Filamin A is involved in human intrahepatic cholangiocarcinoma aggressiveness and progression

Intrahepatic cholangiocarcinoma (iCCA) is a primary liver tumour, characterized by poor prognosis and lack of effective therapy. The cytoskeleton protein Filamin A (FLNA) is involved in cancer progression and metastasis, including primary liver cancer. FLNA is cleaved by calpain, producing a 90 kDa fragment (FLNACT) that can translocate to the nucleus and inhibit gene transcription. We herein aim to define the role of FLNA and its cleavage in iCCA carcinogenesis.


| INTRODUC TI ON
Cholangiocarcinoma (CCA) represents a heterogeneous group of rare and aggressive epithelial cancers that can arise at any point of the biliary tree and are characterized by poor outcomes.CCA is classified based on its anatomical site of origin into distal (dCCA), peri-hilar (pCCA) or intrahepatic (iCCA).The different anatomic locations of CCA correspond to varied etiologies of the disease and can have implications for diagnosis, surgical planning and resectability. 1 Incidence of iCCA is constantly increasing and its mortality rate is extremely high, due to the aggressivity of the disease and the lack of efficient treatments. 1,2Even though some risk factors have been identified, diagnosis is incidental in the majority of the patients and no screening program is available.Although surgical resection is still the first line of treatment, only a minority of patients, 12%-40%, are resectable at the time of diagnosis. 3,4iCCA is a highly chemoresistant tumour with a 5-year survival rate persistently below 10%.Systemic therapeutic options are currently limited, mainly due to the insufficient understanding of its pathogenesis.Target therapies for tumour subtypes with genetic mutations have been introduced into clinical practice, but are only potentially feasible for 15% of patients.Further, chemoimmunotherapy as first-line therapy has recently demonstrated a benefit in overall survival; however, objective response rates remain at 26.7% with a high incidence of adverse events. 5Therefore, there is an urgent need to develop valid therapeutic alternatives for iCCA.
The cytoskeleton protein Filamin A (FLNA) is involved in cancer progression and metastasis. 6Indeed, FLNA, the most widely-expressed protein among the three filamin isoforms, is overexpressed in multiple types of tumours, including liver cancer. 7,8In particular, FLNA expression predicts the recurrence of hepatocellular carcinoma (HCC) after hepatectomy. 9Despite the expression of FLNA having been reported in CCA, its impact on iCCA carcinogenesis is still unknown. 10,11e three members of the mammalian filamin family are filamins A, B and C. The FLNA gene is located on the X chromosome, whereas human FLNB and FLNC are on autosomal chromosomes 3 and 7, respectively.12 All three filamins are widely expressed during development.FLNA and FLNB are ubiquitously expressed throughout the body, 13,14 while FLNC expression is predominantly restricted to skeletal and cardiac muscle cells.15 FLNA acts as a scaffolding molecule for over 90 known protein-binding partners involved in receptor activation, inflammation, cell proliferation and cell motility.15 Indeed, as a mediator of cytoskeleton reorganization, FLNA regulates focal adhesion disassembly at the leading edge of motile cells and coordinates GTPase signalling factors leading to the formation of lamellipodia and filopodia.16 The full-length FLNA is a 280 kDa protein consisting of an N-terminal actin-binding domain and a rod domain of 24 homologous Ig-like repeats.Each repeat has 96 amino acids, interrupted by 30 amino acid hinge (H) domains between repeats 15 and 16 (H1)   and between repeats 23 and 24 (H2).12,17 Filamin A is cleaved by calpain at the H1 and H2 domains, 12 producing a 90 kDa carboxyl-terminal fragment (FLNA CT ) that can translocate to the nucleus.17 The calpain 1/2 inhibitor, Calpeptin, can inhibit FLNA cleavage.18 In this respect, a dual role of FLNA in cancer has been proposed: in the cytoplasm, full-length FLNA promotes the development of metastasis, while for FLNA undergoing proteolysis, the nuclear FLNA CT inhibits ribosomal RNA gene transcription and confers susceptibility to therapeutic interventions. 19These data indicate that the difference in the subcellular localization of FLNA may dictate its role in cancer development.20 Different studies have focused on the FLNA and FLNA CT on the proliferation and migration of primary iCCA cells (n = 3) and HuCCT1 cell line using silencing and Calpeptin, a calpain inhibitor.We observed that FLNA silencing decreased cell proliferation and migration and Calpeptin was able to reduce FLNA CT expression in both the HuCCT1 and iCCA cells (p < .05 vs. control).

