Abstract:
Pancreatic cancer (PC), a highly
malignant tumor of the digestive system with poor therapeutic response
and low survival rates. In recent
years, immunotherapy have developed rapidly and achieved substantial
results in many malignant neoplasms. However, responses to immunotherapy
in PC are rare and its immunosuppressive and
desmoplastic
tumor microenvironment (TME)
composes an important impediment to their efficacy in PC.
Tumor-associated neutrophils
(TANs) play a crucial role in the PC microenvironment, exerting a
profound influence on PC immunotherapy through establishing a robust
stromal shelter and restraining immune cells to assist PC cells in
immune escape, which may subvert the current situation of immunotherapy
for PC. The purpose of this review is to offer a thorough summary of the
latest progress in comprehending the involvement of TANs in PC
desmoplastic and immunosuppressive functions, as well as to emphasize
the potential therapeutic consequences of focusing on TANs in the
immunotherapy of this destructive ailment. Last but not least, we have
provided an outlook for the future of TANs in PC immunotherapy.
Keyword: Tumor-associated neutrophil, Neutrophil extracellular
trap, Pancreatic cancer, Immunotherapy,Immune-checkpoint-inhibitor
therapy
1: Introduction
Pancreatic ductal adenocarcinoma (PDAC) is regarded as one of the most
perilous and demanding malignancies in clinical practice[1], which
has surpassed breast cancer to become the third primary contributor to
cancer-related fatalities, with projections indicating it will soon
become the second most prevalent cause of cancer-related mortality[2,
3]. The 5-year survival rate of PDAC is only 11%[4], due to the
subtle initial symptoms of the majority of PDAC cases, resulting in
patients being diagnosed during advanced stages of the illness[5].
Currently, surgery and chemotherapy are the most effective clinical
treatment for most PDAC patients. Nevertheless, the surgical
intervention is improbable to affect the result for individuals with
advanced diseases in the late stages or in the local area. Additionally,
over the last ten years, FOLFIRINOX and the pairing of gemcitabine and
nab-paclitaxel (two new chemotherapy programs) have shown specific
efficacy, but the anticipated improvements in survival and limitations
on treatment-related toxicities are not as effective as
expected[6-8]. In the face of pancreatic cancer (PC) treatment,
immunotherapy may give patients hope.
Immunotherapy known as immune-checkpoint-inhibitors (ICIs), including
anti-PD-1/anti-PD-L1, have significantly enhanced results for
individuals diagnosed with melanoma and non-small cell lung
cancer[9]. Certain subsets of B cell leukemia or lymphoma have shown
remarkable clinical responses through the use of adoptive cell therapy,
such as CAR-T therapy utilizing chimeric antigen receptor T
cells[10]. Although immunotherapy has made impressive progress, the
efficacy in the immunotherapy of PDAC is disappointing. Apart from the
less than 1% of patients who have microsatellite instability high
(MSI-H) tumors, PDAC is mostly resistant to immunotherapies approved by
the FDA[11], largely because the fact that PDAC has an
immunologically ’cold’ tumor microenvironment (TME) where myeloid cells
are abundant and CD8+ T cells are usually absent[12]. This helps the
pancreatic tumor evade immune surveillance and anti-tumor disorders.
Furthermore, PC is characterized by elevated desmoplasia and severe
hypoxia, which are common pathological traits and major factors
contributing to the limited efficacy of immunotherapy in treating PC. To
overcome these challenges, there has been a growing interest in
addressing the immunosuppressive TME and transforming it from a ’cold’
to a ’hot’ environment. In this particular scenario, myeloid cells,
specifically neutrophils, are regarded as a potential focus for the
future of PC immunotherapy.
Compared to other types of cancer, tumor-associated neutrophils (TANs)
are immune cells that infiltrate the immunosuppressive PC
microenvironment in significant numbers. TANs can form NETs to promote
pancreatic cancer progress[13]. They facilitate immunosuppression
and desmoplastic function to interfere with immunotherapy through
interaction with other TME cells, upgrading expression of PD-L1/PD-1 and
metabolic reprogramming. Therefore, scientists have progressively
redirected their attention towards targeting TANs in order to enhance
the efficacy of PC immunotherapy, which holds immense promise.
2. Transition of neutrophils to
TANs
2.1: Neutrophils: origin and recruitment to
PC
Neutrophils are derived from precursor cells of the myeloid lineage that
are situated in the bone marrow and other tissues outside the marrow,
such as the spleen. Throughout the early stages of differentiation, the
myeloid progenitors maintain their inclination to differentiate into
both the monocyte/macrophage lineage and the neutrophil lineage, in
addition to the eosinophils and basophils[14, 15].
