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Article Open Access

POSTN+ fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling

  • Authors:
    • Chenyang Zhang
    • Si Chen
    • Chenghui Qian
    • Wanqi Lv
    • Qian Zhang
    • Yanjin Wang
    • Yuqiong Wu
    • Xue Liu
  • View Affiliations / Copyright

    Affiliations: Shanghai Key Laboratory of Craniomaxillofacial Development and Diseases, Fudan University, Minhang, Shanghai 201102, P.R. China, Department of Oral Pathology, Dalian Stomatological Hospital, Shahekou, Dalian 116021, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 62
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    Published online on: February 2, 2026
       https://doi.org/10.3892/or.2026.9067
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Abstract

Carcinoma‑associated fibroblasts (CAFs) exhibit notably heterogeneity and are closely implicated in immune checkpoint blockade (ICB) resistance in head and neck squamous cell carcinoma (HNSCC). However, the specific subtypes and mechanisms involved remain to be elucidated. By analyzing two single‑cell RNA sequencing datasets (GSE103322 and GSE139324) and The Cancer Genome Atlas (TCGA)‑HNSCC dataset, two distinct fibroblasts subtypes were identified: Perostin (POSTN)‑ and POSTN+ fibroblasts. A comparison with reported markers revealed that extracellular matrix‑related markers were highly expressed in POSTN+ fibroblasts. In addition, fibroblast activation protein and POSTN expression were positively associated with macrophage infiltration and predicted ICB resistance in TCGA‑HNSCC dataset. Immunogold labeling confirmed the enrichment of POSTN on the membrane surface of CAF‑derived small extracellular vesicles (sEVs) and it was indicated that these POSTN+ sEVs may promote THP‑1‑derived macrophages polarization toward the M2 phenotype. Additionally, sEVs derived from CAFs with POSTN knockdown reduced bone morphogenetic protein (BMP) 4 expression in macrophages, thereby inhibiting M2 polarization through the BMP receptor 2/Smad pathway. Collectively, these findings revealed that a POSTN+ fibroblasts fosters an immunosuppressive microenvironment via sEV‑mediated macrophage polarization, nominating POSTN as a potential therapeutic target to overcome ICB resistance in HNSCC.
View Figures

Figure 1

POSTN+FAP+
fibroblasts contributed to the resistance against ICB in HNSCC. (A)
The datasets of GSE103322 and GSE139324 were integrated for further
analysis. A total of five clusters were identified previously based
on reported biomarkers, including B cell, epithelial, fibroblasts,
myeloid and T cells. (B) Vlnplots demonstrated the expression
levels of POSTN across five clusters. (C) Fibroblasts re-clustered
into nine clusters. (D) Vlnplots visualized the expression levels
of POSTN in the nine clusters. (E) POSTN− fibroblasts
and POSTN+ fibroblasts were annotated based on POSTN
expression levels in fibroblasts subtypes. (F) Heatmap displayed
the expression levels of cell-type specific biomarkers (myCAF,
iCAF, apCAF and eCAF) in POSTN− fibroblasts and
POSTN+ fibroblasts. (G) POSTN showed a significant
correlation with FAP in the TCGA-HNSCC (***P<0.001; Spearman's
rank correlation). (H) High expression of POSTN and FAP was
associated with elevated TIDE scores. Blue and red bars
representing higher and lower expression levels, respectively. (I)
H&E and IHC analysis of FAP and POSTN expression in OSCC cases
(n=40) and normal controls (n=14). Scale bar, 100 and 20 µm. Right:
IOD/Area of FAP and POSTN in the normal and OSCC tissues were
quantified. Statistical significance was determined by unpaired
two-tailed Student's t-test, **P<0.01; ***P<0.001. Error bars
represent the mean ± SEM. POSTN, perostin; FAP, fibroblast
activation protein; ICB, immune checkpoint blockade; HNSCC, head
and neck squamous cell carcinoma; TIDE, tumor immune dysfunction
and exclusion; H&E, hematoxylin and eosin; IHC,
immunohistochemical; OSCC, oral squamous cell carcinoma; IOD,
integrated option density.

