apy molecules are emerging for cancer treatment, such
as monoclonal antibodies against immune checkpoint
factors, vaccines and adoptive T cell therapy.
<table><caption>**Table 1.** Non-exhaustive list of immune evasion mechanisms of CTCs and their analysis methods.</caption><thead><tr><th>Mechanisms</th><th>Type of analysis</th><th>References</th></tr></thead><tbody><tr><td>MHC-I downregulation</td><td>Micrometastatic carcinoma CK18-positive cells colabeled with monoclonal antibody W6/32 directed to a framework antigenic determinant of MHC class I heavy chains associated with β2-microglobulin</td><td>[79]</td></tr><tr><td>MHC-I « pseudonormal »</td><td>In vitro flow cytometry, immunofluorescent staining, confocal microscopy, and immunoelectron microscopy</td><td>[81]</td></tr><tr><td>MHC-I dysfunction by cytokeratins expression</td><td>Co-immunoprecipitation, combined with LC–tandem mass spectrometry and immunoblotting on carcinoma cell line derived from a subclavicular lymph node metastasis</td><td>[82]</td></tr><tr><td>MIC A/B underregulation</td><td>Flow cytometry, ELISA and RT-PCR of tumors cells incubated with platelets</td><td>[93]</td></tr><tr><td>CD47 upregulation</td><td>Gene expression analyses on CTCs from colorectal cancer</td><td>[85]</td></tr><tr><td>FasL expression</td><td>Flow cytometry on peripheral CD95 (FAS)-positive T-helper cells from CTC-positive patients</td><td>[89]</td></tr><tr><td>PD-L1 expression</td><td>CellSearch on metastatic breast cancer patients</td><td>[95]</td></tr></tbody></table>

Monoclonal antibodies against immune checkpoint components inhibit cancer cell-induced immunosuppression. By expressing some ligands on their surface, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) or PD-L1, cancer cells can maintain T cells in an inactive state. PD-L1 is often overexpressed on cancer cells and in the tumor microenvironment. The US Food and Drug Administration (FDA) has approved some immune checkpoint inhibitors (ICIs) against PD-L1, such as pembrolizumab and nivolumab, and against CTLA-4, such as ipilimumab. Pembrolizumab and nivolumab are currently indicated for head and neck squamous cell carcinoma, melanoma, non-small cell lung cancer, and Hodgkin's lymphoma. Ipilimumab was approved for melanoma in 2011 [96]. However, many patients will not respond to ICIs. For instance, pembrolizumab (against PD-L1) monotherapy shows response rates of ≥35% in patients with advanced gastric/gastroesophageal junction cancer [97], advanced small-bowel adenocarcinoma [98], advanced melanoma [99], advanced cervical squamous cell

cancer [100] and advanced non-clear cell renal cell carcinoma [101]. Similarly, nivolumab (against PD-L1) displays response rates of almost 30% in patients with advanced non-small cell lung cancer [102], metastatic urothelial carcinoma [103] and advanced hepatocellular carcinoma [104]. Ipilimumab (against CTLA 4) shows even lower response rates: ~20% in advanced melanoma [105,106]. Cancer cells often acquire mutations that inactivate the JAK1 and JAK2 pathways, thus partly explaining resistance to anti-PD-1 therapies [107]. Moreover, the human leukocyte antigen (HLA)-I genotype has a major influence on the response rates to ICIs [108]. Although most patients do not respond to these immunotherapies, therapeutic antibodies against PD-1, PD-L1 or CTLA4 have markedly increased progression-free survival and overall survival in various cancers, especially when combined with chemotherapy [109–112].

