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Vol.:(0123456789)
Human Cell
https://doi.org/10.1007/s13577-024-01024-7
RESEARCH ARTICLE
Prognostic significance andimmune escape implication
oftumor‑infiltrating neutrophil plasticity inhuman head andneck
squamous cell carcinoma
XiaokeZhu1 · YuHeng1· DuoZhang1· DiTang1· JianZhou1· HanqingLin1· JingyuMa1· XupingDing2· LeiTao1·
LimingLu2
Received: 20 August 2023 / Accepted: 27 December 2023
© The Author(s) under exclusive licence to Japan Human Cell Society 2024
Abstract
Tumor-infiltrating neutrophils play a crucial role in the progression of head and neck squamous cell carcinoma (HNSCC).
Here, we aimed to statistically quantify the plasticity of HNSCC-infiltrating N2/N1 neutrophils and examine its impacts on
survival and immune infiltration landscape. A retrospective study of 80 patients who underwent curative surgical resection
for HNSCC between 2014 and 2017 was conducted in this study. HNSCC-infiltrating neutrophil phenotypes were classified
using immunofluorescence staining, and the N2/N1 neutrophil plasticity was evaluated via the ratio of N2/N1 neutrophils. We
then assessed the correlations between N2/N1 neutrophil plasticity, clinicopathological characteristics, and immune infiltra-
tion landscape using rigorous statistical methods. Infiltration variations of N1 and N2 neutrophils were observed between
the tumor nest (TN) and tumor stroma (TS), with TN exhibiting higher N2 neutrophil infiltration and lower N1 neutrophil
infiltration. High ratios of N2/N1 neutrophils were correlated with advanced TNM stage, large tumor size and invasion of
adjacent tissue. High infiltration of N2 neutrophils was associated with decreased overall and relapse-free survival, which
were opposite for N1 neutrophils. The independent prognostic role of N2/N1 neutrophil plasticity, particularly within the
TN region, was confirmed by multivariate analyses. Moreover, the ratio of N2/N1 neutrophils within the TN region showed
correlations with high CD8+ T cells infiltration and low FOXP3+ Tregs infiltration. We identify HNSCC-infiltrating N2/
N1 neutrophil plasticity as a crucial prognostic indictor which potentially reflects the tumor microenvironment (TME) and
immune escape landscape within HNSCC tissues. Further investigations and validations may provide novel therapeutic
strategies for personalized immunomodulation in HNSCC patients.
Keywords Head and neck squamous cell carcinoma· Tumor-infiltrating neutrophils· Neutrophil plasticity· Prognoses·
Immune infiltration landscape· Immunosuppressive microenvironment
Abbreviations
ANT Adjacent normal tissues
CI Confidence interval
H&E Haematoxylin & eosin
HGF Hepatocyte growth factor
HPV Human papillomavirus
HR Hazard ratio
IF Immunofluorescence
IHC Immunohistochemistry
MMP9 Matrix metalloproteinase 9
NETs Neutrophil extracellular traps
OS Overall survival
PGE2 Prostaglandin E2
RFS Relapse-free survival
TANs Tumor-associated neutrophils
TME Tumor microenvironment
Xiaoke Zhu and Yu Heng contributed equally to this work.
* Lei Tao
doctortaolei@163.com; lei.tao@fdeent.org
* Liming Lu
lulunew2003@163.com; youlanda2009@sjtu.edu.cn
1 Department ofOtolaryngology, Shanghai Key Clinical
Disciplines ofOtorhinolaryngology, Eye Ear Nose
andThroat Hospital, Fudan University, 83 Fenyang Road,
Shanghai200031, People’sRepublicofChina
2 Department ofImmunology andMicrobiology,
Shanghai Institute ofImmunology, Shanghai Jiao Tong
University School ofMedicine, Shanghai200025,
People’sRepublicofChina
X.Zhu et al.
TN Tumor nest
TS Tumor stroma
HNSCC Head and neck squamous cell carcinoma
TMA Tissue microarray
Introduction
Head and neck squamous cell carcinomas (HNSCC) repre-
sent the sixth most common malignancy worldwide, encom-
passing cancers of oral cavity, pharynx and larynx [1]. The
pathogenesis of HNSCC can be attributed to a range of etio-
logical factors, including alcohol abuse, tobacco consump-
tion, and infection with human papillomavirus (HPV) [2,
3]. Although significant advancements have been made in
the diagnosis and treatment of HNSCC, the 5-year overall
survival (OS) rate for locally advanced HNSCC remains
suboptimal at approximately 50%, with even poorer progno-
ses observed in HPV-negative HNSCC cases [4, 5]. Recent
studies have elucidated the complex tumor microenviron-
ment (TME), which comprises a heterogeneous infiltrating
immune cell and stromal cell compartment that interacts
with neighboring tumor cells [6–8]. These interactions
are crucial for the growth, progression, and metastasis of
HNSCC [6].
HNSCC is recognized for its immunogenic nature, and
several studies have reported favorable outcomes of immu-
notherapy using immune checkpoint inhibitors (e.g., anti-
PD1/PDL1 blockade) in the treatment of a particular sub-
set of HNSCC patients [9, 10]. Nevertheless, the response
rate to anti-PD1 immunotherapy remains relatively low
at approximately 20%, potentially due to the variation in
immune cell infiltration landscape among patients [5, 6].
This underscores the pressing need to identify specific
HNSCC-infiltrating cell subtypes correlated with prognoses
and develop multimode therapeutic strategies for remodel-
ling the HNSCC TME.
Accumulating evidences highlight the crucial role of
tumor-infiltrating macrophages as a key immune cell popu-
lation within the TME, exhibiting significant plasticity in
both M2-polarized pro-tumoral and M1-polarized antitu-
moral phenotypes [11, 12]. As another critical immune cell
population within the TME, tumor-associated neutrophils
(TANs) also substantially contribute greatly to the HNSCC
microenvironment, with both peripheral neutrophils and
TANs implicated in tumor progression [13–15]. Despite
this, the precision role of TANs remains to be elucidated;
some studies have associated TANs with favorable outcomes
[16], while others have linked them to poor prognosis [7,
17]. TANs can exert antitumoral function by modulating
IL12 production in macrophages and orchestrating antitumor
immunity [16]. Conversely, TANs may also facilitate tumor
progression and metastasis through directly promoting
tumor cell proliferation, angiogenesis, immunosuppres-
sion, and extracellular matrix remodeling and extravasation
[17–19]. Therefore, the neutrophil diversity and plasticity
of tumors may undertake the two-sided functions of TANs
within the TME.