Neutrophils undergo amplification
and maturation in the bone marrow, proceed into the circulatory system,
and then migrate to the pancreatic tumor, eventually infiltrating the PC
micro-environment[16]. Although neutrophils are typically regarded
as cells with a brief lifespan, they actually have a circulation
half-life of around 8 hours. Nevertheless, certain research indicates
that their lifespan in the circulation is approximately 5.4
days[17-19], which provides the time necessary for TANs to play an
immunosuppressive role in pancreatic cancer.
The entry of neutrophils into the circulatory initiates through
alterations in endothelial cells and proceeds through various stages:
attachment to the blood vessel lining, rolling, adhesion, crawling, and
ultimately transmigration[20]. Neutrophils initially rely on
selectin for rolling near the vascular edge before transitioning to
integrin-mediated adhesion, enabling a firm attachment to endothelial
cells[21, 22]. Subsequently, neutrophils exit the vascular
endothelium by attaching to platelet endothelial cell adhesion
molecules, which can be found on both neutrophils and endothelial
cells[23]. Neutrophils, upon arrival at the vascular basement
membrane, utilize substances like collagenase to break it down,
demonstrating a liking for areas with reduced expression of
extracellular matrix components in order to penetrate the adjacent
tissues. Neutrophils then navigate through intercellular signals between
pericytes, maneuvering along the cell surface until they locate openings
that allow them to exit the vasculature. Once extravasated, neutrophils
display directed movement aligned with the concentration gradient of
chemical signals, ultimately gathering at PC. The chemokine of the CXC
family, is one of the core chemical signals of attracting neutrophils to
PC microenvironment, jointly regulated by granulocyte colony-stimulating
factor(G-CSF) and interleukin-7(IL17). Neutrophils transitioning
functions in humans are mediated by a minimum of seven chemokines
(CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 (IL8)) and two
receptors (CXCR1, CXCR2). Specifically, neutrophils express CXC receptor
CXCR1 and CXCR2, which interact with the CXC family chemokines released
by tumor sources[24]. Monomers and dimers of chemokines from the CXC
family are present, and their conformation can be altered reversibly
upon receptor binding. These changes are associated with both G-protein
and β-arrestin signaling pathways. Activation of G-protein signaling
triggers the activation of different effectors, like calcium channels
and phospholipase C. Conversely, β-arrestin functions as a versatile
adapter and is linked to numerous signaling centers, such as MAP kinase
and tyrosine kinase pathways. Neutrophil migration is facilitated by the
dynamic remodeling of actin through both G-protein and β-arrestin
signaling pathways. During the migration towards PC, neutrophils are
induced by the concentration gradient of CXCR2 ligand, leading them from
lower to higher concentrations[25]. The regulation of neutrophils
proliferation and maturation is significantly influenced by G-CSF.
Simultaneously, the presence of G-CSF diminishes the presence of CXCR4
and its corresponding ligand CXCL12, consequently controlling the
movement of neutrophils[26]. Subsequently, G-CSF collaborates with
CXCR2 and CXCL2 to facilitate the mobilization of neutrophils. Moreover,
IL-17, highly effective in attracting neutrophils, enhances the
production of different chemokines, such as G-CSF, IL6, CCL2, and CXCR2,
ultimately impacting the movement of neutrophils[27-29].
During the recruitment and infiltration of neutrophils, PC cells play a
key role which is mainly derived from the PC cells producing cytokines.
Tumor cells from PDAC are able to attract neutrophils through the
release of CXCL-16, CXCL-8 and CXCL5[30, 31]. The immersion of
neutrophils is aided by the release of IL-1β from PC cells[32]. The
control of these chemokines is highly reliant on the binding of the
nuclear factor-k-gene (NF-κB). In PC cells, the inhibition of KRAS/MEK
triggers the activation of NF-κB signaling, resulting in the release of
CXCL5. Consequently, this leads to an enhanced recruitment of
neutrophils[33]. Conversely, the downregulation of NF-κB signaling
leads to reduced infiltration of MDSCs through the action of CXCL2 and
CXCL5 at low levels[34]. The production of cytokines can be
attributed to various factors, including the impact of inherent genes
and proteins originating from PC cells. The gain-of-function mutation of
the Trp53 gene which suppresses tumors, in PC cells enhances the
chemokines CXCL2 and CXCL5 production, resulting in the recruitment and
infiltration of neutrophils[35]. PDAC cells upregulated SPRY1 gene
expression to control protein derivatives that interacted with ubiquitin
carboxy-terminal hydrolase L1, leading to the activation of the NF-κB
signaling pathway and ultimately enhancing the expression of
CXCL12[36]. For inherent proteins, the deficiency of PC
cell-intrinsic SETD2, a histone H3K36 trimethyltransferase, which is an
inherent protein, enhanced the PI3K-AKT pathway and led to the
overexpression of CXCL1 and GM-CSF, thus contributing to the recruitment
of neutrophils[37]. Reg3g, a soluble small protein expressed in PC
cells, can increase the secretion of IL-10 and transforming growth
factor-β (TGF-β) to facilitate the recruitment of MDSCs[38]. In
addition to influencing the production of chemokines, PC cells enhance
the formation of channels between PDAC cells and neutrophils by
upregulating the gap junction protein beta 3, thereby facilitating the
transfer of cAMP from cancer cells to neutrophils, which promotes the
infiltration[39]. Overall, these discoveries emphasize the intricate
connections between PC cells and neutrophils, offering understanding
into the mechanisms that drive the transformation of neutrophils into
TANs.