Cancer vaccines involve injecting tumor antigens combined with an adjuvant to stimulate DCs. These vaccines can be based on peptides, RNA, DNA, or even directly matured DCs. The aim is to stimulate the patients' adaptive immunity to prevent tumor development. The efficacy of these vaccines relies on the strong activation of CD4⁺ and CD8⁺ T cells, and their infiltration into the tumor. However, many patients do not respond or develop resistance to anti-tumor vaccines. Loss of HLA class 1 expression is one of the main mechanisms of resistance to therapeutic vaccines by hiding tumor antigens [113]. The tumor microenvironment also contributes to resistance mechanisms, notably by interfering with T-cell activation and by secreting immunosuppressive cytokines [114,115]. This leads to low response rates, particularly in patients with melanoma (2.6%) [116]. In the meta-analysis by Nagorsen and Eckhard, the response rate to therapeutic vaccine-based immunotherapies for colorectal cancer was 0.9% [117]. More recently, the combination of the mRNA-based vaccine RO7198457 with the anti-PD-L1 monoclonal antibody atezolizumab has shown a response rate up to 30% in patients with advanced solid cancers [118], a result comparable to that obtained with the antibody alone. Similarly, the combination of the mRNA-based vaccine 4157 and pembrolizumab showed a 30% response rate in patients with advanced or metastatic cancer [119]. These data suggest that combining these therapeutic vaccines with ICIs does not increase the response rate compared with ICIs alone.

Immunotherapies also include the injection of immunoactivating cytokines, such as IL-2, leading to T-cell activation and proliferation. In patients with melanoma, these therapies show a response rate of
12% as monotherapy and 16% in combination with vaccines [120]. Similarly, the combination of IL-2 with the peptide-based gp100 vaccine displays a response rate of 16% [121].

The aim of these approaches is to induce the anti-cancer immune response, but there are also immunotherapies with direct anti-cancer properties. The transfer of genetically modified T cells, expanded ex-vivo to target specific tumor antigens in the patient, is an important area of immunotherapy research. The development of CAR-T cells represents one of the most promising approaches in this field. Its success has been widely confirmed in patients with hematological cancers [122–124], but remains to be validated in solid cancers. In a meta-analysis of 22 studies published in 2018, the response rate to CAR-T cell therapies was 9% in solid tumors [125]. The administration of CAR-T cells in 17 patients with HER2-positive sarcomas led to a positive response in 4 patients and in 3 of them the tumor could be removed [126]. One explanation for these low response rates is that tumors acquire mutations leading to loss of expression of the CAR-T cell targets [127]. New bispecific CAR-T cells have been developed to limit this immune escape [128]. Overall, the tumor microenvironment is the main barrier to the efficacy of CAR-T cells in solid tumors. Moreover, administration of CAR-T cells is associated with high toxicity: cytokine-release syndrome, neurotoxicity, off-targeted effects, and acute respiratory distress syndrome [129].

Today, new classes of antibodies are undergoing rapid preclinical and clinical development. For instance, by targeting different antigens, bispecific antibodies offer the possibility of assisting and promoting tumor cell destruction by the immune synapse [130]. In 2022, tebentafusp was approved for the treatment of metastatic uveal melanoma. This bispecific antibody targets the gp100 peptide exposed on the surface of HLA-A*02:01 and CD3 [131]. In a phase 3 clinical trial involving 378 patients, tebentafusp showed greater progression-free survival (31% vs. 19%) than conventional treatments (pembrolizumab, ipilimumab, or dacarbazine) [132] and also longer overall survival [132]. In 2022, the European Medicines Agency (EMA) granted market authorization for the bispecific antibody teclistamab for relapsed/refractory multiple myeloma. It targets both the CD3 receptor on T cells and the membrane protein B-cell maturation antigen overexpressed on myeloma cells. In 165 patients with myeloma refractory to three lines of therapy enrolled in a phase 1–2 clinical trial, the overall response rate to teclistamab was 63%, and the mean progression-free survival was 11.3 months [133]. Today, bispecific

antibodies offer broader prospects, and many of them are currently in clinical trials for both solid and hematological cancers.

In conclusion, immunotherapies face many challenges and the response rates rarely exceed 30%. Combining these therapies with chemotherapy or radiotherapy increases these response rates, but the discovery of immune evasion mechanisms remains a key factor for predicting and improving the efficacy of immunotherapies.