Conclusions:
These findings demonstrate that FLNA is involved in human iCCA progression and calpeptin strongly decreased FLNA CT expression, reducing cell proliferation and migration.

K E Y W O R D S
calpeptin, cholangiocarcinoma, cleavage, cytoskeleton, liver cancer

Lay Summary
Filamin A is involved in human Intrahepatic Cholangiocarcinoma progression and its high expression correlates with a worse disease-free survival.RNA sequencing revealed a significant enrichment of cell proliferation, motility and spreading pathways in Intrahepatic Cholangiocarcinoma with high Filamin A expression.
Calpeptin treatment on Intrahepatic Cholangiocarcinoma primary cells strongly decreased Filamin A expression, reducing cell proliferation and migration.function of full-length FLNA, while little is known about the impact of FLNA cleavage and FLNA CT 's role in tumorigenesis.
In this study, we aimed to establish whether FLNA is involved in iCCA progression and aggressiveness.To that end, we evaluated the potential correlation between FLNA expression and the survival of patients affected by iCCA.Moreover, in vitro experiments were performed to investigate the role of this cytoskeleton protein in cell proliferation and cell mobility, focusing on the role of FLNA CT in the iCCA cells.

| Study population
The study was approved by the Independent Ethics Committee of the IRCCS Humanitas Research Hospital in Rozzano (Milan, Italy), and conformed to the ethical guidelines of the Declaration of Helsinki (approval no.146/20).Informed consent was obtained from all subjects involved in the study.
A retrospective cohort of 82 patients who underwent hepatic resection for iCCA at the IRCCS Humanitas Research Hospital between January 2004 and December 2018 was analysed.All patients had pathology-proven iCCA and were without chemotherapy treatment before resection.Hepatitis B-or C-positive patients as well as cirrhosis were excluded.Samples were selected from areas without macroscopic evidence of necrosis or haemorrhage.Resected iCCA patients were followed up every 3 months, as per protocol in our Center, or until death and major events were recorded.Further, iCCA primary cells were prospectively obtained from 6 iCCA surgical resections.Characteristics of the patients enrolled in the study are listed in Table 1.

| Immunohistochemistry
FLNA expression was evaluated by immunohistochemistry on paraffin-embedded tumour tissue sections.After deparaffinization and rehydration of the sections, antigen demasking was performed in 10 mm sodium citrate buffer (pH 6.0) at 98°C (Dako).Endogenous peroxidase activity and unspecific binding sites were blocked with a Peroxidase-Blocking Solution for 15 min at room temperature (Dako).Slides were washed and incubated with an anti-FLNA antibody (Millipore; MAB1678; 1:1000) overnight.Envision peroxidase (HRP)-conjugated secondary antibody (Dako) was applied for 30 min, and the positivity was developed with 3,3′-diaminobenzidine (Dako).Tissue sections were counterstained with Mayer's haematoxylin.As negative controls, the primary antibody was omitted.Images were captured by light microscopy using an Olympus BX51 microscope.
Cases were reviewed during a multi-head session at the microscope by two expert pathologists and immunoreactivity was graded according to previously described immunohistochemical scores. 21ese scores were then correlated with a prognostic parameter of disease-free survival (DFS) by using survival analyses.