In a manner akin to tumor-infiltrating macrophages,
Fridlender etal. [20] reported that TANs can be polarized
into two distinctive phenotypes: pro-tumoral N2 neutro-
phils and antitumoral N1 neutrophils. N2 neutrophils pro-
mote tumor cell proliferation and contribute to angiogen-
esis through the secretion of cytokines such as neutrophil
elastase, prostaglandin E2 (PGE2), and matrix metallopro-
teinase 9 (MMP9) [21]. They also induce immunosuppres-
sion by upregulating the expression of immune checkpoints
(e.g., PD-L1 and VISTA) [7, 19], and recruiting immuno-
suppressive cells into the TME (e.g., regulatory T cells) [22].
Although TANs have been shown to play a prognostic role
in tumors [7], the plasticity and implications of HNSCC-
infiltrating N2/N1 neutrophils remains uncertain.
In this study, we aimed to investigate the plasticity of
HNSCC-infiltrating N2/N1 neutrophils within the TME and
explore its correlation with the infiltration of other immune
cells. Moreover, we sought to determine the prognostic role
of HNSCC-infiltrating N2/N1 neutrophils. To our knowl-
edge, this is the first study to evaluate the prognostic impli-
cations of the plasticity of HNSCC-infiltrating N2/N1 neu-
trophils in HNSCC patients undergoing curative resection.
Material andmethods
Study population andtissue microarrays
In this study, we recruited a cohort of HNSCC patients at our
medical center who underwent curable surgical treatments
between June 2014 and September 2017. The inclusion and
exclusion criteria for candidates were stringent, requiring
a de novo pathohistological diagnosis of HNSCC without
prior treatment and the absence of distant metastasis. This
study adhered to the principles of the Declaration of Helsinki
and received approval from the Medical Research Council
of the Eye & ENT Hospital, Fudan University, Shanghai,
China (No. KJ2008-01). Furthermore, participation in the
study was consented by all patients.
To examine the plasticity of HNSCC-infiltrating N2/
N1 neutrophils within the TME, tissue microarray (TMA)
samples were collected from HNSCC patients, consisting
of 80 pairs of tissue specimens, including HNSCC tissues
and adjacent normal tissues (ANT). The HNSCC TMAs
were serially sliced, with each TMA section contain-
ing 4-μm-thick tissues. Our study collected the following
clinical and pathological data: age, sex, smoking and drink-
ing history, history of vocal leukoplakia, primary subsite,
Prognostic significance andimmune escape implication oftumor‑infiltrating neutrophil…
pathological grade, tumor size, clinical stage, metastatic
lymph node status, and follow-up data.
Immunohistochemistry andimmunofluorescence
Haematoxylin & eosin (H&E), immunohistochemistry (IHC)
staining and immunofluorescence (IF) staining were per-
formed on formalin-fixed and paraffin-embedded sections of
TMA, as previously describe [7, 11]. H&E staining was uti-
lized to evaluate routine histological features and delineate
the tumor stroma (TS) and tumor nest (TN) areas (Supple-
mentary Fig.1), which were confirmed by two board-certi-
fied pathologists. Following the manufacturer’s instructions,
IHC staining was performed using the specified antibod-
ies: anti-CD3 (Abcam, ab16669, 1:150), anti-CD4 (Abcam,
ab133616, 1:500), anti-CD8(Abcam, ab217344, 1:2000),
anti-FOXP3(Abcam, ab20034, 1:500). Three high-power
fields (HPF) of version at 400× magnification were used to
estimate the average number of cells per HPF.
For IF staining, the primary antibodies used were anti-
CD66b (BioLegend, 305102, clone G10F5, 1:100), anti-
CD206 (Abcam, ab252921, 1:1000), anti-iNOS (Abcam,
ab283655, 1:200) monoclonal antibodies, which were
incubated at 4℃ overnight. Subsequently, TMA sections
were incubated in the dark for 60min with Cy3 (Abcam,
ab97035, 1:200), Alexa Fluor 488 (Abcam, ab150077,
1:200), and Cy5 (Abcam, ab6564, 1:200). Finally, DAPI
(Solarbio, C0060) was used for a 20-min incubation. The
results were visualized using a fluorescence microscope
(Olympus). CD66b+CD206+iNOS− neutrophils and a few
CD66b+CD206+iNOS+ neutrophils were defined as HNSCC-
infiltrating N2 neutrophils, while CD66b+CD206−iNOS+
neutrophils and a few CD66b+CD206−iNOS− neutrophils
were defined as HNSCC-infiltrating N1 neutrophils (Sup-
plementary Fig.2) [23]. The average numbers of N2 and N1
neutrophils per HPF in the TN, TS, combined tumor area
and ANT, respectively, were counted across three separate
400× HPF.
Study outcomes
The primary outcome of this study was overall survival
(OS), calculated from the date of curative surgery to all-
cause mortality or the last follow-up. The secondary out-
come was relapse-free survival (RFS), defined as the interval
from surgery to any disease recurrence (local, regional, or
distant).
Statistical analysis
All statistical analyses were performed using GraphPad
version 7.0 and SPSS software version 24.0. Categorical
variables were presented as frequency (percentage), and
continuous variables were presented as median (range). The
optimal cutoff values of the immune cells were determined
using the X-tile software, with the cutoff value of N2/N1
plasticity set at 1. Chi-square (χ2) tests were employed to
evaluate the associations between HNSCC-infiltrating N2/
N1 neutrophils and clinicopathological characteristics, while
Spearman’s correlation analyses were performed to assess
the relationships between two continuous variables. Survival
was presented using the Kaplan–Meier curves and compared
using the log-rank tests. Univariate and multivariate analyses
were conducted to assess the independent prognostic role of
variables. Sensitivity analyses were performed by restricting
cases to patients diagnosed with laryngeal SCC and those
who were older than 65years. Statistical significance was
deemed when the p value < 0.05.
Results
Patient characteristics
All patients included in this study were male, with a signifi-
cant proportion having a history of smoking (n = 56, 70%)
and drinking (n = 43, 53.8%). The majority of patients had
tumors originating from the larynx (n = 61, 76.3%), while
the remainder had tumors from the hypopharynx (n = 19,
23.8%). A total of 62 (77.5%) patients presented with
advanced T stage (i.e., T3–4), while 41 (51.2%) had adverse
N stage (i.e., N1–2). The detailed demographic and clinico-
pathological features of the cases were presented in Table1.