Figure 2

Association of
POSTN+FAP+ fibroblasts with the immune
landscape in HNSCC. (A) POSTN and FAP expression associated with
the level of immune cell infiltration in the TCGA-HNSCC (Spearman's
rank correlation). (B) A significant positive correlation was
observed between the expression of POSTN/FAP and CD80/CD163 in the
TCGA-HNSCC (***P<0.001; Spearman's rank correlation). (C)
Co-expression of POSTN (green) and CD163 (orange) in HNSCC cases
(n=4). Top: images of HNSCC tissues (Scale bar, 100 µm). Bottom:
images of HNSCC tissues (Scale bar, 50 µm). (D) High and low
immunity subtypes were identified by unsupervised hierarchical
clustering. (E) The distribution of stromal, immune, ESTIMATE,
TumorPurity and POSTN scores in high and low immunity subtypes. (F)
The infiltration abundances of 22 immune cells across
POSTN−/POSTN+ fibroblasts. Statistical
significance was determined by unpaired two-tailed Student's
t-test, ns, not significant; *P<0.05; **P<0.01;
***P<0.001. Error bars represent the mean ± SEM. POSTN,
perostin; FAP, fibroblast activation protein; TCGA, The Cancer
Genome Atlas; HNSCC, head and neck squamous cell carcinoma.

Figure 3

POSTN is located on the surface of
sEV-derived from CAFs. (A) Flowchart of sEVs isolation process. (B)
The morphology and structure of sEVs examined by TEM. Scale bar,
100 nm. (C) The expression of biomarkers of sEVs were examined by
western blotting. CD9, CD81 and ALIX were used as positive
biomarkers for sEVs, while Calnexin was used as a negative
biomarker. GAPDH was used as internal control. (D) Nanoflow
cytometer examined the particles concentration and mean size
distribution of sEVs. (E) The expression levels of POSTN in CAFs
and their sEVs were measured. CD9 and CD81 were used as positive
markers of sEVs (n=3 per group). GAPDH was used as an internal
control. (F) Western blotting analysis was performed the expression
levels of POSTN in density gradient fractionation of sEV-derived
CAF-S6. CD9 and CD81 were used as positive markers of sEVs. (G) TEM
images of immunogold-labeled POSTN on CAF-S5/-S6 sEVs (Scale bar,
100 nm). (H) ELISA examination of POSTN in CAF-S5/-S6 sEVs (n=3 per
group). Statistical significance was determined by unpaired
two-tailed Student's t-test, **P<0.01; ***P<0.001. Error bars
represent the mean ± SEM. For source data for blotting assays, see
Fig. S5. POSTN, perostin; sEV,
small extracellular vesicles; CAFs, carcinoma-associated
fibroblasts; TEM, transmission electron microscopy.

Figure 4

sEV-derived CAFs induced macrophage
M2 polarization. (A) The expression levels of POSTN in CAF-S5/-S6
and their sEVs transfected with shNC and shPOSTN were examined by
western blotting (n=3 per group). CD9 and CD81 were used as
positive markers of sEVs. Left: Representative images. Right:
Quantitative analysis of POSTN in different groups. (B) Particles
concentration and size distribution of sEV-derived CAF-S5/-S6
transfected with shNC and shPOSTN were analyzed by Nanoflow
cytometer. (C) Representative image of macrophages derived from
THP-1 treated with PMA (50 ng/ml; scale bar, 100 µm). (D) The mRNA
expression of CD68 was analyzed by RT-qPCR. (E) Internalization of
PKH26-labeled sEV-derived CAF-S5/-S6 transfected with shNC and
shPOSTN by macrophages was examined by flow cytometer at 2 and 6 h
after incubation (n=3 per group). Quantitative analysis of the MFI
of PKH26-labeled sEVs internalized by macrophages for 2 and 6 h,
respectively. The mRNA expression of CD80, CD86, CD163 and CD206
expression in macrophages were treated with sEV-derived CAF-S5/-S6
transfected with shNC and shPOSTN were analyzed by (F) RT-qPCR, (G)
flow cytometry and (H) western blotting. Statistical significance
was determined using a one-way ANOVA test. ns, not significant;
*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Error bars
represent the mean ± SEM. For source data for blotting assays, see
Fig. S5. sEV, small extracellular
vesicles; CAFs, carcinoma-associated fibroblasts; POSTN, perostin;
sh, short hairpin; NC, negative control; RT-qPCR, reverse
transcription-quantitative PCR; MFI, mean fluorescence
intensity.

