Circulating tumor cell detection and single‐cell analysis using an integrated workflow based on ChimeraX®‐i120 Platform: A prospective study

Circulating tumor cell (CTC) analysis holds great potential to be a noninvasive solution for clinical cancer management. A complete workflow that combined CTC detection and single‐cell molecular analysis is required. We developed the ChimeraX^®‐i120 platform to facilitate negative enrichment, immunofluorescent labeling, and machine learning‐based identification of CTCs. Analytical performances were evaluated, and a total of 477 participants were enrolled to validate the clinical feasibility of ChimeraX^®‐i120 CTC detection. We analyzed copy number alteration profiles of isolated single cells. The ChimeraX^®‐i120 platform had high sensitivity, accuracy, and reproducibility for CTC detection. In clinical samples, an average value of > 60% CTC‐positive rate was found for five cancer types (i.e., liver, biliary duct, breast, colorectal, and lung), while CTCs were rarely identified in blood from healthy donors. In hepatocellular carcinoma patients treated with curative resection, CTC status was significantly associated with tumor characteristics, prognosis, and treatment response (all P < 0.05). Single‐cell sequencing analysis revealed that heterogeneous genomic alteration patterns resided in different cells, patients, and cancers. Our results suggest that the use of this ChimeraX^®‐i120 platform and the integrated workflow has validity as a tool for CTC detection and downstream genomic profiling in the clinical setting.

Abbreviations

ADABOOST

AdaBoost classification trees

AFP

alpha‐fetoprotein

AUC

areas under the curve

BC

breast cancer

BCLC

barcelona clinic liver cancer

BHL

benign hepatic lesion

CCD

charge‐coupled device

CHB

chronic hepatitis B

CK

cytokeratin

CNA

copy number alteration

CNLC

Chinese staging for liver cancer

CRC

colorectal cancer

CTC

circulating tumor cell

CTM

circulating tumor microemboli

CV

coefficient of variation

DAPI

4’,6‐diamidine‐2’‐phenylindole dihydrochloride

EpCAM

epithelial cell adhesion molecule

FPR

false‐positive rate

GBM

stochastic gradient boosting

HCC

hepatocellular carcinoma

HD

healthy donor

ICC

intrahepatic cholangiocarcinoma

LC

liver cirrhosis

LCA

lung cancer

LOD

limit of detection

PBS

phosphate‐buffered saline

PCR

polymerase chain reaction

RF

random forest

ROC

receiver operating characteristic

SVM

support vector machines

TCGA

The Cancer Genome Atlas

TPR

true‐positive rate

TTR

time to recurrence

WBC

white blood cell

WGA

whole‐genome amplification

WGS

whole‐genome sequencing

XGB

extreme gradient boosting

     

Long non‐coding RNA RACGAP1P promotes breast cancer invasion and metastasis via miR‐345‐5p/RACGAP1‐mediated mitochondrial fission

Long non‐coding RNAs (lncRNAs) are emerging as key molecules in various cancers, yet their potential roles in the pathogenesis of breast cancer are not fully understood. Herein, using microarray analysis, we revealed that the lncRNA RACGAP1P, the pseudogene of Rac GTPase activating protein 1 (RACGAP1), was up‐regulated in breast cancer tissues. Its high expression was confirmed in 25 pairs of breast cancer tissues and 8 breast cell lines by qRT‐PCR. Subsequently, we found that RACGAP1P expression was positively correlated with lymph node metastasis, distant metastasis, TNM stage, and shorter survival time in 102 breast cancer patients. Then, in vitro and in vivo experiments were designed to investigate the biological function and regulatory mechanism of RACGAP1P in breast cancer cell lines. Overexpression of RACGAP1P in MDA‐MB‐231 and MCF7 breast cell lines increased their invasive ability and enhanced their mitochondrial fission. Conversely, inhibition of mitochondrial fission by Mdivi‐1 could reduce the invasive ability of RACGAP1P‐overexpressing cell lines. Furthermore, the promotion of mitochondrial fission by RACGAP1P depended on its competitive binding with miR‐345‐5p against its parental gene RACGAP1, leading to the activation of dynamin‐related protein 1 (Drp1). In conclusion, lncRNA RACGAP1P promotes breast cancer invasion and metastasis via miR‐345‐5p/RACGAP1 pathway‐mediated mitochondrial fission.