HNSCC‑infiltrating N2/N1 neutrophil plasticity
intheTME ofHNSCC
According to the indication of H&E staining, the num-
ber of CD66b+CD206+iNOS− N2 neutrophils and
CD66b+CD206−iNOS+ N1 neutrophils in the TN, TS, com-
bined tumor area and ANT were calculated, respectively
(Fig.1A, Supplementary Fig.2) [23]. Our study observed
a higher median number of N1 neutrophils (8 [range, 1–31]
in HNSCC vs. 4 [range, 0–13] in ANT) and N2 neutrophils
(4 [range, 0–41] vs. 0 [range, 0–4]) in HNSCC tissue than in
the ANT, which were confirmed by both unpaired and paired
analyses (Supplementary Fig.3, Supplementary Table1).
The ratio of HNSCC-infiltrating N2/N1 neutrophils was also
higher in HNSCC tissue (0.5 [range, 0–4.2]) than that of
ANT (0, [range 0–2]).
The count of HNSCC-infiltrating N1 neutrophils was higher
than that of N2 neutrophils in the TS region (p < 0.0001) and
combine tumor area (p = 0.0023), while the number of two
neutrophil phenotypes were comparable in the TN (p = 0.8411)
as indicated by both unpaired and paired analyses (Fig.1B–D).
The number of N1 neutrophils was highest in the TS, while
X.Zhu et al.
the number of N2 neutrophils and the ratio of N2/N1 neu-
trophils were highest in the TN (Fig.1E–G). The number of
total CD66b+ neutrophils were comparable in the TN and TS
regions (p = 0.5421, Fig.1H).
Correlations betweentheplasticity ofN2/N1
neutrophils andclinicopathological features
The cutoff values of the HNSCC-infiltrating N1 and N2 neu-
trophils in the TN were determined as 3 and 2, respectively.
This resulted in the categorization of 35 cases into the TN high
HNSCC-infiltrating N1 subgroup and 43 patients into the TN
high HNSCC-infiltrating N2 subgroup. Notably, high infiltra-
tion of HNSCC-infiltrating N1 neutrophils in the TN was sig-
nificantly related to primary tumor subsite (p = 0.022), T stage
(p = 0.026), as well as history of diabetes (p = 0.037) but not
with other features (Supplementary Table2). Conversely, the
high infiltration of HNSCC-infiltrating N2 phenotype in TN
was significantly related only to smoking history (p = 0.016)
and N stage (p = 0.026, Supplementary Table3). In the TS
region, the determined cutoff values of the HNSCC-infiltrat-
ing N2 and N1 phenotypes were 1 and 5, respectively. This
resulted in 39 patients being classified as TS high HNSCC-
infiltrating N1 subgroup and 41 patients as TS high HNSCC-
infiltrating N2 subgroup. No significant correlations were
identified between high infiltration of TS N1 neutrophils
and other factors, while the high infiltration of TS N2 neu-
trophils displayed a notable correlation with smoking history
(p = 0.036, Supplementary Table2,3). In terms of combined
tumor area, the threshold values of the HNSCC-infiltrating
N1 and N2 phenotypes were 7 and 4, respectively. Correlation
analyses revealed that high infiltration of N2 neutrophils in
combined tumor area was significantly related solely to smok-
ing history (p = 0.032, Supplementary Table2,3).
Furthermore, the HNSCC-infiltrating N2/N1 plasticity
was assessed by the N2/N1 ratio to account for variations
between N2 and N1 neutrophils. Patients with an N2/N1
ratio exceeding 1 were classified into the high N2/N1 ratio
subgroup. Correlation analyses (Table2) revealed that a high
N2/N1 ratio within the TN was significantly associated with
smoking history (p = 0.009), T stage (p = 0.004), N stage
(p = 0.005), TNM stage (p = 0.011), tumor size (p = 0.033),
and invasion of adjacent tissue (p = 0.006). A high N2/N1
ratio within the TS was significantly related only to N stage
(p = 0.024), and a high ratio of N2/N1 phenotypes in the
combined tumor area was associated with smoking history
(p = 0.034), primary site (p = 0.031), and N stage (p = 0.006).
Relationship betweentheinfiltration ofN2/N1
neutrophils andother immune cells inHNSCC tissue
The TMA sections were additionally stained with CD3, CD4,
CD8 and FOXP4 to gain a more profound understanding
Table 1 Demographic and clinicopathological characteristics of the
patients with HNSCC
HNSCC head and neck squamous cell carcinoma, LNs lymph nodes,
Tre g regulatory T cell
Variables
Total, n 80
Age at diagnosis, median (range) 63 (46–80)
Smoking history, n (%)
No 24 (30%)
Yes 56 (70%)
Drinking history, n (%)
No 37 (46.2%)
Yes 43 (53.8%)
Diabetes, n (%)
No 69 (86.3%)
Yes 11 (13.2%)
Hypertension, n (%)
No 57 (71.3%)
Yes 23 (28.7%)
History of vocal leukoplakia, n (%)
No 77 (96.3%)
Yes 3 (3.7%)
Primary site, n (%)
Larynx 61 (76.3%)
Hypopharynx 19 (23.8%)
T stage, n (%)
T1–2 18 (22.5%)
T3–4 62 (77.5%)
N stage, n (%)
N0 39 (48.8%)
N1–2 41 (51.2%)
TNM stage, n (%)
I–II 11 (13.8%)
III 69 (86.2%)
Tumor size, n (%)
≤ 3.5cm 40 (50.0%)
> 3.5cm 40 (50.0%)
Differentiation, n (%)
Well or moderately 71 (88.8%)
Poorly 9 (11.3%)
Number of metastatic LNs, n (%)
0 39 (48.8%)
1 14 (17.4%)
> 1 27 (33.8%)
Invasion of adjacent tissue, n (%)
No 25 (31.3%)
Yes 55 (68.2%)
HNSCC-infiltrating CD3+ T cells, median (range) 100 (7–301)
HNSCC-infiltrating CD4+ T cells, median (range) 76 (5–223)
HNSCC-infiltrating CD8+ T cells, median (range) 18 (1–151)
HNSCC-infiltrating FOXP3+ Treg cells, median
(range)
8 (0–167)
Prognostic significance andimmune escape implication oftumor‑infiltrating neutrophil…
N1 N2
0
5
10
15
20
25
Cell count / HPF
N1 N2
0
10
20
30
40
Cell count / HPF
ns
N1 N2
0
5
10
15
20
25
Cell count / HPF
N1 N2
0
10
20
30
40
Cell count / HPF
ns
Tumor
S
trom
a
Tu
m
o
r N
est
N1 N2
0
10
20
30
40
50
Cell count / HPF
N1 N2
0
10
20
30
40
50
Cell count / HPF
C
o
m
b
in
ed
T
u
m
o
r Ar
ea
TN TS ANT
0
5
10
15
20
25
Cell count / HPF
TN TS ANT
0
5
10
15
20
25
Cell count / HPF
TN TS ANT
0
10
20
30
40
Cell count / HPF
TN TS ANT
0
10
20
30
40
Cell count / HPF
TN TS ANT
0
10
20
30
40
50
Cell count / HPF
ns
TN TS ANT
0
10
20
30
40
50
Cell count / HPF
ns
TN TS ANT
0
2
4
6
8
10
N2/N1 Ratio
TN TS ANT
0
2
4
6
8
10
N2/N1 Ratio
N1
N
2
Total Neutrophils
N2
/
N1 Ratio
B
C
D
E
G
F
H
50 50 50
50 50 50
50 50 50
50 50 50
50 50 50
200 200 200 200 200 200
A
Fig. 1 Identification of the HNSCC-infiltrating N2/N1 neutro-
phils plasticity. Representative immunofluorescence staining (A) of
HNSCC-infiltrating N2 neutrophils (yellow arrow) and N1 neutro-
phils (white arrow); paired and unpaired comparisons of the counts of
HNSCC-infiltrating N2/N1 neutrophils in TN (B), TS (C), and com-
bined tumor area (D). Paired and unpaired comparisons of HNSCC-
infiltrating N1 neutrophil counts (E), N2 neutrophil counts (F), N2/
N1 ratios (G) and total neutrophil counts (H) within different regions
(TN vs. TS vs. ANT). ****p < 0.0001, ***p < 0.001, **p < 0.01, ns
p > 0.05. TN Tumor nest, TS tumor stroma, ANT adjacent normal tis-
sue, HPF high-power field
X.Zhu et al.