2.2: Polarization of neutrophils to
TANs
Neutrophils, the initial cells to reach locations of emerging
inflammation, have a crucial function in inflammation associated with
cancer, often referred to as the ”wound that never heals”. However,
continuous infiltration of neutrophils is a distinctive feature of
long-lasting inflammation and adds to the damage of tissues.
Consequently, due to the inflammatory environment in the tumor cells,
neutrophils undergo stimulation and polarization into various epithelial
types. Based on the anti-tumorigenic and pro-tumorigenic function of
neutrophils, it is widely classified in N1 and N2 types by the academic
community, including the proinflammatory protumor, anti-inflammatory
protumor, proinflammatory antitumor, anti-inflammatory
antitumor[40]. Specific surface markers have been identified in
order to distinguish between the two types. N1 neutrophils exhibit
elevated levels of ICAM-1 and CD95 expression, while displaying reduced
CD182 expression[25]. Conversely, N2 neutrophils demonstrate
diminished levels of ICAM-1 and CD95 expression, while displaying
elevated CD182 expression[25]. The drivers of neutrophils
polarization are complex, involving many cytokines and signal pathways.
TGF-β functions as an immunosuppressive compound within the tumor
microenvironment, causing neutrophils to adopt a protumorigenic
phenotype (N2) type[41]. Blocking TGF-β with the TGF-β receptor
inhibitors SM16, LY2157299, and AGEN1423 result in the accumulation of
neutrophils exhibiting an antitumor phenotype (N1).
I interferon (IFN) induced the
phenotype changes in both mouse and human neutrophils from N2 type to N1
type [42]. There is a high probability that the polarization of N1
and N2 represents an antagonistic signaling pathway involving TGF-β and
type 1 IFN cytokines. Nevertheless, the precise system has yet to be
validated. In addition, In recent years,
HIF-1 α,
BHLHE40, A2A adenosine receptor,
S100A9, TLR4, Lipocalin-2(LCN2), adenosine triphosphate(ATP) and
adenosine(ADO) have been shown to correlate with the polarization of N1
and N2[43-49]. S100A9 disrupts the NF-kB signaling pathway, reducing
the activity of the pro-inflammatory N1 phenotype[47]. A2AAR
together with A2BAR promotes N2 at the expense of N1 activation[49].
The current limitations of TANs studies are due to the absence of
technical proficiency and the intricacy of the populations involved. The
primary emphasis is on the subsequent domains: (1) The categorization of
TANs primarily relies on the M1 and M2 classification of tumor
associated macrophages (TAMs). However, this simple binary partitioning
is far from meeting the current research on TANs. Reassuringly, MDSC has
been recently found as an immunosuppressive neutrophil to broaden the
polarization family of TANs. Furthermore, the rapid progress in
single-cell sequencing techniques has enhanced our comprehension of TAN
polarization. (2) TANs have the ability to assume either the
anti-tumorigenic ’N1’ phenotype or the pro-tumor ’N2’ phenotype
depending on their function, and extensive studies have been conducted
to understand the mechanisms behind promoting N2 polarization.
Nevertheless, the N1 characteristics of TANs exhibit their capacity to
combat tumors, a trait that we must not disregard. Sadly, few studies
related to promoting neutrophils polarization to N1.Targeting the
polarization of TANs to inhibit the transition to N2 and promote it to
N1 is the future of PC immunotherapy. Therefore, studies about N1 need
to be studied further.
Fig.1 The process of neutrophils transforming to TANs
Neutrophils undergo amplification and maturation in the bone marrow,
enter the circulatory system, and then migrate to the PC, infiltrate the
micro-environment, eventually polarize to N1 and N2. The figure was
created using Figdraw
(www.figdraw.com).