| iCCA primary cell isolation and culture
iCCA cells were isolated from 6 iCCA surgical resections, as previously described 22,23 ; Briefly, iCCA tissues were enzymatically dissociated in 1 mg/mL collagenase IV (Sigma-Aldrich) at 37°C for 45 min.Subsequently, cells were strained using a strong nylon mesh with 70 μm micron pores designed for isolating primary cells to consistently obtain a uniform single-cell suspension.antibiotics (Sigma-Aldrich).A schematic overview of the isolation procedure is depicted in Figure S1A.The purity of the isolated CCA primary cells was evaluated before being employed in further experiments, as previously described by our group (Figure S1B). 24The primary cells were used between passage one and passage four following their isolation.
HuCCT1 cells, a human intrahepatic cholangiocarcinoma cell line (JCRB, Osaka, Japan), were grown in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine and antibiotics.Cell lines were cultured at 37°C in a 5% CO 2 atmosphere.

| FLNA silencing
Preliminary experiments to determine the optimal concentration of siRNAs and the kinetics of FLNA silencing were performed.
A negative control siRNA (Thermo Fisher Scientific, AM4611, C-siRNA)-a non-targeting sequence without significant homology to human, mouse or rat transcript sequences -was used in each experiment.Western blotting was performed for each experiment to verify the inhibition of FLNA expression after silencing.As previously described, 8 FLNA gene silencing was performed in HuCCT1 cells using a human-specific FLNA pre-designed siRNA (Invitrogen,

| Proliferation assay
Cell proliferation was assessed by colourimetric measurement of 5-Bromo-2′-deoxyuridine (BrdU; Thermo Fisher Scientific) incorporation during DNA synthesis in proliferating cells, according to the manufacturer's instructions, as previously reported by. 21Briefly, after 24 h of serum starvation, cells were incubated for 24 h with calpeptin (10, 50 and 100 μM; Sigma-Aldrich) in HuCCT1 and calpeptin 100 μM in iCCA cells.Afterwards, the cells were incubated with BrdU for 2 h (cell lines) or 24 h (primary cultures) to allow BrdU incorporation into newly synthesized cellular DNA.Each experiment was done in triplicate and all experiments were repeated at least three times.

| Cell viability assay
Cell viability was assessed in HuCCT1 cells and CCA primary cells by Absorbance was read at 570 nm in a 96-well plate in the Synergy H4 plate reader (Fisher Scientific).Each experiment was done in triplicate and all experiments were repeated at least three times.

| Subcellular fractionation
HuCCT1 cells were lysed in RIPA-A buffer (0.3% Triton™ X-100, 50 mM Tris-HCl pH 7.4 and 1 mM EDTA; Sigma-Aldrich) containing protease inhibitors cocktail with rotation at 4°C for 30 min, as previously described. 25Cell extracts were centrifuged at 12 000 g at 4°C for 10 min to pellet nuclei and the supernatant was saved (cytoplasmic fraction).
The nuclei pellet was resuspended in RIPA-B buffer containing protease inhibitors cocktail.Cell lysates were centrifuged at 12 000 g at 4°C for 10 min and the supernatant was saved (nuclear fraction).Vinculin and Histone 3 were used as cytosolic and nuclear markers, respectively.

| Cell migration assay
To analyse the role of FLNA in cell migration, HuCCT1 and iCCA primary cells FLNA silenced or treated with calpeptin 100 μM, were plated in serum-free media in a 24-well plate containing polycarbonate membrane inserts (CytoSelect™ Cell Migration Assay Kit Cell Biolabs, Inc.), according to the instruction of the manufacturer.
Media containing 10% FBS was used as a chemoattractant.
After 24 h of incubation at 37°C, migratory cells were first dissociated from the membrane and then stained with the CyQuant® GR Dye (Cell Biolabs Inc).Finally, each extracted sample was quantified by measuring fluorescence with a fluorescence plate reader at 480 nm/520 nm.
Coverslips were mounted on glass slides with one drop of liquid mounting medium (ProLong® Gold Antifade Mountant, ThermoFisher).
All images were collected using SP8 Confocal Microscope (Leica).