Table 2 Associations between HNSCC-infiltrating N2/N1 neutrophils ratio and clinicopathological features among patients with HNSCC
Supplementary Table2 and 3 show the associations between HNSCC-infiltrating N2 or N1 neutrophils and clinicopathological features
HNSCC head and neck squamous cell carcinoma, LNs lymph nodes, Treg regulatory T cell
Bold = P value meets definition for statistical significance
Variables Tumor nest Tumor stroma Combined tumor area
Low N2/N1 High N2/N1 p value Low N2/N1 High N2/N1 p value Low N2/N1 High N2/N1 p value
Age at diagnosis 0.388 0.266 0.716
< 65/ ≥ 65 28/25 17/10 39/27 6/8 35/26 10/9
Smoking history 0.009 0.158 0.034
No/yes 21/32 3/24 22/44 2/12 22/39 2/17
Drinking history 0.098 0.384 0.346
No/yes 28/25 9/18 32/34 5/9 30/31 7/12
Diabetes 0.625 0.429 0.768
No/yes 45/8 24/3 56/10 13/1 53/8 16/3
Hypertension 0.690 0.188 0.396
No/yes 37/16 20/7 45/21 12/2 42/19 15/4
History of vocal leukoplakia 0.988 0.416 0.691
No/yes 51/2 26/1 63/3 14/0 59/2 18/1
Primary site 0.151 0.822 0.031
Larynx/Hypopharynx 43/10 18/9 50/16 11/3 50/11 11/8
T stage 0.004 0.916 0.152
T1–2/T3–4 17/36 1/26 15/51 3/11 16/45 2/17
N stage 0.005 0.024 0.006
N0/N1–2 32/21 7/29 36/30 3/11 35/26 4/15
TNM stage 0.011 0.429 0.219
I–II/III 11/42 0.27 10/56 1/13 10/51 1/18
Tumor size (cm) 0.033 0.556 0.431
≤ 3.5/ > 3.5 31/22 9/18 34/32 6/8 32/29 8/11
Differentiation 0.471 0.692 0.909
Well or moderately/poorly 48/5 23/4 50/7 12/2 54/7 17/2
Number of metastatic LNs 0.492 0.169 0.387
0/1/ > 1 29/8/16 11/5/11 36/9/21 4/4/6 32/8/21 8/5/6
Invasion of adjacent tissue 0.006 0.812 0.096
No/yes 22/31 3/24 21/45 4/10 22/39 3/16
Tumor-associated CD66b+
neutrophils
0.699 0.208 0.079
Low/high 24/29 11/16 31/35 4/10 30/31 5/14
HNSCC-infiltrating CD3+ T
cells
0.237 0.239 0.793
Low/high 24/29 16/11 35/31 5/9 31/30 9/10
HNSCC-infiltrating CD4+ T
cells
0.582 0.014 0.361
Low/high 26/27 15/12 38/28 3/11 33/28 8/11
HNSCC-infiltrating CD8+ T
cells
0.001 0.941 0.005
Low/high 23/30 22/5 37/29 8/6 29/32 16/3
HNSCC-infiltrating FOXP3+
Tregs
0.030 0.222 0.120
Low/high 35/18 11/16 40/26 6/8 38/23 8/11
Prognostic significance andimmune escape implication oftumor‑infiltrating neutrophil…
of immune cytolysis and suppression mechanisms within
HNSCC tissue. The median number in the TMA was 100
(range 7–301) for CD3+ cells, 76 (range 5–223) for CD4+
T cells, 18 (range 1–151) for CD8+ T cell, and 8 (range
0–167) for FOXP3+ Treg cells (Table1). The optimal cutoff
values were determined as 100 for CD3+ cells, 80 for CD4+
T cells, 20 for CD8+ T cells, and 15 for FOXP3+ Tregs.
Interestingly, a higher number of CD8+ T cell was corre-
lated with improved survival (OS: p < 0.0001, HR 0.115,
95% IC: 0.034–0.384; RFS: p = 0.0111, HR 0.338, 95% CI
0.140–0.817). In contrast, elevated infiltration of FOXP3+
Tregs was associated with worse survival (OS: p = 0.0019,
HR 3.502, 95% IC: 1.528–8.023; RFS: p = 0.0051, HR
3.538, 95% CI 1.530–8.182, Fig.2, Supplementary Table4).
However, no correlations were observed between survival
and CD4+ T cells or CD3+ T cells (Fig.2, Supplementary
Table4).
High infiltration of HNSCC-infiltrating N2 phenotype,
especially within TN, was associated with low CD8+ T
cells infiltration and high FOXP3+ Tregs infiltration (Sup-
plementary Fig.4), while no significant correlations were
identified for HNSCC-infiltrating N1 phenotype (Supple-
mentary Fig.5). Moreover, a high N2/N1 ratio in the TN
was significantly related to low CD8+ T cells infiltration
(p = 0.001) and high FOXP3+ Tregs infiltration (p = 0.030),
as evidenced by χ2 tests (Table2) and further confirmed
through unpaired t tests (Fig.3) and Spearman’s correla-
tion analyses (Fig.3). In addition, our study evaluated the
correlation between HNSCC-infiltrating N2/N1 neutrophil
plasticity and the ratios of CD8/Treg, CD8/CD3 and Treg/
CD3, which were all significantly associated with survival
(Supplementary Fig.6–8). These intriguing findings sug-
gest that a high N2/N1 ratio may be related to lower CD8/
CD3 ratio, lower CD8/Treg ratio and higher Treg/CD3
ratio, with more significant associations observed within
the TN (Supplementary Fig.6–8). These correlations may
potentially indicate that HNSCC-infiltrating N2/N1 neu-
trophil plasticity reflects the immune escape landscape of
the TME.