3: Neutrophil extracellular
trap(NET)
NET, which is a net-shaped structure composed of nucleic or granular DNA
fibers, polymorphic and chromosome-binding cytoproteins, and particulate
proteins, is formed by neutrophils in the extracellular space. NET
formation follows a regulated and specialized cell death process called
NETosis[50]. NET formation comprises key steps such as nuclear
enlargement, breakdown of the nuclear envelope, merging of nucleic acids
and granule proteins within a spacious intracellular vacuole, discharge
of nuclear content into the cytoplasm, and ultimately, cell membrane
disintegration[51]. Recently, Tumor-derived protein tissue inhibitor
of metalloproteinases-1 (TIMP1) has been studied and found to be a new
mechanism that forms NET in PDAC[52], binding to CD63 on neutrophils
thereby triggering ERK phosphorylation to form NET, which is important
because TIMP1 is overexpressed and secreted in PDAC development[53].
Another mechanism involves the loss of KDM6A and the activation of the
receptor for advanced glycation end products (RAGE), both of which
induce the generation of NET in PDAC[54, 55]. The failure of
immunotherapy in PC can be attributed to various factors, and one such
factor is the involvement of NETs in tumor progression, which includes
PC immunoevasion and crosstalk in the immune system and TME
cells[56] (this will be discussed in the next paragraph). Therefore,
metformin and chloroquine as emerging inhibitors of NET have been seen
as the future of optimizing immunotherapy [57, 58]
4: TANs lead to failure of immunotherapy for pancreatic
cancer
Immune cell entry into the tumour microenvironment is crucial in
anti-tumour immunity for recognizing and eliminating PC cells. However,
the existence of TANs promotes the development of a robust
stromal barrier by interaction
with TME cells thereby hindering this process. Consequently, immune cell
infiltration is impeded, resulting in the failure of immunotherapy.
Moreover, even when immune cells manage to enter the PC
microenvironment, the immunosuppressive factors and hypoxic
microenvironment present suppress their activity[59]. TANs
contribute to this immune evasion mechanism by
enhancing PD-L1 and PD-1
expression, and inducing metabolic reprogramming in hypoxic conditions.
These factors collectively contribute to the overall failure of PC
immunotherapy.
4.1: Interaction with other TME
cells
Pancreatic cancer exhibits a substantial and compact desmoplastic
stroma, which makes up 70–90% of the tumor’s overall volume[60].
The network is intricate and comprises different types of extracellular
matrix (ECM) and stromal cells, like cancer-associated fibroblasts
(CAFs) and pancreatic stellate cells (PSCs), along with
vascular-associated smooth muscle cells, pericytes, endothelial cells,
mesenchymal stem cells, immune cells such as tumor-associated
macrophages (TAMs), neurons, and blood vessels[61, 62].This review
focuses on the interaction between TANs and PSCs, CAFs, TAMs in PC
microenvironment.
Pancreatic stellate cells
(PSCs)
Pancreatic stellate cells (PSCs), which are distinct to the pancreas,
can usually be found in a dormant condition on the outer surface of the
acinus[63]. The differentiation of PSC into myofibroblastic and
inflammatory subtypes is greatly affected by soluble cytokines released
by PC cells[64]. In the desmoplasia, myofibroblastic PSCs play a
significant role, forming a physically protective barrier around PC
cells, providing immunological protection against recognition[65].
Likewise, inflammatory PSCs generate hyaluronic acid (HA), which forms a
physical barrier by inducing external pressure, thereby decreasing the
infiltration of immune cells into the PC microenvironment[66, 67].
PSCs in the orthotopic PDAC model caused the enlargement of TANs,
resulting in heightened infiltration of TANs within the tumor[68].
The combination of PSCs and PC cells greatly amplified this impact. In
contrast, the release of TANs DNA can stimulate the proliferation of
PSCs by activating the signal from the receptor for advanced glycation
end products (RAGE), and this role of DNA in promoting stellate cell
proliferation is eliminated when the receptor is absent[69]. IL-1β
plays a crucial part in the clash between the PSCs and the TANs. In
PDAC, TANs secrete IL-1β to induce PSCs activation enhancements and then
PSCs can also further secrete
IL-1β to recruit/activate TANs, as demonstrated by a study conducted by
Joao Incio and his colleagues[70]. In addition, TANs can alter the
micro-structures of the base of pancreatic cancer by reprogramming
stellar cells[71], and the related mechanism is that TANs regulate
the expression of α-SMA in stellar cells.