Abbreviations

CDS

coding sequence

ceRNAs

competitive endogenous RNAs

Drp1

dynamin‐related protein 1

FFPE

formalin‐fixed paraffin‐embedded

lncRNAs

long non‐coding RNAs

miRNAs

microRNAs

RACGAP1

Rac GTPase activating protein 1

TCGA

The Cancer Genome Atlas

     

A novel cell‐free DNA methylation‐based model improves the early detection of colorectal cancer

Screening for early‐stage disease is vital for reducing colorectal cancer (CRC)‐related mortality. Methylation of circulating tumor DNA has been previously used for various types of cancer screening. A novel cell‐free DNA (cfDNA) methylation‐based model which can improve the early detection of CRC is warranted. For our study, we collected 313 tissue and 577 plasma samples from patients with CRC, advanced adenoma (AA), non‐AA and healthy controls. After quality control, 187 tissue DNA samples (91 non‐malignant tissue from CRC patients, 26 AA and 70 CRC) and 489 plasma cfDNA samples were selected for targeted DNA methylation sequencing. We further developed a cfDNA methylation model based on 11 methylation biomarkers for CRC detection in the training cohort (area under curve [AUC] = 0.90 (0.85–0.94]) and verified the model in the validation cohort (AUC = 0.92 [0.88–0.96]). The cfDNA methylation model robustly detected patients pre‐diagnosed with early‐stage CRC (AUC = 0.90 [0.86–0.95]) or AA (AUC = 0.85 [0.78–0.91]). Here we established and validated a non‐invasive cfDNA methylation model based on 11 DNA methylation biomarkers for the detection of early‐stage CRC and AA. The utilization of the model in clinical practice may contribute to the early diagnosis of CRC.     

TGFβ promotes YAP‐dependent AXL induction in mesenchymal‐type lung cancer cells

The acquisition of chemoresistance remains a major cause of cancer mortality due to the limited accessibility of targeted or immune therapies. However, given that severe alterations of molecular features during epithelial‐to‐mesenchymal transition (EMT) lead to acquired chemoresistance, emerging studies have focused on identifying targetable drivers associated with acquired chemoresistance. Particularly, AXL, a key receptor tyrosine kinase that confers resistance against targets and chemotherapeutics, is highly expressed in mesenchymal cancer cells. However, the underlying mechanism of AXL induction in mesenchymal cancer cells is poorly understood. Our study revealed that the YAP signature, which was highly enriched in mesenchymal‐type lung cancer, was closely correlated to AXL expression in 181 lung cancer cell lines. Moreover, using isogenic lung cancer cell pairs, we also found that doxorubicin treatment induced YAP nuclear translocation in mesenchymal‐type lung cancer cells to induce AXL expression. Additionally, the concurrent activation of TGFβ signaling coordinated YAP‐dependent AXL expression through SMAD4. These data suggest that crosstalk between YAP and the TGFβ/SMAD axis upon treatment with chemotherapeutics might be a promising target to improve chemosensitivity in mesenchymal‐type lung cancer.

Abbreviations

AUC

area under the curve

AXL

AXL receptor tyrosine kinase

BCL2

B‐cell lymphoma 2

CTD2

cancer target discovery and development

CTGF

connective tissue growth factor

DEG

differentially expressed genes

DOXO

doxorubicin

EMT

epithelial–mesenchymal transition

Eto

etoposide

FDA

Food and Drug Administration

ITGB3

integrin beta‐3

MAPK

mitogen‐activated protein kinase

MMP2

matrix metalloproteinase‐2

MMP9

matrix metalloproteinase‐9

mRNA

messenger RNA

NF‐κB

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

SBE

SMAD binding element

SERPINE1

serpin family E member 1

siRNA

small interfering RNA

ssGSEA

single‐sample gene set enrichment analysis

TCGA

The Cancer Genome Atlas

TGFβ

transforming growth factor beta

YAP

Yes‐associated protein

YAP8SA

mutants of inhibitory phosphorylation site at eight serine to Alanine of YAP

ZEB1

zinc finger E‐box binding homeobox 1

ZEB2

zinc finger E‐box‐binding homeobox 2

     

Comprehensive cross‐platform comparison of methods for non‐invasive EGFR mutation testing: results of the RING observational trial

Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next‐generation sequencing (NGS)‐based methods, three high‐sensitivity PCR‐based platforms, and two FDA‐approved methods were compared using 72 plasma samples, from EGFR‐mutant non‐small‐cell lung cancer (NSCLC) patients progressing on a first‐line tyrosine kinase inhibitor (TKI). NGS platforms as well as high‐sensitivity PCR‐based methodologies showed excellent agreement for EGFR‐sensitizing mutations (K = 0.80–0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86–0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false‐positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs.