Absent Present
CD8
Treg
CD3
CD4
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low CD8
High CD8
P < 0.0001
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low Treg
High Treg
P = 0.0019
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low CD4
High CD4
P = 0.4902
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low CD3
High CD3
P = 0.1189
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low CD8
High CD8
P = 0.0111
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low Treg
High Treg
P = 0.0051
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low CD4
High CD4
P = 0.8383
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low CD3
High CD3
P = 0.5060
CD
8
T
reg
C
D
4
CD
3
BA
C
D
E
50 50
50 50
50 50
50 50
Fig. 2 Representative immunohistochemistry staining for CD8+
T cells, Tregs, CD4+ T cells and CD3+ T cells (A). Kaplan–Meier
curves for OS and RFS by the infiltration variation of CD8+ T cells
(B), Tregs (C), CD4+ T cells (D) and CD3+ T cells (E). RFS relapse-
free survival, OS overall survival
X.Zhu et al.
0246810
0
50
100
150
200
Ratio of N2/N1
Count of CD8+ T cells / HPF
Pearson r = -0.2827
P value = 0.0111*
0246
0
50
100
150
200
Ratio of N2/N1
Count of CD8+ T cells / HPF
Pearson r = 0.0234
P value = 0.8368
012345
0
50
100
150
200
Ratio of N2/N1
Count of CD8+ T cells / HPF
Pearson r = -0.1668
P value = 0.1391
0246810
0
50
100
150
200
Ratio of N2/N1
Count of FOXP3+ Treg cells / HPF
Pearson r = 0.2253
P value = 0.0445*
0246
0
50
100
150
200
Ratio of N2/N1
Count of FOXP3+ Treg cells / HPF
Pearson r = 0.2023
P value = 0.0719
012345
0
50
100
150
200
Ratio of N2/N1
Count of FOXP3+ Treg cells / HPF
Pearson r = 0.2187
P value = 0.0498
0246810
0
50
100
150
200
Ratio of N2/N1
Count of CD4+ T cells / HPF
Pearson r = 0.0452
P value = 06905
0246
0
100
200
300
Ratio of N2/N1
Count of CD4+ T cells / HPF
Pearson r = 0.2064
P value = 00662
012345
0
100
200
300
Ratio of N2/N1
Count of CD4+ T cells / HPF
Pearson r = 0.1276
P value = 02593
0246810
0
50
100
150
200
Ratio of N2/N1
Count of CD3+ T cells / HPF
Pearson r = -0.0322
P value = 07769
0246
0
100
200
300
Ratio of N2/N1
Count of CD3+ T cells / HPF
Pearson r = 0.1577
P value = 01624
012345
0
100
200
300
Ratio of N2/N1
Count of CD3+ T cells / HPF
Pearson r = 0.0650
P value = 05668
T
umor nes
t
T
umor stroma
Combined tumor area
0
50
100
150
200
0
50
100
150
200
ns
0
50
100
150
200
0
50
100
150
200
0
50
100
150
200 ns
0
50
100
150
200
0
50
100
150
200
250
ns
0
50
100
150
200
250
0
50
100
150
200
250
ns
0
100
200
300
400
ns
0
100
200
300
400
ns
0
100
200
300
400 ns
CD
8
T
re
g
C
D
4
CD
3
C
D8 Tre
g
C
D
4
C
D
3
C
D
8
Treg
C
D
4
CD
3
T
u
m
o
r n
est
T
umor stroma
L
ow N2
/
N1
H
i
g
h N2/N
1
Low N2/N1
High N2/N
1
Low N2/N
1
Hi
g
h N2/N
1
Combined tumor are
a
A
B
C
D
E
F
Prognostic significance andimmune escape implication oftumor‑infiltrating neutrophil…
Independent prognostic role ofHNSCC‑infiltrating
N2/N1 neutrophils inHNSCC
The Kaplan–Meier plots (Fig.4) and univariate analyses
(Fig.5) revealed that a higher infiltration of HNSCC-
infiltrating N1 neutrophils within the TN was signifi-
cantly associated with improved OS (HR, 0.262; 95%
CI 0.105–0.651; p = 0.004) and RFS (HR, 0.335; 95%
CI 0.140–0.800; p = 0.014). Conversely, worse OS and
RFS were observed in patients with a higher infiltration
of HNSCC-infiltrating N2 neutrophils in TN (OS: HR,
2.454; 95% CI 1.071–5.623; p = 0.034; RFS: HR, 1.906;
95% CI 0.848–4.284; p = 0.119). When assessing sur-
vival based on N2/N1 ratio, significant distinctions were
noticed between high and low N2/N1 subgroups within the
TN (OS: HR, 10.470; 95% CI 4.157–26.369; p < 0.0001;
RFS: HR, 5.092; 95% CI 2.261–11.469; p < 0.0001)
and in the combined tumor area (OS: HR, 6.211; 95%
CI 2.846–13.554; p < 0.0001; RFS: HR, 4.541; 95% CI
2.080–9.914; p < 0.0001).
The multivariate analyses (Fig.5, Supplementary
Table5–7) demonstrated that after adjusted for prognostic
risk factors (i.e., tumor size, T stage, N stage, TNM stage,
HNSCC-infiltrating CD8+ T cells and HNSCC-infiltrating
FOXP3+ Treg cells, Supplementary Table4), low infiltra-
tion of HNSCC-infiltrating N1 neutrophils within the TN
(adjusted HRos, 0.355; 95% CI 0.132–0.950; p = 0.039),
high infiltration of HNSCC-infiltrating N2 neutrophils
within the TN (adjusted HRos, 3.069; 95% CI 1.144–8.233;
p = 0.026), high N2/N1 neutrophils ratio in the TN
(adjusted HRos, 4.458; 95% CI 1.384–14.366; p 0.012)
and in combined tumor area (adjusted HRos, 3.373; 95%
CI 1.269–8.965; p = 0.015) were all verified as independ-
ent prognostic factors for worse OS. Regarding RFS, low
infiltration of HNSCC-infiltrating N1 neutrophils within
the TN (adjusted HRRFS, 0.342; 95% CI 0.132–0.885;
p = 0.027), high N2/N1 neutrophils ratio within the TN
(adjusted HRos, 3.088; 95% CI 1.171–8.140; p = 0.023) and
the combined tumor area (adjusted HRos, 3.350; 95% CI
1.194–9.400; p = 0.022) were considered as independent
prognostic factors for worse RFS (Fig.5, Supplementary
Table5–7), after adjusted for corresponding RFS-related
risk factors (Supplementary Table4). The results of sensi-
tivity analysis were in alignment with our primary findings
(Supplementary Table8, 9).