Cancer-associated fibroblasts
(CAFs)
CAF, like PSCs, can also generate a stromal barrier to hinder the
infiltration of immune cells, but the diffidence is that there is
limited investigation on the interaction between TANs and CAFs in PC,
primarily concentrating on NETs and CAFs. TANs generating NET adjust the
activity of CAFs and also driven by CAFs. Shin Takesue team applied
treatment with DNase I, a NET inhibitor, suppressed the activation of
CAFs in PDAC mice[72]. Conversely, CAFs released Amyloid β in the
vicinity through CD11b on TANs within the PDAC, thereby triggering NET
formation in a ROS and PDA4-dependent manner[73]. According to the
same study, NET stimulates CAF expansion, contractility, and matrix
component deposition supportive of PDAC growth.
Tumor-associated macrophages
(TAMs)
The previous article described the
recruitment of TANs, and the
recruitment of TAM is also inseparable from the G-CSF and CXC families,
and the related channels also seem to be intervention. For example, the
activated neutrophils that release IL-8 (CXCL8) and TNF-α activate in
the inflammation site and recruit macrophage cells [74]. In terms of
polarization, TGF-β not only promotes N2 polarization but also promotes
M2 polarization, where neutrophils recruit macrophages prior to their N2
polarization. It suggests that TAMs are closely associated with TANs in
a TME. TAMs and TANs are also noteworthy in relation to the development
of stromal barrier in PC. In co-cultivation, TANs and TAMs generated
elevated amounts of oncostatin M (OSM) and IL-11, respectively, compared
to single cultivation[75]. Reprogramming OSM-OSMR signals can induce
fibrous cell formation, thereby enhancing PC growth[76]. Not only
TAMs, but also TANs, secrete OSM to stimulate tumors[77]. However,
TANs and TAMs may be mutual inhibiting relationships in PC. Timothy’s
team conducted therapeutic responses and related studies in mice with
established PDAC tumors with small molecular CCR2 inhibitors and CXCR2,
respectively, in combination with chemotherapy. CXCR2 neutrophils were
found to be elevated in the blood, bone marrow and tumor of human PDAC
patients and associated with low overall survival rates, while hormone
mice showed an increase in CXCR2 neutrophils after treatment targeting
CXCR2 TAMs. Instead, preventing CXCR2 granulocyte mobilization in PDAC
may result in an increase in TAMs [30].
4.2: The immunosuppressive functions of TANs in
PC
Observations of interactions between TANs and T cells are evident in
cases of pancreatic cancer. CD17 and γδT cells can enhance the
recruitment of neutrophils by secreting IL-4[78]. Moreover, the rise
in the ratio of cells generating IFNγ within the activated subsets of
CD4 and CD8 T cells might play a role in the transformation of TAN from
N2 to N1[79].On the contrary, TANs suppress immune cells. The
removal of TANs from PC can additionally hinder the growth of CD8 T
cells and promote the growth and demise of the CD56 T cell[80]. The
powerful immunosuppressive function of TANs is the most direct factor
for PC immunotherapy failure. Our main emphasis is on presenting the
expression of PD-L1/PD-1 and the metabolic reprogramming of TANs in PC.
PD-1,PD-L1 on TANs
The role of PD-L1/PD-1 in tumor immune evasion is important as it
hinders the activation of RAS-ERK1/2 and PI3K/AKT signaling pathways.
This inhibition leads to the suppression of downstream molecules PI3K,
PLCγ2, and ERK, which in turn inhibits the proliferation of T
lymphocytes[81, 82]. Additionally, it reduces IL-2 production and
glucose metabolism[83], resulting in long-term growth
arrest[84]. In general, PD-L1 is found on cells that belong to the
hematopoietic lineage, which encompasses activated T cells, B cells,
monocytes[85, 86], dendritic cells[87], and macrophages[88].
Lymphocytes and myeloid cells naturally express PD-1 on their cell
membranes. It should be emphasized that neutrophils and NETs have the
ability to express PD-L1 and PD-1 as well[89]. TANs and NETs
contribute to immune evasion by interacting in a PD-L1/PD-1-dependent
way, a phenomenon well acknowledged in PC. Xu Wang groups conducted in
vitro experiments to assess the proliferation of cytotoxic T lymphocytes
(CTLs). The findings suggested that the existence of P2RX1-deficient
neutrophils significantly suppressed the proliferation of CTLs.