| RNA-sequencing: data generation and analysis
Total RNA was extracted from iCCA primary cells (n = 6) using the Single Cell RNA Purification Kit (Norgen Biotek), following the manufacturer's recommendations.Subsequently, total RNA was subjected to poly(A) mRNA sequencing.Libraries were constructed by SMARTer-Stranded Total RNA Kit (Clontech), according to the manufacturer's instructions.Sequencing was performed with the NextSeq 500 (Illumina).All libraries were sequenced in paired-end mode (75-bp length).
Raw reads were preprocessed for adapter trimming and quality check was assessed using the FastQC tool (http:// www.bioin forma tics.babra ham.ac.uk/ proje cts/ fastqc).Reads were aligned to the reference genome (Ensembl Homo sapiens release GRC38) using the STAR algorithm v.2.7.9. 26Differential expression analysis was performed using the Generalized Linear Model approach implemented in the R/Bioconductor edgeR package (R version 4.1.0;edgeR version 3.36.0). 27Only genes with a cut-off expression >1 CPM in at least three samples were included in the analysis.Table S1 includes the complete list of differentially expressed genes and statistical information (p-value and FDR).Functional enrichment analysis was performed using Ingenuity Pathway Analysis software (IPA v01-13; Qiagen) and Gene Set Enrichment Analysis (GSEA v4.1.0).
To compare iCCA primary cells with HuCCT1 cells, we exploited a public dataset of RNA-sequencing of hepatobiliary cancer cell lines. 28Our analysis focused on the FLNA gene expression in HuCCT1 cells, revealing that this cell line exhibited significantly elevated FLNA gene expression levels (TPM = 1624.7)compared to other cell lines.Subsequently, we investigated whether the up-regulated genes in our dataset were found to be highly expressed in the HuCCT1 cell line.For this purpose, we focused on genes belonging to the IPA pathways of 'Cellular Movement' enriched in our dataset (n = 54 genes).For HuCCT1 cells, we considered expressed and protein-coding genes (TPM≥1; n = 9399 genes).Our next step involved dividing the comprehensive list of 9399 genes into quartile groups applying expression thresholds and evaluating their intersection with our gene list (n = 54 genes).The statistical significance of the intersections was estimated through the hypergeometric test in R (phyper function; Table S1).

| Statistical analysis
The The clinical and demographic characteristics of this cohort of patients are summarized in Table 1.To investigate the significance of the different expressions of FLNA in iCCA, the clinical parameters of iCCA patients were correlated with the combined immunoscore for FLNA, evaluated as previously described. 21deed, iCCA patients were divided into low and high filamin expression according to the median value of the immunoscore distribution (low FLNA expression: immunoscore <3; high FLNA expression: immunoscore ≥3).As shown in Figure 1C, the median DFS in patients with low expression of FLNA was significantly increased in comparison with patients with high expression of FLNA (27 months vs. 10 months).Similarly, 5-year DFS was 30.8% vs. 10.9% (p = .009),in low-and high-FLNA patients, respectively.

| Cholangiocarcinoma primary cells express both full-length FLNA and the cleaved fragment (FLNA CT ) and it is localized in the cytoplasm
To investigate the functional role of FLNA in iCCA, primary tumour cells were isolated from surgically resected tumours.We selected three primary iCCA cells according to different filamin expressions (low, medium and high) observed by immunohistochemistry.These primary iCCA cells express FLNA as a full-length protein (280 kDa) and the cleaved (90 kDa) fragment FLNA CT , as well as for HuCCT1 cells (Figure 2A).
Since the localization of FLNA could affect the behaviour of the tumour, we evaluated its expression in HuCCT1 cells and iCCA primary cells by immunofluorescence.As shown in Figure 2B, FLNA is localized in the cytoplasm, confirming the previous results obtained with immunohistochemistry.Then, to detect FLNA CT expression in the cytoplasm and nucleus, we performed a subcellular fractionation assay.Interestingly, we observed that FLNA CT has cytoplasmic localization in both CCA primary cells and HuCCT1 (Figure 2C).These data suggest that cytoplasmatic localization of both full-length FLNA and FLNA CT might be crucial for the behaviour of iCCAs.