Discussion
This study first evaluated the plasticity of HNSCC-infiltrat-
ing N2/N1 neutrophils within the TME and their prognos-
tic implications. Our findings support the notion that the
plasticity of HNSCC-infiltrating neutrophils, particularly
the ratio of N2/N1 neutrophils, serves as an independent
prognostic indicator for HNSCC and is related to adverse
clinical and pathological features. Furthermore, the plastic-
ity HNSCC-infiltrating N2/N1 neutrophils, especially within
TN, may potentially reflect the immune escape and immu-
nosuppressive state of TME.
Accumulating evidences suggests that neutrophils play
a pivotal role in modulating tumor onset and progression
[17]. They exhibit antitumoral functions, such as function-
ing as antigen-presenting cells [24], directly killing tumor
cells [25, 26], and mediating antibody-mediated trogoptosis
[27]. Neutrophils are also implicated in promoting tumor
progression by secreting MMP9, BV8 [17], hepatocyte
growth factor (HGF) [28], or by undergoing the process of
NETosis [29]. Consequently, these evidences raise the pos-
sibility of phenotypic plasticity and functional versatility
that dictate their response to TME cues [30, 31]. Various
markers, including CD66b, MPO, CD15 and HE staining,
have been employed to identify HNSCC-infiltrating neu-
trophils. CD66b is indispensable and crucial for neutrophil
transmigration and adhesion and is consistently expressed
in all stages of neutrophil differentiation [32]. Therefore,
our study utilized CD66b staining to identify neutrophils. In
addition, compelling evidences from previous studies have
identified distinct neutrophil phenotypes in cancer patients
[33]. However, markers for N1 and N2 neutrophil pheno-
types are not yet to be determined. Thus, it remains neces-
sary to identify appropriate molecular markers for N1 and
N2 neutrophils, respectively. Recent studies [23, 34] have
reported the effective use of CD206 and iNOS in identi-
fying neutrophil phenotypes, with their effects analogous
to their distinctive properties in macrophages [11]. In our
study, the expressions of CD206 and iNOS were assessed in
tissue-infiltrating CD66b+ neutrophils and were proven to be
suitable for identifying neutrophils with distinct prognostic
functions, indicating the diversity and plasticity of HNSCC-
infiltrating N2/N1 neutrophils within the TME.
Previous researches have demonstrated that tumor cells
could secrete numerous cytokines that facilitate the recruit-
ment of neutrophils, including members of the C-X-C
chemokine family, growth factor, IL-6, and GM-CSF [35,
36]. Furthermore, neutrophils exhibit high plasticity and
have remarkable adaptive abilities in response to differ-
ent TME cues. Fridlender etal. reported that transforming
growth factor-beta (TGF-β) could drive the polarization of
neutrophils toward a protumor phenotype (N2), whereas
Fig. 3 Comparisons of HNSCC-infiltrating CD8+ T cells, Tregs,
CD4+ T cells and CD3+ T cells between low and high ratio of N2/N1
neutrophils in TN (A), TS (B) and combined tumor area (C). Correla-
tions between HNSCC-infiltrating CD8+ T cells, Tregs, CD4+ T cells
and CD3+ T cells and the ratio of N2/N1 neutrophils in TN (D), TS
(E) and combined tumor area (F). TN tumor nest, TS tumor stroma,
HPF high-power field
◂
X.Zhu et al.
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N1
High N1
P = 0.0017
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N1
High N1
P = 0.8830
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N1
High N1
P = 0.1841
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N2
High N2
P = 0.0283
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N2
High N2
P = 0.0548
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N2
High N2
P = 0.0708
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N2/N1
High N2/N1
P < 0.0001
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N2/N1
High N2/N1
P = 0.1114
0102030405060
0
20
40
60
80
100
Months
Overall survival rate, %
Low N2/N1
High N2/N1
P < 0.0001
Tu
m
o
r n
es
t
T
umor stroma
A
B
C
C
ombined tumor are
a
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N1
High N1
P = 0.0126
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N1
High N1
P = 0.8615
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N1
High N1
P = 0.2990
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N2
High N2
P = 0.1033
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N2
High N2
P = 0.3534
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N2
High N2
P = 0.3735
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N2/N1
High N2/N1
P < 0.0001
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N2/N1
High N2/N1
P = 0.2772
0102030405060
0
20
40
60
80
100
Months
Cumulative Incidence of relapse, %
Low N2/N1
High N2/N1
P < 0.0001
T
umor nes
t
T
umor stroma
C
ombined tumor are
a
D
E
F
Fig. 4 Kaplan–Meier plots for OS in HNSCC patients by the HNSCC-infiltrating N2/N1 neutrophil plasticity within TN (A), TS (B) and combined tumor area (C). Kaplan–Meier plots for OS
in patients with HNSCC by the plasticity of HNSCC-infiltrating N2/N1 neutrophils within TN (D), TS (E) and combined tumor area (F). RFS relapse-free survival, OS overall survival, TN
tumor nest, TS tumor stroma
Prognostic significance andimmune escape implication oftumor‑infiltrating neutrophil…
0510 15 20
HROS(95% CI)
T
umor nes
t
Tumor-infiltrating N1
Low
Hi
g
h
Tumor-infiltratin
g
N2
Low
High
N2/N1 ratio
Low
H
ig
h
Co
m
b
in
ed
tu
m
o
r
a
r
ea
Tumor-infiltratin
g
N1
Low
H
i
g
h
Tumor-infiltratin
g
N2
Low
Hi
g
h
N2/N1 r
at
i
o
Low
H
i
gh
0.262(0.105-0.651)
2.454(1.071-5.623)
10.470(4.157-26.369)
0.945(0.443-2.017)
2.154(0.965-4.804)
1.989(0.838-4.718)
0.600(0.280-1.286)
2.027(0.926-4.436)
6.211(2.846-13.554)
0.004
0.034
< 0.0001
0.883
0.061
0.119
0.189
0.077
< 0.0001
0510 15 20
Adjusted HROS(95% CI)†
0.355(0.132-0.950)
3.069(1.144-8.233)
4.458(1.384-14.366)
0.874(0.351-2.173)
2.485(0.725-8.513)
3.010(1.072-8.450)
0.572 0.216-1.515
1.61 0.540-4.794
3.373 1.269-8.965)
0.039
0.026
0.012
0.772
0.148
0.036
0.261
0.393
0.015
HR
OS
(
95
%
C
I)
Adj
uste
d
HR
OS
(95
%
C
I)†
Tumor strom
a
Tumor-infiltratin
g
N1
Low
H
i
g
h
Tumor-in
f
iltrating N2
L
ow
Hi
g
h
N2/N1 ratio
Low
Hi
g
h
0.945
(
0.443-2.017
)
2
.154
(
0.965-4.804
)
1.989(0.838-4.718
)
0
.