Abbreviations

BEAMing

beads, emulsion, amplification, and magnetics

cfDNA

circulating free DNA, cell‐free DNA

cobas

cobas^® EGFR Mutation Test v2 (Roche Diagnostics)

ctDNA

circulating tumor DNA

CUSUM

cumulative sum

ddPCR

droplet digital polymerase chain reaction

dPCR

digital polymerase chain reaction

EGFR

epidermal growth factor receptor

FFPE

formalin‐fixed, paraffin‐embedded

ICC

intraclass correlation coefficient

MAF

mutant allele frequency

NGS platforms

Ion S5™ XL and GeneRead™

NGS

next‐generation sequencing

NSCLC

non‐small‐cell lung cancer

PNA‐Q‐PCR

peptic nucleic acid probe‐based real‐time polymerase chain reaction

Therascreen

Therascreen EGFR Plasma RGQ PCR Kit (QIAgen)

TKI

tyrosine kinase inhibitor

     

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial‐to‐mesenchymal transition and lipid‐derived energy production in prostate cancer cells

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid‐binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial‐to‐mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid β‐oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12''s role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP‐PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

Abbreviations

AR

androgen receptor

ATP

adenosine triphosphate

CN

copy number

CPT1

carnitine palmitoyltransferase I

CS

citrate synthase

EMT

epithelial–mesenchymal transition

ET

electron transfer‐state

FABP

fatty acid‐binding protein

LD

lipid droplet

OA

oleic acid

PCa

prostate cancer

PPAR

peroxisome proliferator‐activated receptor

PPRE

peroxisome proliferator‐activated receptor response element

TZD

thiazolidinediones

     

Circ_0008039 supports breast cancer cell proliferation,migration, invasion,and glycolysis by regulating the miR‐140‐3p/SKA2 axis

Circular RNAs (circRNAs) have been shown to modulate gene expression and participate in the development of multiple malignancies. The purpose of this study was to investigate the role of circ_0008039 in breast cancer (BC). The expression of circ_0008039, miR‐140‐3p, and spindle and kinetochore‐associated protein 2 (SKA2) was detected by qRT‐PCR. Cell viability, colony formation, migration, and invasion were evaluated using methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, colony formation assay, and transwell assay, respectively. Glucose consumption and lactate production were measured using commercial kits. Protein levels of hexokinase II (HK2) and SKA2 were determined by western blot. The interaction between miR‐140‐3p and circ_0008039 or SKA2 was verified by dual‐luciferase reporter assay. Finally, a mouse xenograft model was established to investigate the roles of circ_0008039 in BC in vivo. We found that circ_0008039 and SKA2 were upregulated in BC tissues and cells, while miR‐140‐3p was downregulated. Knockdown of circ_0008039 suppressed BC cell proliferation, migration, invasion, and glycolysis. Moreover, miR‐140‐3p could bind to circ_0008039 and its inhibition reversed the inhibitory effect of circ_0008039 interference on proliferation, migration, invasion, and glycolysis in BC cells. SKA2 was verified as a direct target of miR‐140‐3p and its overexpression partially inhibited the suppressive effect of miR‐140‐3p restoration in BC cells. Additionally, circ_0008039 positively regulated SKA2 expression by sponging miR‐140‐3p. Consistently, silencing circ_0008039 restrained tumor growth via increasing miR‐140‐3p and decreasing SKA2. In conclusion, circ_0008039 downregulation suppressed BC cell proliferation, migration, invasion, and glycolysis partially through regulating the miR‐140‐3p/SKA2 axis, providing an important theoretical basis for treatment of BC.