| RNA sequencing reveals that FLNA is involved in iCCA primary cell proliferation and motility
We performed RNAseq analysis on 6 iCCA primary cells, selected according to the immunoscore.These iCCA cells were categorized into two groups: the high-FLNA group (characterized by IHC scores ranging from 3 to 9) and the low-FLNA expression group (defined by IHC scores of 0-2).Our results revealed a significant correlation between the quantity of FLNA protein, as assessed by IHC and mRNA expression levels, thereby supporting the accuracy of the immunoscore in reflecting FLNA expression in iCCA patients.The patient's characteristics are summarized in Table 3.We used two different strategies to investigate the pathways that are modulated in our condition.We applied IPA on the list of 661 differentially expressed genes from RNAseq, to identify enriched functions that are modulated in High versus Low FLNA samples (Figure 2D; Table S2).
The GSEA analysis was applied to the total gene list using the Correlation approach to identify gene sets belonging to C2 collection significantly correlated with the expression of the FLNA gene along our samples.The analysis revealed a negative correlation with gene sets related to proliferation and epithelial-to-mesenchymal transition (Figure 2F).
Furthermore, in order to ascertain the comparability of primary CCA cells displaying high FLNA expression and HuCCT1 cells in terms of their transcriptional profiles, we employed a publicly available dataset. 28We focused on genes related to "Cellular Movement" in our gene list (n = 54 genes) and highly expressed in HuCCT1.Out of the 54 up-regulated genes, 28 genes were found to be expressed above the 75th percentile in the HuCCT1 dataset.This observation yielded a p-value of 1.959553 −05 , as determined by the hypergeometric test.The remaining 26 genes were distributed below the 75th percentile, showing no statistically significant association (p > .05;Table S1).
These results confirm the similar transcriptional profile between our high-FLNA group and HuCCT1 cells.

| FLNA promotes cell proliferation and migration of iCCA primary cells
To investigate the role of FLNA in tumour progression, we evaluated the effect of FLNA silencing.As shown in Figure 3A, FLNA silencing (reaching about 85% reduction of FLNA expression) strongly reduced full-length FLNA and FLNA CT expression both in iCCA primary cells and the HuCCT1 cell line.To further investigate the involvement of FLNA in cell proliferation, we performed a BrdU cell assay on both HuCCT1 cells and the three iCCA primary cells silenced for FLNA.As shown in Figure 3B, FLNA knockdown significantly decreased BrdU incorporation in HuCCT1 cells (−54% ± 19%, p < .01 vs.
Furthermore, we investigated the involvement of FLNA in iCCA cell motility.To overcome the limitations in the use of primary cells, such as limited lifespan, the motility assay was performed on HuCCT1 and in the highest FLNA-expressing iCCA primary cells (iCCA 3).

| FLNA integrity plays a key role in iCCA cell proliferation and migration
To investigate the role of FLNA integrity on iCCA cell proliferation, we used calpeptin treatment on iCCA primary cells.Calpain is an enzyme that hydrolyses FLNA producing a 90 kDa fragment (FLNA CT ), we therefore investigated the effect of calpeptin, a chemical inhibitor of calpain on FLNA CT