Figure 5

BMP4 activates BMPR2/Smad signaling
to induce macrophage M2 polarization. (A) PCA analysis of
macrophages were treated with sEV-derived CAF-S6 transfected with
shNC and shPOSTN in three replicate times. (B) Venn-diagram of DEGs
between shPOSTN sEV vs. CTRL and shPOSTN sEVs vs. shNC sEVs. (C)
Heatmap showing the top DEGs in each group. (D) Volcano plot
showing BMP4 downregulation in macrophages treated with shPOSTN
sEVs vs. shNC sEVs. (E) GSEA analysis of hallmark pathways in
macrophages treated with sEV-derived CAF-S6 transfected with either
shPOSTN or shNC. (F) GO enrichment analysis was performed to
identify pathways associated with the representative DEGs. (G) The
bubble plot displays the top 20 markedly enriched KEGG pathways for
the DEGs. (H) The mRNA expression of BMP4 in macrophages induced
sEVs derived from CAF-S5/-S6 transfected with shNC and shPOSTN were
analyzed by RT-qPCR. (I) The mRNA expression of TNF-α, IL-6, TGF-β
and IL-10 in macrophages stimulated with BMP4 at concentrations of
0, 50 and 100 ng/ml. (J) The expression of CD163 and CD206 in
macrophages treated with BMP4 at concentrations of 0, 100 ng/ml
examined by flow cytometry. (K) The level of BMPR2, pSmad1/5/9,
Smad 5 and Smad 9 in macrophages treated with BMP4 (100 ng/ml) for
48 h were examined by western blotting (n=3 per group). (L) The
level of pSmad1/5/9, Smad5, Smad9, CD163 and CD206 in macrophages
treatment with or without LDN193189 inhibitor (n=3 per group).
Statistical significance was determined using a one-way ANOVA test,
ns, not significant *P<0.05; **P<0.01; ****P<0.0001. Error
bars represent the mean ± SEM. Source data for blotting assays, see
Fig. S5. PCA, principal component
analysis; sEV, small extracellular vesicles; sh, short hairpin; NC,
negative control; DEGs, differentially expressed genes; POSTN,
perostin; sEV, small extracellular vesicles; GSEA, gene set
enrichment analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of
Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR;
pSmad, phosphorylated Smad.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang C, Chen S, Qian C, Lv W, Zhang Q, Wang Y, Wu Y and Liu X: POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling. Oncol Rep 55: 62, 2026.
APA
Zhang, C., Chen, S., Qian, C., Lv, W., Zhang, Q., Wang, Y. ... Liu, X. (2026). POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling. Oncology Reports, 55, 62. https://doi.org/10.3892/or.2026.9067
MLA
Zhang, C., Chen, S., Qian, C., Lv, W., Zhang, Q., Wang, Y., Wu, Y., Liu, X."POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling". Oncology Reports 55.4 (2026): 62.
Chicago
Zhang, C., Chen, S., Qian, C., Lv, W., Zhang, Q., Wang, Y., Wu, Y., Liu, X."POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling". Oncology Reports 55, no. 4 (2026): 62. https://doi.org/10.3892/or.2026.9067
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang C, Chen S, Qian C, Lv W, Zhang Q, Wang Y, Wu Y and Liu X: POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling. Oncol Rep 55: 62, 2026.
APA
Zhang, C., Chen, S., Qian, C., Lv, W., Zhang, Q., Wang, Y. ... Liu, X. (2026). POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling. Oncology Reports, 55, 62. https://doi.org/10.3892/or.2026.9067
MLA
Zhang, C., Chen, S., Qian, C., Lv, W., Zhang, Q., Wang, Y., Wu, Y., Liu, X."POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling". Oncology Reports 55.4 (2026): 62.
Chicago
Zhang, C., Chen, S., Qian, C., Lv, W., Zhang, Q., Wang, Y., Wu, Y., Liu, X."POSTN<sup>+</sup> fibroblast‑secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling". Oncology Reports 55, no. 4 (2026): 62. https://doi.org/10.3892/or.2026.9067
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