883
0
.
061
0
.11
9
0
.874
(
0.351-2.173
)
2
.485(0.725-8.513)
3.010
(
1.072-8.450
)
0
.772
0
.14
8
0
.
036
0510 15 20
HR
RFS
(95% CI)
T
u
m
o
r n
est
Tumor-infiltratin
g
N
1
Lo
w
Hi
gh
Tumor-infiltratin
g
N2
L
ow
Hig
h
N2/N1 rati
o
L
ow
Hig
h
Co
m
b
in
ed tu
m
o
r
a
r
ea
Tumor-in
f
iltratingN
1
L
ow
Hig
h
Tumor-in
f
iltrating N2
L
ow
Hi
gh
N2
/
N1 rati
o
Lo
w
Hi
gh
0.335(0.140-0.800)
1.906(0.848-4.284)
5.092(2.261-11.469)
0.863(0.399-1.870)
1.445(0.663-3.149)
1.659(0.663-4.148)
0.638(0.294-1.384)
1.372(0.634-2.970)
4.541(2.080-9.914)
0.014
0.119
< 0.0001
0.709
0.355
0.279
0.255
0.422
< 0.0001
0510 15 20
Adjusted HR
RFS
(95% CI)‡
0.342(0.132-0.885)
2.295(0.717-7.339)
3.088(1.171-8.140)
1.034(0.424-2.518)
0.873(0.286-2.664)
1.419(0.459-4.386)
0.736(0.287-1.885)
1.146(0.391-3.364)
3.350(1.194-9.400)
0.027
0.162
0.023
0.942
0.812
0.544
0.523
0.804
0.022
HR
R
F
S
(95
%C
I
)
Adj
uste
dHR
RF
S
(95
%C
I
)
‡
T
umor stroma
Tumor-infiltrating N
1
L
o
w
Hi
g
h
Tumor-in
f
iltrating N2
Lo
w
Hi
g
h
N2/N1 r
at
i
o
Lo
w
Hig
h
0.863
(
0.399-1.870
)
1.445
(
0.663-3.149
)
1.659
(
0.663-4.148
)
0
.7
09
0
.
3
55
0
.27
9
1.034(0.424-2.518
)
0.873(0.286-2.664
)
1.419(0.459-4.386
)
0
.
9
4
2
0
.
8
1
2
0
.54
4
B
A
Fig. 5 Univariate and multivariate COX regression estimating the
prognostic value of HNSCC-infiltrating N2/N1 plasticity within dif-
ferent regions for OS (A) and RFS (B). †The independent prognostic
value of HNSCC-infiltrating N2/N1 neutrophil plasticity for OS was
estimated through multivariate COX regression adjusted for primary
site, T stage, N stage, TNM stage, tumor size, HNSCC-infiltrating
CD8+ T cells and HNSCC-infiltrating FOXP3+ Tregs, which were
selected through univariate COX regression models (Supplementary
Table4); ‡The independent prognostic value of HNSCC-infiltrating
N2/N1 neutrophil plasticity for RFS was estimated through multi-
variate COX regression adjusted for smoking history, primary site,
T stage, N stage, tumor size, HNSCC-infiltrating CD8+ T cells and
HNSCC-infiltrating FOXP3+ Tregs, which were selected through
univariate COX regression models (Supplementary Table4); Supple-
mentary Table5–7 showed the corresponding HRs for each variables
in multivariate COX regression models
X.Zhu et al.
TGF-β neutralization could induce the accumulation of neu-
trophils with an antitumor phenotype (N1) [20]. Previous
studies have also reported the abundance of TGF-β expres-
sion within HNSCC and the benefit of TGF-β blockade
in improving survival [37], which may correlate with our
finding that the infiltration of N2 neutrophils was higher in
HNSCC tissue than that in adjacent normal tissue. Notably,
we discovered that the infiltration pattern of N2/N1 neu-
trophils varied between the TN and TS, with a relatively
higher number of N2 neutrophils and a lower number of N1
neutrophils localized in the TN region compared to the TS.
Accordingly, the ratio of HNSCC-infiltrating N2/N1 neutro-
phils was also higher in the TN region than that in the TS.
Our study revealed that patients with a smoking history
tended to have a high ratio of HNSCC-infiltrating N2/N1
neutrophils in TN, consistent with previous studies that
smoking-induced chronic inflammation and nicotine expo-
sure could promote neutrophil polarization to the N2 phe-
notype [23, 38]. Furthermore, patients with higher N2/N1
ratios, particularly in TN, were found to be at an advanced
TNM stage, with larger tumor size and invasive growth into
adjacent tissue. The reason why increased N2 neutrophils
infiltrate advanced HNSCC requires additional discussion.
One possible explanation is that advanced HNSCC can
secrete more specific cytokines to homing N2 neutrophils,
like members of the C-X-C chemokine family and IL6 as
mentioned above [35, 36]. In addition, advanced HNSCC
may produce increased factors to drive the polarization of
neutrophils toward N2 phenotype [37]. Notably, prior studies
have reported that HNSCC-infiltrating N2 neutrophils could
promote tumor growth and invasion through the production
of neutrophil extracellular traps (NETs) and the secretion of
ROS, MMP9, HGF, myeloperoxidase, and oncostatin M [39,
40]. Thus, we hypothesis that a positive feedback loop may
exist between tumor progression and the accumulation of N2
neutrophils within tumor, which needs further investigation
in our future studies. That is, the development and growth
of HNSCC can attract the homing of N2 neutrophils or drive
the polarization of neutrophils toward N2 phenotype, and
in turn, the increased N2 neutrophils within HNSCC can
promote the progression of tumor via various pro-tumor
mechanisms. Further studies are warranted to elucidate this
association in an effort to find potential clues for future can-
cer therapy.
Increasing evidences have highlighted the significance of
cancer immune landscape in modulating tumor progression,
patient prognosis and immunotherapy effectiveness [41, 42].