| DISCUSS ION
iCCA is a rare and aggressive cancer that often presents at an advanced stage, making it difficult to treat.It is associated with a poor prognosis, with a five-year survival rate of less than 10%. 1 There is currently no effective treatment for iCCA, and research efforts are focused on identifying new therapeutic targets and developing novel treatments. 2 FLNA is a cytoskeletal protein that has been implicated in the development and progression of various cancers, including HCC. 9 It is involved in the regulation of actin cytoskeleton dynamics and plays an important role in cell migration, adhesion and signalling.
Our study demonstrates that FLNA is overexpressed in human iCCA and is associated with increased cell migration and invasion.
Importantly, FLNA expression levels significantly correlate with DFS, suggesting that it may be a useful biomarker for disease progression and prognosis.
Although the mechanisms by which FLNA contributes to iCCA progression are not yet fully understood, our study provides significant pathogenetic insights.First, transcriptomic data suggest that FLNA may play a role in the activation of signalling pathways that promote cell survival, proliferation and metastatization in iCCA.Further, knockdown of FLNA expression using siRNA inhibits the migration and invasion of human primary iCCA cells in vitro.Similarly, the knockdown of FLNA has been associated with decreased metastasis formation and proliferation in human melanoma cells and pulmonary neuroendocrine tumours. 8,31In this respect, nanoparticle-mediated FLNA siRNA delivery systems could be proposed as a potential therapeutic strategy for iCCA progression. 32rther, we observed that both full-length FLNA and FLNA CT are located in the cytoplasm of iCCA primary cells.4][35] Cytoplasmic FLNA is often highly overexpressed in metastatic cancers, 36,37 on the contrary, after FLNA proteolysis, FLNA CT can translocate to the nucleus, where it regulates transcription and inhibits tumour growth and metastasis. 20ck of nuclear FLNA CT is considered a mechanism of cancer progression. 37Thus, our data lead us to speculate that the absence of nuclear localization may confer aggressiveness to iCCA and promote tumour development.This was further supported by calpeptin treatment on iCCA primary cells, which decreased cell proliferation and inhibited cell viability.These results are in agreement with a previous study conducted in different tumour cells, where FLNA CT has been shown to be involved in tumour cell proliferation, migration, invasion and colony formation. 18cordingly, recent data suggest that targeting calpain activity with specific inhibitors seems to be a useful strategy for limiting the development and invasiveness of primary tumours. 38To note, combining calpeptin 100 μM with low-dose chemotherapy proves significantly more effective in inhibiting cell viability compared to using calpeptin 100 μM or GEM/CDDP alone.This highlights calpeptin's capacity to influence the susceptibility of CCA cells to chemotherapy." Finally, FLNA was reported to regulate MMP-9 expression, suppress prostate cancer cell migration and correlate with Snail-induced epithelial-mesenchymal transition (EMT) in colorectal cancer. 35,36Since FLNA was found to be associated with Smads and to regulate transforming growth factor β1 (TGFβ1), while calpeptin was able to suppress the activation of the TGFβ1-Smad2/3 signalling pathway, with a crucial role in lung EMT, 39,40 we can speculate that FLNA/calpeptin action may regulate iCCA progression through the TGFβ1-Smad signalling pathway.
Even though our study lacks in vivo validation, the main strength of this study is the use of primary iCCA cells isolated from resected patients with different levels of FNA expression.
4392420) and the Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668019) according to the manufacturer's instructions.Briefly, 400 000 cells and 20 000 cells were seeded into 6-well plates and 96-well plates, respectively.Lipofectamine 2000 (5 or 0.5 μL) was diluted in Opti-MEM (Invitrogen).45 nM FLNA siRNA was also diluted in Opti-MEM in a separate test tube and then added to the Lipofectamine 2000 Reagent mix (1:1 ratio).The final mix was incubated for 5 min at room temperature.Subsequently, HuCCT1 or iCCA cells were incubated with the mixture for 72 h.

1 |
results are expressed as mean ± SD.Descriptive statistics were carried out, and the Shapiro-Wilk normality test was employed to assess normality.An unpaired two-tailed Student's test was used to detect the significance between two series of data.One-way ANOVA analysis was used to compare three or more groups, followed by Dunnett's post hoc comparisons test.For the analysis of iCCAs' cell proliferation, two-way ANOVA followed by Sidak's multiple comparisons test was used.Survival was estimated by the Kaplan-Meier method and comparisons were performed by the Log-Rank test.The Cox proportional hazard model was used to identify independent prognostic factors for survival.These factors include age, sex, aetiology, quality of the underlying liver, serum Ca-19.9, serum bilirubin level, serum cholinesterase level, platelet count, size of the tumour, number of tumours, microvascular invasion, macrovascular invasion, tumour grading, extent of hepatectomy, type of hepatectomy and type of surgical margin.To avoid the problem of model overfit-ting and to limit its optimistic performance, we tailored the statistical model using bootstrap resampling.29,30Data are presented using hazard ratios (HRs), subdistribution hazard ratio (sHR), 95% confidence intervals (CIs), P-values and survival plots.A p-value <.05 was considered significant for all tests.Computations were performed using the GraphPad Prism software (La Jolla, CA, USA, v. 8.0) and IBM-SPSS statistics software (Chicago, IL, USA, v. Filamin A expression in iCCA tissues correlates with DFS The immunoexpression of FLNA was analysed and graded in 82 patients who underwent hepatic resection for iCCA in January 2004 and December 2018 taking into account both the percentage of positive cells (0%-30% = 1, 31%-60% = 2, 61%-100% = 3) and the staining intensity (1 = weak; 2 = medium intensity; and 3 = strong immunoreactivity).As shown in Figure1A, FLNA had variable expression in different samples, primarily displaying a cytoplasmic localization within tumour cells.
1 (A) FLNA expression in iCCA.Tissue was stained with mouse monoclonal anti-FLNA antibody (brown staining) and counterstained with haematoxylin (blue staining): (i) low FLNA expression; (ii) medium FLNA expression; (iii) high FLNA expression; Scale bar = 50 μm.(B) the table and graph bar show the iCCA patients' percentage for each combined score.(C) Five-year disease-free survival of 82 patients resected for iCCA stratifying based on FLNA expression in the intratumoral tissue compartment.As shown, there is a statistically significant difference (p = .009)between low-FLNA and high-FLNA patients.