Nevertheless, the suppressive impact was counteracted upon the
introduction of an anti-PD-1 neutralizing antibody. In addition,
neutrophils lacking P2RX1 were observed to effectively suppress the
cytotoxic activity of OVA-specific CTL. Similarly, suppression was also
subsequently reversed upon the introduction of Anti-PD-1
antibody[90]. Importantly, PDAC liver metastases systematically
attract neutrophils that lack P2RX1, leading to the development of an
immunosuppressive microenvironment in PDAC liver metastases through the
involvement of PD-L1/PD-1[91]. NET is no exception, NETs in PDAC
contain the immunosuppressive ligand PD-L1, which exerts an exhausting
and dysfunctional effect on T cells[92, 93]. Mechanistically, tumor
cells exhibiting elevated glycolysis rates mechanistically discharged
lactic acid into their vicinity, potentially leading to heightened PD-1
expression on TANs[94]. The mechanisms of TANs expressing PD-L1 are
diverse. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a
crucial transcription factor that controls multiple cellular reactions
to environmental stresses[95]. It plays a significant role in
regulating the metabolism of monoxide, purines, fatty acids, and the TCA
cycle[96]. In PDAC, TANs increase reactive oxygen species (ROS)
production to boost Nrf2 function and inhibiting Nrf2 with a chemical
antagonist can increase the expression of PD-L1 on TANs[90].
Furthermore, autophagy is expected to enhance the expression of PD-L1 on
TANs in PDAC. The impact of autophagy on the expression of PD-L1 on
neutrophils has been examined in numerous studies[97, 98], yet the
specific mechanism in PDAC remains uncertain. The research team led by
Shenghong Yang discovered that the absence of autophagy due to the
removal of ATG5 resulted in an increase in the expression of PD-L1 in
PDAC[99]. This suggests that the deletion of ATG5 can activate TBK1,
leading to elevated levels of CCL5[100], that recruited T cells to
produce IFNγ, thereby fueling PD-L1 upregulation.
Currently, the only information available is that TANs facilitate immune
evasion of PC cells by performing immunosuppressive activities via the
PD-L1/PD-1 pathway, while the specific mechanisms behind this process
are still unknown. However, TANs can hinder the activity of T-cells
through a PD-L1-PD-1 reliant mechanism[101-104] in other certain
malignancies. Moreover, they can impede the NK cells’ immune response
against tumors by interacting with the PD-L1 / PD-1 axis[105] and
even potentially modulating the self- toxicity of tumor cells through
binding with PD-1[89]. In conclusion, relevant research in PC is
urgently needed. Contrary to popular belief, a recent investigation
carried out by Keyu Li proposed a contrasting point of view. Multi-omic
analyses were conducted in the research, examining paired pre- and
post-treatment PDAC samples obtained from a platform neoadjuvant study
involving GM-CSF-secreting allogeneic PDAC vaccine (GVAX) vaccine with
or without nivolumab (PD-1).The findings indicated that while TANs have
a greater influence on immune regulation in PDAC TMEs, there was no
observed correlation between PD-L1 TANs and OS in the treatment
arm[106].This implies that in the future, we may need to shift our
focus towards PD-1 TANs; however, there is currently limited research on
this topic.
Metabolic reprogramming
Metabolic reprogramming refers to the changes in energy metabolism of
cells to enable their survival in harsh environments. TANs’ function is
influenced by glucose metabolism, lipid metabolism, tricarboxylic acid
cycle, and amino acid metabolism[107]. This review primarily
concentrates on the regulation of immune cell function by TANs,
specifically emphasizing metabolic reprogramming in glucose and amino
acid metabolism.
Glucose metabolism
The strong fibroinflammatory tissue contributes to making tumors
deprived of oxygen in PC, changing metabolic pathways within the tumor
and resulting in unfavorable survival. TANs have a glycolytic character
and demonstrate a robust commitment to anaerobic glycolysis for energy
production and to support their effector functions[108]. TANs are
capable of performing their immunosuppressive role due to the storage
and buildup of glycogen in neutrophils[109], allowing them to
function effectively in a hypoxic PC microenvironment with restricted
oxygen and glucose availability.
The progression of PC and immunosuppression are promoted by HIF-1α, a
crucial factor that regulates cell adaptation to hypoxia[110, 111].