CD8+ T cells have been shown to induce pyroptosis in tumor
cells through the release of TNF-α and IFN-γ [43], with
long-term survivors exhibiting higher densities of CD8+ T
cells in HNSCC tissue as evidenced by IHC and IF staining
[13]. these findings align with our observation that increased
CD8+ T cells infiltration correlates with improved survival
outcomes. Conversely, higher densities of FOXP3+ Tregs
have been linked to poorer survival outcomes due to the
expression of CTLA4 [44], which is also consistent with
our observations. We noted an increased number of FOXP3+
Tregs and a decreased number of CD8+ lymphocytes infil-
trating HNSCC tissue, accompanied by a high infiltration
of N2 phenotype or a high ratio of N2/N1 neutrophils in
TN. Moreover, we identified a negative relationship between
the HNSCC-infiltrating N2/N1 ratio and the CD8/CD3 and
CD8/Treg ratios, as well as a positive correlation between
the HNSCC-infiltrating N2/N1 ratio and the Treg/CD3 ratio.
These intriguing findings may be attributable the complex
interplay between immune cells within the HNSCC TME.
It is important to note that neutrophils primarily promote
tumor growth and immune escape through their inhibitory
effects on T lymphocytes [36]. HNSCC-infiltrating neu-
trophils have been shown to suppress the proliferation and
function of CD8+ T cells via PD-L1 and PD-1 binding [7]
or the release of immunosuppressive cytokines, such as argi-
nase 1 and ROS [45]. Meanwhile, Mishalian etal. reported
that HNSCC-infiltrating neutrophils secrete the chemokine
CCL17, which plays a crucial role in recruiting FOXP3+
Tregs [22]. Contrarily, a recent study demonstrated that neu-
trophil infiltration can enhance the prognostic significance
of CD8+ T cells infiltration in cancer, suggesting a potential
role in promoting antitumor immunity [46]. The intricate
interactions and crosstalk between HNSCC-infiltrating neu-
trophil phenotypes and T cells warrant further investigation
to elucidate novel therapeutic approaches for HNSCC.
In this study, we primarily assessed the prognostic
implications of HNSCC-infiltrating N2/N1 neutrophil
plasticity in HNSCC patients. Elevated N2 neutrophils
infiltration and a higher ratio of N2/N1 neutrophils in TN
significantly were correlated with poorer OS and RFS
in HNSCC patients, as indicated by the Kaplan–Meier
curves. Thereafter, univariate and multivariate COX
regression analyses identified the plasticity of HNSCC-
infiltrating N2/N1 neutrophils as an independent prognos-
tic predictor. One possible underlying mechanism involves
the interaction between neutrophils and tumor cells in TN,
which may induce the neutrophil polarization toward the
N2 phenotype and, in turn, directly facilitate tumor cell
proliferation [17]. In addition, neutrophils in TN may
release neutrophil extracellular traps, which could impede
T cell penetration and disrupt contact between immune
cytotoxic cells and tumor cells [47, 48]. Consequently,
incorporating N2/N1 neutrophil plasticity in TN into
the TNM staging system might provide a more accurate
prognostic classification for HNSCC patients. Moreover,
several studies [49, 50] have reported the role of HNSCC-
infiltrating neutrophils and NETs in mediating resistance
to immunotherapy. Therefore, further assessments of
the correlations between the HNSCC-infiltrating N2/N1
Prognostic significance andimmune escape implication oftumor‑infiltrating neutrophil…
neutrophils plasticity, particularly in TN, and treatment
responses may yield valuable insights for selecting optimal
therapeutic strategies.
Despite the findings of our study, several limitations
warrant further discussion. First, the single-center and ret-
rospective nature of this study may constrain the general-
izability of our results, necessitating validation through
larger, multicenter, and prospective cohorts. Second, we
only included patients with laryngeal or hypopharyngeal
SCC, and future studies should investigate the prognos-
tic implications of HNSCC-infiltrating N2/N1 neutrophil
plasticity in other HNSCC subsites. Third, the underly-
ing mechanisms by which the HNSCC TME polarizes
HNSCC-infiltrating neutrophils towards N2 phenotype
remain unclear. Further studies should focus on elucidat-
ing the mechanisms of N2/N1 neutrophils plasticity, espe-
cially in TN, and their specific roles in HNSCC.
All in all, our study demonstrated the existence of
HNSCC-infiltrating N2/N1 neutrophils plasticity in HNSCC.
HNSCC-infiltrating N2/N1 neutrophil plasticity carries sub-
stantial implications regarding immune evasion and prog-
nostic outcomes. Moreover, HNSCC-infiltrating N2 neu-
trophils within the TN may serve a pivotal function in the
HNSCC TME. Further studies exploring neutrophils polari-
zation, especially within the TN region, and its associations
with immunopathological characteristics in HNSCC may
yield invaluable insights for therapeutic strategies.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13577- 024- 01024-7.
Acknowledgements We thank all members of Professor Lu’s Labo-
ratory at Shanghai Institute of Immunology and Shanghai Jiaotong
University School of Medicine for their technical assistance.
Author contributions LLM, LT, and DZ designed the experiments.
XKZ and YH contributed to executing the described experiments. DT
and JZ was responsible for the bioinformatic analysis of the data. LLM,
LT, XKZ, and JYM analyzed the data. XKZ and XPD contributed to
the preparation of this manuscript, and all authors aided in editing the
final manuscript.
Funding This study was supported by of the Major Clinical Research
Project of Shanghai Shen-kang Hospital Clinical Development
Center under Grant (Grant numbers: SHDC2020CR6011); the Sci-
ence and Technology Innovation Project of Shanghai Shen-kang
Hospital Clinical Development Center under Grant (Grant numbers:
SHDC12015114); the National Natural Science Foundation of China
under Grant (Grant numbers: 81772878, 82003178); the Shanghai
Municipal Key Clinical Specialty under Grant (Grant numbers: shslc-
zdzk00801); the Science and Technology Committee of Shanghai
under Grant (Grant numbers: 20MC1920200); the Science and Tech-
nology Commission of Shanghai Municipality under Grant (Grant
numbers: 20Y11902200); the Shanghai Anti-Cancer Development
Foundation under Grant (Grant numbers: H6001-008); and the Train-
ing Program of the Excellent Doctors of Fudan University under Grant
(Grant numbers: QT00140).
Data availability Data will be made available on request.
Declarations
Conflict of interest The authors have no relevant financial or non-fi-
nancial interests to disclose.
Ethics approval This study adhered to the principles of the Declaration
of Helsinki and received approval from the Medical Research Council
of the Eye & ENT Hospital, Fudan University, Shanghai, China (No.
KJ2008-01).
Patient consent for publication Consent obtained directly from
patient(s).
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