## 6. CTC challenges

CTCs are among the most aggressive cancer cells. They are the key players in the metastatic cascade, but little is known about their immune evasion mechanisms. Their interactions with different cell types in the blood compartment often enable them to increase their chances of survival. Targeting CTCs during the different steps of the metastatic cascade could contribute to limit metastasis formation. Therefore, it is essential to identify the mechanisms of CTC immune escape. As most of the known mechanisms involve interactions of proteins expressed on the cell surface (e.g. CD47, PD-L1) and some are regulated by various post-translational modifications (PTMs), genomic tools are not sufficient to identify new immune escape mechanisms.

Several groups have already performed global proteomic analyses of CTCs using different methodological approaches. Sinkala et al. [134] used a microfluidic western blot method for a multiplex protein panel to analyze single CTCs from patients with estrogen receptor-positive breast cancer. Moreover, proteomic profiling of 1–5 CTCs allowed defining a panel of prostate cancer-specific proteins and discriminating between CTCs and white blood cells [135]. In head and neck squamous cell carcinoma, proteomic analysis of CTCs by high-dimensional single-cell mass cytometry allowed the identification of common CTC subgroups. Their proteomic profiles could be exploited to develop new biomarkers and for patient stratification for immune checkpoint inhibitor treatment [136]. Proteomic analysis by MS has been used to analyze CTCs from mice xenografted with breast cancer cells [137].

Nevertheless, a key step in assessing CTC heterogeneity using omic methods is to increase their number because they are extremely rare in the blood compartment. The culture and expansion of CTCs *in vitro* provides quantitative (a large number of CTCs) and qualitative (metastasis-initiating CTCs) added value [138]. In addition, to overcome the low CTC availability in blood samples, it is possible to increase the
volume of blood collected by leukapheresis. In this
way, liters of blood can be screened [139]. The identifi-
cation of new mechanisms of CTC immune evasion
will require the proteomic study of their surface, open-
ing a new and promising avenue for the discovery of
new immunotherapies.

7. Proteomic tools to assess CTC surfaceome

**7.1. Immunopeptidomics**

Research in CTC biology, immune evasion mecha-
nisms and therapeutics targets has long been hampered
by the rarity of these events and the fact that the only
FDA-approved system, the CellSearch® system, uses
fixed cells. Few permanent CTC lines have been estab-
lished for colon, breast and lung cancer, paving the
way for new approaches [140,141]. These CTC lines
represent a rare source of metastasis-initiating cells.
These cells, which have the potential to self-renew and
expand *in vitro*, have all the properties needed to
develop secondary tumors in patients. Proteomic tools
can be very useful to study the surfaceome of these
CTCs in order to understand their escape mechanisms
and to develop new treatments.

Immunopeptidomics defines the study of the immu-
nopeptidome (i.e. the peptides presented by the MHC
molecules) mainly using LC and MS-based methods
[142]. This has already allowed obtaining major
insights into the biosynthesis of peptides, right up to
their binding to HLA molecules, that will be briefly
summarized here. Peptide presentation on the surface
of individual human cells is a dynamic and constant
process and provides a picture of the cell health state
[143]. When a cell is infected or had undergone trans-
formation, its immunopeptidome profile changes and
this is sensed by the immune system. Peptides are pre-
sented to the immune system using two pathways: (a)
The antigen processing and presentation machinery
pathway degrades intracellular ubiquitinated proteins
via the nuclear and cytosolic proteasomes. Then, the
short peptides (8–15 amino acids) are taken to
the endoplasmic reticulum via transporter associated
with antigen processing (TAP) molecules, where they
bind to HLA class I molecules (Fig. 2). These com-
plexes are then exported to the cell surface to be
scanned by the TCR expressed on CD8⁺ T cells [144].
(b) The second pathway for peptide presentation on
the cell surface involves the degradation of endocytic
extracellular proteins in the endosome-lysosome com-
partment. These peptides (up to 25 amino acids) are
then loaded onto HLA class II molecules and exported

to the cell surface. Surface presentation of these com-
plexes to CD4⁺ T cells is exclusively done by profes-
sional antigen-presenting cells, including DCs, B cells,
macrophages and thymic epithelial cells [145]. In can-
cer cells, the presented peptides (neo-peptides or neo-
antigens) may be the result of various gene alterations,
such as non-synonymous mutations that modify the
protein sequence, gene fusions leading to fusion pro-
teins, cancer-specific alternative mRNA splicing or
aberrant PTMs [144].