Abbreviations

ANOVA

analysis of variance

BC

breast cancer

circRNAs

circular RNAs

DMSO

dimethyl sulfoxide

ECAR

extracellular acidification rate

ECL

enhanced chemiluminescence

FBS

fetal bovine serum

HK2

hexokinase II

MEGM

mammary epithelial growth medium

miR‐140‐3p

microRNA‐140‐3p

MTT

methylthiazolyldiphenyl‐tetrazolium bromide

PBS

phosphate‐buffered saline

PRKAR1B

protein kinase A regulatory subunit R1‐beta

SD

standard ± deviation

SKA2

spindle and kinetochore‐associated protein 2

     

TRIP13 promotes metastasis of colorectal cancer regardless of p53 and microsatellite instability status

Overexpression of TRIP13, a member of the AAA‐ATPase family, is linked with various cancers, but its role in metastasis is unknown in colorectal cancer (CRC). In the current study, we investigated the role TRIP13 in experimental metastasis and its involvement in regulation of WNT/β‐catenin and EGFR signaling pathways. Evaluation of formalin‐fixed paraffin‐embedded (FFPE) and frozen tissues of adenomas and CRCs, along with their corresponding normal samples, showed that TRIP13 was gradually increased in its phenotypic expression from adenoma to carcinoma and that its overexpression in CRCs was independent of patient''s gender, age, race/ethnicity, pathologic stage, and p53 and microsatellite instability (MSI) status. Moreover, liver metastases of CRCs showed TRIP13 overexpression as compared to matched adjacent liver tissues, indicating the biological relevance of TRIP13 in CRC progression and metastasis. TRIP13 knockdown impeded colony formation, invasion, motility, and spheroid‐forming capacity of CRC cells irrespective of their p53 and MSI status. Furthermore, xenograft studies demonstrated high expression of TRIP13 contributed to tumor growth and metastasis. Depletion of TRIP13 in CRC cells decreased metastasis and it was independent of the p53 and MSI status. Furthermore, TRIP13 interacted with a tyrosine kinase, FGFR4; this interaction could be essential for activation of the EGFR‐AKT pathway. In addition, we demonstrated the involvement of TRIP13 in the Wnt signaling pathway and in the epithelial–mesenchymal transition. Cell‐based assays revealed that miR‐192 and PNPT1 regulate TRIP13 expression in CRC. Additionally, RNA sequencing of CRC cells with TRIP13 knockdown identified COL6A3, TREM2, SHC3, and KLK7 as downstream targets that may have functional relevance in TRIP13‐mediated tumor growth and metastasis. In summary, our results demonstrated that TRIP13 promotes tumor growth and metastasis regardless of p53 and MSI status, and indicated that it is a target for therapy of CRC.

Abbreviations

CIN

chromosomal instability

CRC

colorectal cancer

EMT

epithelial–mesenchymal transition

FFPE

formalin‐fixed, paraffin‐embedded

LEF

lymphoid enhancer factor

MS

microsatellite

MSI

microsatellite instable

MSS

microsatellite stable

NSG

NOD/SCID/IL2γ receptor‐null

NT

nontargeting

SAC

spindle assembly checkpoint

TCF

T‐cell factor

TRIP13

thyroid hormone receptor interactor 13

UAB

University of Alabama at Birmingham

     

A cancer‐associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss‐of‐function

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27^Kip1, a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer‐associated germinal p27^Kip1 variant, namely glycine9‐>arginine‐p27^Kip1 (G9R‐p27^Kip1) identified in a parathyroid adenoma. We unexpectedly found that G9R‐p27^Kip1 lacks the major tumor suppressor activities of p27^Kip1 including its antiproliferative and pro‐apoptotic functions. In addition, G9R‐p27^Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild‐type p27^Kip1‐transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R‐p27^Kip1 on S12, a residue normally never found modified in p27^Kip1. The novel S12 phosphorylation appears responsible for the loss of function of G9R‐p27^Kip1 since S12AG9R‐p27^Kip1 recovers most of the p27^Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D‐p27^Kip1 recapitulates G9R‐p27^Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin‐dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27^Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post‐translational modifications due to generation of novel consensus sequences by cancer‐associated missense mutations.