F I G U R E 4
Production of FLNA CT was inhibited after treatment with Calpeptin.(A, B) Immunoblots of full-length FLNA and FLNA CT in HuCCT1 and iCCA 3 cells treated with calpeptin (100 μM).Quantification of densitometric readings of the immunoblot bands is shown in the right panel.FLNA CT /vinculin ratios in calpeptin-treated cells were normalized to their respective untreated control group.Experiments were repeated at least 3 times.Values represent mean (±SD).Unpaired two-tailed Student's t-test, *p < .05 and **p < .01versus control.
Future developments of this work should aim to investigate the role of FLNA on the tumour microenvironment.In conclusion, our study demonstrates that FLNA, an important protein involved in the regulation of actin cytoskeleton dynamics and cell migration, is overexpressed in iCCA and may play a role in the development and progression of this deadly liver cancer.Further research is needed to fully understand the mechanisms by which FLNA contributes to iCCA and to develop effective FLNA-targeted therapies for this disease.

F I G U R E 5
Treatment with calpeptin inhibited cell proliferation, cell viability and cell migration.(A) After 24 h of calpeptin treatment at different concentrations (10, 50 and 100 μM), a BrdU incorporation assay was performed on HuCCT1 cells.Values represent mean (±SD), ***p < .01 and ****p < .001versus control.Statistical analysis was performed with one-way ANOVA followed by Dunnett's post hoc comparisons test.(B) Calpeptin 100 μM decreased cell proliferation of primary iCCA 3 cells.Experiments were repeated at least three times.Unpaired two-tailed Student's t-test, ****p < .001versus control.(C, D) HuCCT1 and primary iCCA cells were incubated with calpeptin (100 μM) for 48 h.Cell viability was assessed by MTT.Absorbance was read at 570 nm in the Synergy H4 plate reader.Each experiment was done in triplicate and all experiments were repeated at least three times.Values represent mean (±SD).*p < .05,**p < .01versus control.(E, F) HuCCT1 and iCCA 3 cells were plated in serum-free media with or without calpeptin 100 μM.After 24 h of incubation at 37°C, migratory cells were stained and quantified by measuring fluorescence with a fluorescence plate reader at 480 nm/520 nm.Data from triplicate experiments are presented as mean ± SD values.Unpaired two-tailed Student's t-test, *p < .05,**p < .01versus control.(G) CCA primary cells were treated with calpeptin (100 μM), or gemcitabine 0.2 μM (GEM) and cisplatin 0.04 μM (CDDP), or gemcitabine 1 μM and cisplatin 0.2 μM, or combined with calpeptin (100 μM), for 48 h.Cell viability was assessed by MTT.Absorbance was read at 570 nm in the Synergy H4 plate reader.Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparisons test.Values represent mean (±SD).*p < .05,**p < .01,***p < .001,****p < .001.

TA B L E 1
Demographic and clinical characteristics of the iCCA cohort examined.
Univariate and multivariableCox regression models for the assessment of baseline factors associated with DFS.
TA B L E 2