Therefore, the immunopeptidome is a modulator of
the immune response, and understanding how peptides
are edited is fundamental for the analysis of the inter-
actions between cancer and immune cells. Peptide edit-
ing is regulated by numerous PTMs through enzymes
that can be deregulated in cancer. These PTMs can
lead to tumor-specific immunogenic peptides. Phos-
phorylation, glycosylation and citrullination are the
most widely studied PTMs. In cancer cells, MHC-I
and MHC-II molecules can present phosphorylated
peptides [146,147] that activate CD8⁺ and CD4⁺
T cells [148,149]. The conversion of an arginine residue
to citrulline (citrullination) by deamination is mediated
by protein arginine deiminase (PADI) family members
[150,151]. Some studies suggest that the citrullinome is
a rich source of cancer neo-antigens that are involved
in immune response [151]. Citrullinated peptides are
predominantly presented by MHC-II molecules via
autophagy that is induced by tumorigenesis [152].
Then, these peptides can stimulate the Th1 response
[152]. Therefore, neo-antigens are therapeutic candi-
dates to boost the anti-tumor immune response
[153,154]. In addition, PTMs of peptides presented by
the MHC-I and -II molecules are widely implicated in
anti-tumor immune recognition, but only few of these
PTMs have been studied. In the case of CTCs, no
immunopeptidome data is available yet.

Given that the immunopeptiome is involved in
immune recognition, its analysis is implicated in the
development of immunotherapies. Proteomic tools are
used to identify TAAs and tumor-specific antigens
(TSAs) that can be targeted by immunotherapies
[155,156]. TSAs are ideal targets because they are not
expressed on healthy cells. There are many sources of
TSAs: cancer-induced aberrant PTMs, cryptic pep-
tides, variants resulting from the use of a non-
canonical open reading frame or a reading frame shift,
endogenous retroelements, and defective ribosome
products [156]. Strategies to identify these peptides are
based first on the analysis of next-generation sequenc-
ing data, including exome sequencing, RNA sequenc-
ing and ribosome sequencing. Ribosome profiling
defines the mRNA regions that are translated into
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** MHC class I antigen presentation pathway. Peptide presentation on the major histocompatibility complex I (MHC-I) surface is a multi-step process. After transcription, the RNA of a given protein is taken up by the 30S and 60S ribosomal subunits to initiate translation. Errors in translation lead to the formation of defective ribosomal products (DRiPs). Unfolded or inactive proteins are ubiquitinated and sent to the proteasome where they are degraded into peptides. The proteasome also has a non-genetically encoded ligation activity that leads to ligated peptides. Peptides are then translocated into the lumen of the endoplasmic reticulum (ER) by a transporter associated with antigen presentation (TAP). MHC-I molecules are stabilized and conformed by chaperone proteins, such as tapasin, calreticulin and ERp57. Peptides without affinity for MHC-I molecules are taken up by ER aminopeptidase associated with antigen processing (ERAAP). Once the peptide is bound to MHC-I molecules, the complex is delivered to the membrane to be recognized by the TCR of CD8⁺ T cells. Created with BioRender.com. DRiPs, defective ribosomal products; ERAAP, ER aminopeptidase associated with antigen processing; MHC, major histocompatibility complex; TAP, transporter associated with antigen presentation; TCR, T-cell receptor.</figcaption></figure>

proteins. Then, many bioinformatic tools are available to predict whether a given peptide is likely to be present on the surface. These tools are generally based on the probability of degradation by the proteasome or the affinity between peptide and HLA molecules and are used to refine the analysis [157–160]. Then, HLA-peptide complexes are isolated by immunoprecipitation using specific antibodies and identified by MS. MS data are compared with protein databases or customized databases using computational searching engines, such as MaxQuant [161]. This strategy for identifying TSAs associated with an immune response is at the basis of the design of innovative therapies, such as peptide-based vaccines and CAR-T cells [162–164].