LDHA, also known as lactate dehydrogenase A, is an enzyme composed of
four subunits. It plays a crucial role in controlling the last stage of
aerobic glycolysis and has the ability to generate L-2 hydroxyglutarate
in low oxygen conditions. This compound hinders the proliferation and
movement of T cells, thereby aiding in the immune evasion of PC
cells[59, 112]. In PC, glucose metabolism helps TANs restrain immune
cells through improving HIF-1α and LDHA expression. The BHLHE40 gene
functions as a link between HIF-1α and LDHA, leading to a notable
increase in the expression of HIF-1α and LDHA, having a suppressive
impact on the production of pro-inflammatory cytokines by CD8 T cells
and the proliferation capacity of lymphocytes[43, 113]. Among these,
by increasing the level of ARG1 in TANs, HIF-1α carries out
immunosuppressive functions[44]. In the meantime, neutrophils
lacking HIF-1α enhanced the anti-cancer function of CTLs and NK cells,
thereby contradicting the immunosuppressive impact of HIF-1α[44]. It
is highly probable that the rapamycin (mTOR) signaling pathway is
responsible for the intricate processes, as the absence of HIF-1α in
mice could lead to heightened mTOR activity in tumor infiltrating CTLs,
resulting in hindered proliferation, cytotoxicity, and granzyme B
expression due to the presence of mTOR inhibitors[114]. Similarly,
TANs exhibit increased LDHA expression, leading to decreased IFNγ and
TNFα expression in CD13 T cells. LDHA is also upregulated in response to
CD3/CD28 activation, resulting in a slight inhibition of T cell
proliferation[43].
Amino acid metabolism
Arginase1 (ARG1), an important enzyme in the urea cycle, breaks down
L-arginine into urea and L-ornithine. This enzyme can limit the immune
response of T cells by depleting arginine, thereby controlling the
immune evasion of PC cells[115], owing to arginine is crucial for
the survival, growth, differentiation, production of cytokines, and
functioning of T cells[116, 117]. Recent research has closely linked
ARG1 to TANs. In PDAC, Jing Zhang and colleagues discovered that a
specific inhibitor of ARG1 could eliminate the inhibition of T cell
proliferation caused by TANs[118]. This indicates that the potent
immunosuppressive effects are mediated through mechanisms related to
ARG1. It is interesting to note that NET has the ability to not only
produce ARG1 but also boost its functionality. The NETs released by TANs
derived from individuals with PDAC establish a microenvironment where
cathepsin S (CTSS) breaks down human ARG1 into various molecular forms
that possess heightened enzymatic activity under normal pH
conditions[119]. Consequently, these enzymatically active aggregates
are formed, leading to the inhibition of T cells.
Fig.2 Interaction between TANs with other TME cells and the
immunosuppressive functions of TANs in PC
TANs promote the development of a robust stromal barrier by interaction
with PSCs, CAFs and TAMs. In addition, TANs enhancing PD-L1 and PD-1
expression, and utilizing metabolic reprogramming in hypoxic conditions
suppress immune cells thereby lead to PC immunoevasion. These are why PC
immunotherapy has failed. The figure was created using Figdraw
(www.figdraw.com).
5: TANs and PC
immunotherapy
Numerous experiments have been carried out to assess the effectiveness
of immunotherapeutic approaches in PDAC, such as immune checkpoint
inhibitors (ICI), adoptive cell transfer, oncolytic virus (OV) therapy,
and agonistic anti-CD40 monoclonal antibody (mAb) therapy, resulting in
notable outcomes. The role of the TANs is closely associated with these
strategies, which is quite fascinating. Studies have shown that
individuals receiving CAR-T cell treatment often encounter decreased
toxicity caused by neutrophils[120], and G-CSF is frequently
employed to handle associated negative responses[121]. Furthermore,
OV treatment enhanced the maturation of DCs and activation of cytotoxic
T cells, resulting in a notable increase in the survival duration of
mice afflicted with PDAC. After the administration of OV-mOX40L, the
combination of OV and OX40L resulted in a synergistic or additive
effect, leading to a decrease in the proportion of N2 neutrophils, a
significant increase in pro-inflammatory N1 neutrophils and an
enhancement of anti-tumor immune responses[122]. Another promising
immunotherapy in PDAC is anti-CD40 mAb. When activated, CD40 is a
cell-surface member of the TNF receptor superfamily that promotes the
activation of antitumor T cells. Consistent with other treatments, TANs
have been linked to reduced survival in patients with PDAC who receive a
combination of an anti-CD40 monoclonal antibody and
gemcitabine[123]. As a result, the focused therapy of TANs has
become increasingly popular in the realm of PC immunotherapy.
5.1: Targeting TANs optimizes PC
immunotherapy
Despite the widespread use of
immunotherapy in clinical practice, it is insufficient on its own to
fully impede the advancement and spread of pancreatic tumors.
Consequently, there is a focus on directing attention towards TANs, as
they have the potential to enhance the effectiveness of immunotherapy
treatments, specifically ICI. The primary approaches for treating PC ICI
that focus on TANs involve suppressing the recruitment and polarization
of TANs, as well as disrupting the immunosuppressive capabilities of
TANs. Currently, the most prevalent treatment is blocking recruitment.