Abbreviations

1D/WB

monodimensional western blotting

2D/WB

two‐dimensional western blotting

CDK

cyclin‐dependent kinase

CHX

cycloheximide

G9R‐p27

glycine9‐>arginine‐p27

IUPs

intrinsically unstructured proteins

mAbs

monoclonal antibodies

MEN

multiple endocrine neoplasia

MENX

multiple endocrine neoplasia X

PTMs

post‐translational modifications

rAbs

rabbit antibodies

TSG

tumor suppressor gene

wt‐p27

wild‐type p27

     

CircRNA EPHB4 modulates stem properties and proliferation of gliomas via sponging miR‐637 and up‐regulating SOX10

Gliomas are the most common type of primary brain tumors. CircRNA ephrin type‐B receptor 4 (circEPHB4) is a circular RNA derived from the receptor tyrosine kinase EPHB4. However, the clinical significance and the specific roles of circEPHB4 in gliomas and glioma cancer stem cells (CSC) have not been studied. Here, we found that circEPHB4 (hsa_circ_0081519) and SOX10 were up‐regulated and microRNA (miR)‐637 was down‐regulated in glioma tissues and cell lines. Consistently, circEPHB4 was positively correlated with SOX10 but negatively correlated with miR‐637. The altered expressions of these molecules were independently associated with overall survival of patients. CircEPHB4 up‐regulated SOX10 and Nestin by directly sponging miR‐637, thereby stimulating stemness, proliferation and glycolysis of glioma cells. Functionally, silencing circEPHB4 or increasing miR‐637 levels in glioma cells was sufficient to inhibit xenograft growth in vivo. In conclusion, the circEPHB4/miR‐637/SOX10/Nestin axis plays a central role in controlling stem properties, self‐renewal and glycolysis of glioma cells and predicts the overall survival of glioma patients. Targeting this axis might provide a therapeutic strategy for malignant gliomas.

Abbreviations

ANOVA

analysis of variance

circEPHB4

circRNA ephrin type‐B receptor 4

circRNA

circular RNA

HK2

hexokinase 2

mRNA

messenger RNA

miRNA

microRNA

PDK1

pyruvate dehydrogenase kinase 1

PI

propidium iodide

PKM2

pyruvate kinase M2

qRT‐PCR

quantitative real‐time polymerase chain reaction

RIP

RNA immunoprecipitation

SD

standard deviation

shcircEPHB4

short hairpin RNA specifically targeting circEPHB4

     

Predicting osimertinib‐treatment outcomes through EGFR mutant‐fraction monitoring in the circulating tumor DNA of EGFR T790M‐positive patients with non‐small cell lung cancer (WJOG8815L)

The WJOG8815L phase II clinical study involves patients with non‐small cell lung cancer (NSCLC) that harbored the EGFR T790M mutation, which confers resistance to EGFR tyrosine kinase inhibitors (TKIs). The purpose of this study was to assess the predictive value of monitoring EGFR genomic alterations in circulating tumor DNA (ctDNA) from patients with NSCLC that undergo treatment with the third‐generation EGFR‐TKI osimertinib. Plasma samples of 52 patients harboring the EGFR T790M mutation were obtained pretreatment (Pre), on day 1 of treatment cycle 4 (C4) or cycle 9 (C9), and at diagnosis of disease progression or treatment discontinuation (PD/stop). CtDNA was screened for EGFR‐TKI‐sensitizing mutations, the EGFR T790M mutation, and other genomic alterations using the cobas EGFR Mutation Test v2 (cobas), droplet digital PCR (ddPCR), and targeted deep sequencing. Analysis of the sensitizing—and T790M—EGFR mutant fractions (MFs) was used to determine tumor mutational burden. Both MFs were found to decrease during treatment, whereas rebound of the sensitizing EGFR MF was observed at PD/stop, suggesting that osimertinib targeted both T790M mutation‐positive tumors and tumors with sensitizing EGFR mutations. Significant differences in the response rates and progression‐free survival were observed between the sensitizing EGFR MF‐high and sensitizing EGFR MF‐low groups (cutoff: median) at C4. In conclusion, ctDNA monitoring for sensitizing EGFR mutations at C4 is suitable for predicting the treatment outcomes in NSCLC patients receiving osimertinib (Clinical Trial Registration No.: UMIN000022076).