Today, MS is the only unbiased method for identifying peptides expressed on the surface of MHC-I and II molecules. The majority of immunopeptidomic studies use data-dependent tandem MS (MS/MS) acquisition (DDA). Although this method is considered ideal for new target discovery, it lacks the sensitivity to detect less abundant ions and displays low reproducibility [164]. Pak et al. [165] demonstrated that using the data-independent acquisition (DIA) method for immunopeptidomic analyses could improve neo-antigen detection.

Combined with next-generation sequencing methods and prediction algorithms, MS offers a powerful tool to identify immune response mechanisms in the
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Glycosylation to avoid natural killer (NK) cell cytotoxicity. Protein glycosylation begins in the endoplasmic reticulum. It involves the addition of carbohydrates to polypeptide chains by glycosyltransferases and oligosaccharyltransferases. This process is deregulated in cancer, and this often leads to the aberrant addition of sialic acid at the end of the chain. The most common sialic acid in humans is N-acetylneuraminic acid (NeuNAc). Sialic acid molecules bind to the sialic acid-binding immunoglobulin-like lectin 7 (SIGLEC-7) receptor on NK cells. Binding induces phosphorylation of the SIGLEC-7 immunoreceptor tyrosine-based inhibitory motif (ITIM). Upon phosphorylation, this domain can recruit phosphatases (SHP1/2) that block the NKG2D activation pathway, thus preventing NK cell activation. Created with BioRender.com. ITIM, immunoreceptor tyrosine-based inhibitory motif; NK, natural killer; SIGLEC, sialic acid-binding immunoglobulin-like lectin.</figcaption></figure>

context of cancer. Specifically, this strategy and the few available CTC lines [141] could be used for immunopeptidomic analyses to bring needed insights into the HLA-peptide complexes and profiles in CTCs.

## 7.2. Surface glycoproteomics

Glycosylation is a key cellular PTM that regulates a wide range of pathophysiological functions. This PTM involves the addition of monosaccharides and complex carbohydrates to proteins through the coordinated action of many different substrate-dependent enzymes in the endoplasmic reticulum and Golgi apparatus. N-linked glycosylation involves the addition of glycans to the nitrogen of asparagine residues, while O-linked glycosylation targets the oxygen atoms of serine and threonine residues. Most glycoproteins are secreted or sent to the surface. Several studies have shown that deregulation of the glycosylation machinery is

associated with tumor development [166–168]. Aberrant or incomplete glycosylation due to overexpression or deregulation of transferases affects cell adhesion to the basal membrane, cell–cell interactions, ECM remodeling, cell proliferation, genome stability and interaction with immune cells [168].

Glycoproteomics describes the system-wide study of the glycosylation profile, glycan structures and modification sites using mainly LC/MS methods to assign a biological function to glycoproteins [22]. The workflow of a glycoproteomic analysis includes several key steps: sample selection, processing, enzymatic digestion, enrichment and MS analysis. Lectins are involved in various biological functions, such as immune defense and cell–cell interactions, and are considered carbohydrate binders [169]. Therefore, lectins are sometimes used to separate glycoproteins from other proteins in LC–MS approaches or to analyze different profiles. Analytical methods using lectins include multi-lectin
affinity chromatography [170], lectin microarray [171] and enzyme-linked lectin assays [172]. The analysis of glycoproteins expressed on the cancer cell surface appears to be relevant for the discovery of new immune evasion mechanisms because some PTMs may be at the core of interactions with the immune system and CTCs.