Fig.3 The strategies of treatment on PC ICI targeting to TANs. (1)
Inhibiting neutrophils recruitment to PC. (2) Inhibiting the formation
of NET. (3) Inhibiting neutrophils polarizing to N2 and promoting them
to N1. (4) Inhibiting TANs immunosuppression-related targets. The figure
was created using Figdraw
(www.figdraw.com).
Blocking the recruitment
The receptor CXCR2 is extensively researched as a site of action for
TANs-targeted PC ICI therapy due to its crucial role in the recruitment
and activation of neutrophils. To mobilize and recruit neutrophils, this
G-protein-coupled receptor interacts with different human CXC
chemokines, such as CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8.
Consequently, targeting CXCR2 can be beneficial in reducing TANs in the
PC microenvironment. Indeed, blocking CXCR2 has been demonstrated to
significantly improve the responsiveness to PC anti-PD1
immunotherapy[124]. Blocking CXCR2 enhanced the quantity of CD3,
CD4, CD8 T lymphocytes and decreased suppressive regulatory T cells,
resulting from the impediment of TANs recruitment[33]. Furthermore,
the suppression of CXCR1[125] and CXCR8[126] further enhances
the efficacy of PC ICI treatment.
Similarly, blocking G-CSF and GM-CSF can optimize PC ICI therapy. By
inhibiting the impact of G-CSF and GM-CSF on neutrophil development and
decreasing the recruitment of TANs to the PC microenvironment,
Lorlatinib effectively hinders the progression of PDAC [127]. By
combining with immune checkpoint inhibition, lorlatinib enhances the
effectiveness of PD-1 inhibition, resulting in increased activation of
CD8+ T cells within PDAC tumors[127]. Furthermore, the combination
of Gemcitabine with anti-CSF1 receptor, anti-PD-1, and anti-PD-1 not
only decreased the recruitment and infiltration of TANs but also
enhanced the antitumor efficacy[128].
Blocking the polarization
By function, TANs have the ability to transition into either the
anti-tumorigenic ’N1’ phenotype or the pro-tumorigenic ’N2’ phenotype.
The current treatment strategy is to target the polarization of TANs to
inhibit the transition to N2 and promote it to N1. TGF-β, a widely
studied polarization target for TANs, can convert TANs to N2 specimens.
The growth of PC is significantly inhibited and the immersion of
anti-tumor M1 macrophage cells in TME is enhanced by testing the TGFβ
receptor I kinase inhibitor in combination with the anti-PD-L1 antibody
to treat PC patients[129, 130]. Furthermore, in comparison to ICI
alone, Irreversible electroporation (IRE) has emerged as a new ablative
method for the clinical management of pancreatic cancer[131]. the
combination of IRE with ICI exhibits encouraging therapeutic efficacy in
both pre-clinical and clinical experiments[132]. TANs regulation
with TGF inhibitors can enhance PC response to IRE and immunotherapy
combined[133]. IFN can promote the polarization of TANs to N1
epithelium, and has been used in PC ICI. TMAO is a metabolite produced
by microorganisms in the gut. A recent study found that TMAO seems to
enhance the type-I IFN pathway, thereby increasing the effectiveness of
checkpoint immunotherapy and improving the survival of mice with
tumors[134].
Blocking the Immunosuppression-related
targets
Until recent years, some immunosuppressive mechanisms of TANs in PC have
been explained. Although, significant researches have been conducted on
targeting immunosuppression-related targets, implementation of these
findings in the clinical setting is still lacking. In the PC setting,
Methotrexate-induced microparticles derived from tumor cells have been
discovered to boost T-cell antitumor reactions by reducing the
expression of PD-1 in TANs. The enhancement of the curative impact of
individual anti-PD-L1 therapy has been proven[94]. Furthermore, when
it comes to NETs, IL17 can recruit neutrophils, induce NET formation,
and eradicate cytotoxic CD8 T cells from the tumor[78]. By
inhibiting IL17 in this process, the sensitivity of immune system
blockage (PD-1, CTLA4) can be enhanced, leading to an improvement in PC
immunotherapy[78]. NETs contain non-cellular ARG1 released by
activated bone marrow cells, which is another significant aspect of PC.
When an ARG1 inhibitor is combined with ICIs, it can reverse the
inhibition of T lymphocytes and restore the function of CD8+T cells in
PDAC tumors within the extracellular milieu[119].
Table 1 Clinical study on targeting TANs optimizing PC ICI