Abbreviations

CIs

confidence intervals

ctDNA

circulating tumor DNA

ddPCR

droplet digital PCR

EGFR

epidermal growth factor receptor

MFs

mutant fractions

NGS

next‐generation sequencing

NSCLC

non‐small cell lung cancer

ORR

overall response rate

OS

overall survival

PD

progressive disease

PFS

progression‐free survival

PR

partial response

SD

stable disease

TKI

tyrosine kinase inhibitor

     

Intratumoral immunosuppression profiles in 11q‐deleted neuroblastomas provide new potential therapeutic targets

High‐risk neuroblastoma (NB) patients with 11q deletion frequently undergo late but consecutive relapse cycles with fatal outcome. To date, no actionable targets to improve current multimodal treatment have been identified. We analyzed immune microenvironment and genetic profiles of high‐risk NB correlating with 11q immune status. We show in two independent cohorts that 11q‐deleted NB exhibits various immune inhibitory mechanisms, including increased CD4+ resting T cells and M2 macrophages, higher expression of programmed death‐ligand 1, interleukin‐10, transforming growth factor‐beta‐1, and indoleamine 2,3‐dioxygenase 1 (P < 0.05), and also higher chromosomal breakages (P ≤ 0.02) and hemizygosity of immunosuppressive miRNAs than MYCN‐amplified and other 11q‐nondeleted high‐risk NB. We also analyzed benefits of maintenance treatment in 83 high‐risk stage M NB patients focusing on 11q status, either with standard anti‐GD2 immunotherapy (n = 50) or previous retinoic acid‐based therapy alone (n = 33). Immunotherapy associated with higher EFS (50 vs. 30, P = 0.028) and OS (72 vs. 52, P = 0.047) at 3 years in the overall population. Despite benefits from standard anti‐GD2 immunotherapy in high‐risk NB patients, those with 11q deletion still face poor outcome. This NB subgroup displays intratumoral immune suppression profiles, revealing a potential therapeutic strategy with combination immunotherapy to circumvent this immune checkpoint blockade.

Abbreviations

11q‐del

11q‐deleted

ADCC

antibody‐dependent cellular cytotoxicity

CDC

complement‐dependent cytotoxicity

COJEC

chemotherapeutic agents cisplatin, vincristine, carboplatin, etoposide, and cyclophosphamide

CTLA‐4

cytotoxic T lymphocyte antigen 4

EFS

event‐free survival

FISH

fluorescence in situ hybridization

HR

hazard ratio

ICI

immune checkpoint inhibitor

IDO1

indoleamine 2,3‐dioxygenase 1

IFN‐γ

interferon‐γ

IL‐10

interleukin 10

INRG

International Neuroblastoma Risk Group

miR

microRNA

MLPA

multiplex ligation‐dependent probe amplification

MMR

mismatch repair

MNA

MYCN amplification

MS

metastatic special stage

MSI

microsatellite instability

NB

neuroblastoma

NCA

numerical chromosome aberrations

NOS

nitric oxide synthase

OS

overall survival

PD‐1

programmed cell death protein 1

PD‐L1

programmed death‐ligand 1

SCA

segmental chromosome aberrations

TAM

tumor‐associated macrophages

Tfh

follicular helper T cells

TGF‐β

tumor growth factor‐β

TMB

tumor mutational burden

TME

tumor microenvironment

TNF‐α

tumor necrosis factor‐α

Treg

regulatory T cells

     

Periostin+cancer‐associated fibroblasts promote lymph node metastasis by impairing the lymphatic endothelial barriers in cervical squamous cell carcinoma

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer‐associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro‐metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin⁺CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin⁺CAFs impaired lymphatic endothelial barriers by activating the integrin‐FAK/Src‐VE‐cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin‐induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin^‐CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF‐enriched cases without LNM. In conclusion, we identified a specific periostin⁺CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast‐targeted interventions that block CAF‐dependent metastasis.

Abbreviations

CAFs

cancer‐associated fibroblasts

CM

conditioned medium

CSCC

cervical squamous cell carcinoma

HDLECs

human dermal lymphatic endothelial cells

LN

lymph node

LNM

lymph node metastasis

LVs

lymphatic vessels

MFI

mean fluorescence intensity

NC

negative control

NOFs

normal fibroblasts

SFM

serum‐free media

TAMs

tumor‐associated macrophages

TEM

transmission electron microscopy

TME

tumor microenvironment