The addition of sialic acid by sialyltransferases at the end of glycans is a PTM often deregulated in breast [173], pancreatic [174], ovarian [175] and lung cancer [176]. Sialic acid plays a pivotal role in immunomodulation and immune evasion of cancer cells (Fig. 3). Sialylated proteins are recognized by sialic acid-binding immunoglobulin-type lectins (SIGLEC) expressed on the surface of immune cells [177]. Specifically, SIGLEC-7 and -9 are expressed on the surface of DCs, NK cells, CD8⁺ T cells, and monocytes. These interactions promote immunosuppression and protect cancer cells. Binding of SIGLEC-7 and -9 to their ligands on pancreatic ductal adenocarcinoma (PDAC) cells directs the differentiation of monocytes into macrophages with an immunosuppressive phenotype [177]. Recently, SIGLEC9 mRNA expression was correlated with poor prognosis and high infiltration of immune cells in various cancer types, such as adrenocortical carcinoma, lung adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme and prostate adenocarcinoma [178]. Läubli et al. [179] showed that blocking SIGLEC-9 expressed on the surface of myelomonocytic cells enhanced the neutrophil anti-tumor activity. Additionally, they found that immune surveillance was improved in mice deficient in Siglec-E, the murine equivalent of SIGLEC-9 [179]. Furthermore, overexpression of sialylated glycans on the surface of B16 melanoma cells affects the balance between effector and regulatory T cells (Treg), facilitating the presence of Treg cells and thus orchestrating immune escape [180]. Sialoglycan interaction with SIGLEC-7 and 9 expressed on the NK cell surface reduces their cytotoxicity [181]. This mechanism allows cancer cells in which MHC-I has been downregulated to escape NK cell-mediated lysis.

The glycosylation profile of cancer cell surface proteins greatly influences the invasion process, notably by modulating their interactions with immune cells. The multifactorial modifications leading to abnormal glycosylation have never been investigated in CTCs, and glycoproteomic studies could provide insights into the different mechanisms used by CTCs to bypass the circulating immune system. Some groups are developing tools for proteomic analysis of single cells isolated from blood samples. The current proteomic methods allow working with a reduced number of cells. For

instance, the LTQ Orbitrap Velos Pro Mass Spectrometer allowed differentiating between CTCs and lymphocytes in blood samples, and to categorize CTCs from different cancer types in function of their profiles [182]. Proteomic methods could be powerful tools to discover new biomarkers and potentially new biological mechanisms.

## 8. Conclusions

Immunotherapies have revolutionized cancer treatment, but there are still too many patients who do not respond to these therapies. Cancer cells develop resistance or simply do not express the therapeutic targets. Moreover, there is no targeted therapy against CTCs and particularly metastasis-initiating CTCs. Yet, these are the most aggressive tumor cells and they are thought to be implicated in the immune evasion mechanisms and therapy resistance due to their phenotypic heterogeneity. The available few CTC lines and proteomic tools, such as immunopeptidomic and glycoproteomic assays, could provide the means for an in-depth analysis of the various mechanisms used by CTCs to evade the immune system and targeted therapies. These emerging and innovative tools combined with these metastasis-initiator CTC lines could give a unique chance to pave the way for understanding CTC immune evasion properties and for the development of therapies targeting specific neo-antigens expressed by CTCs.

## Acknowledgements

We thank Elisabetta Andermarcher for assistance with her comments and proofreading that greatly improved the manuscript. C. Alix-Panabières is supported by la Fondation ARC pour la Recherche sur le cancer, and les Fonds de dotation AFER pour la recherche médicale. The figures were created with BioRender.com.

## Conflict of interest

The authors declare no conflict of interest.

## Author contributions

DM (Conceptualization-Equal, Investigation-Lead, Methodology-Equal, Validation-Equal, Writing—original draft-Lead, Writing—review & editing-Lead), JV (Validation-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), CH (Validation-Equal, Writing—original draft-Equal, Writing—review
& editing-Equal), and CAP (Conceptualization-Equal, Methodology-Equal, Supervision-Lead, Validation-Equal, Writing—original draft-Equal, Writing—review & editing-Equal).

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