FSCN1/ACK1 axis-dependent M2 macrophage polarization fuels colorectal cancer progression

Abstract

BACKGROUND

Fascin actin-bundling protein-1 (FSCN1) is upregulated in colorectal cancer (CRC) and correlated with poor prognosis. However, its role in shaping the tumor-immune microenvironment remains poorly understood.

AIM

To elucidate the mechanism by which FSCN1 regulates macrophage polarization and promotes CRC progression.

METHODS

FSCN1 expression was assessed in human CRC tissues and cell lines. Functional assays evaluated the effects of FSCN1 knockdown on CRC cell proliferation, migration, and invasion in vitro and on tumor growth in a nude mouse xenograft model in vivo. Macrophage polarization was analyzed using conditioned medium from FSCN1-manipulated CRC cells. Mechanistic studies employed rescue experiments with ACK1 overexpression to validate the FSCN1/ACK1 signaling axis.

RESULTS

FSCN1 was significantly upregulated in CRC tissues and positively correlated with M2-type tumor-associated macrophage infiltration. FSCN1 knockdown suppressed CRC cell malignant behavior in vitro and inhibited tumor growth in vivo. Mechanistically, FSCN1 silencing reduced activated Cdc42-associated kinase 1 (ACK1) expression. Conditioned medium from FSCN1-deficient CRC cells reprogrammed macrophages toward an M1-like phenotype, as evidenced by increased CD80, inducible nitric oxide synthase, and interleukin-12 and decreased CD206, arginase 1, and interleukin-10. Crucially, ACK1 overexpression reversed this M1 repolarization, confirming that FSCN1 drives M2 polarization via ACK1. In vivo, FSCN1 knockdown delayed tumor onset, reduced Ki67-positive cells, and reshaped the microenvironment toward a less immunosuppressive state.

CONCLUSION

The FSCN1/ACK1 axis promotes CRC progression by facilitating M2 macrophage polarization, highlighting it as a potential therapeutic target for restoring antitumor immunity in CRC.

Key Words: Colon cancer; Fascin actin-bundling protein-1; Activated Cdc42-associated kinase 1; Macrophage; M2 polarization

Core Tip: This study reveals that fascin actin-bundling protein-1 (FSCN1) promotes colorectal cancer progression by driving M2 macrophage polarization via activated Cdc42-associated kinase 1. FSCN1 knockdown reprograms macrophages toward an anti-tumor M1 phenotype and suppresses tumor growth, identifying the FSCN1/activated Cdc42-associated kinase 1 axis as a therapeutic target for restoring anti-tumor immunity.

INTRODUCTION

Colorectal cancer (CRC) is a malignant tumor of the digestive tract, with a high incidence worldwide. From a gender perspective, colon cancer ranks third and second in the number of new cases of male and female cancer worldwide, respectively, and its death toll ranks third among male and female cancer deaths[1,2]. In China, the number of new cases of colon cancer is second only to lung cancer, making it the second largest cancer. Among the female population, CRC is the second leading cause of cancer-related deaths after lung cancer. In addition, once cancer cell metastasis occurs in patients, their 5-year survival rate significantly decreases, posing a huge threat to life safety[3,4].

Tumor metastasis is driven by not only intricate alterations within cancer cells but also dynamic interactions between these cells and their surrounding microenvironment. The tumor microenvironment (TME), the internal milieu where tumors originate and progress, comprises tumor cells, stromal cells, extracellular matrix, microvasculature, interstitial fluid, and various other components[5]. It is a key factor affecting metastasis. The microenvironment of the primary and metastatic sites of tumors, as well as the immune cells within them, are expected to become better targets for treating tumor metastasis[6]. TME has been proven to be an indispensable part in the occurrence, development, and metastasis of colon cancer. For example, ANKRD22 expression is closely related to the survival time of patients with colon cancer and the abundance of M1 tumor-associated macrophage (TAM) infiltration. ANKRD22 silencing alters the subtype distribution of TAMs, weakens their pro-inflammatory activity, and enhances their immunosuppressive activity. Piceatannol effectively suppresses the invasiveness of SW480 cells by disrupting the positive feedback autocrine/paracrine loop involving transforming growth factor-β1 (TGF-β1) between M2-like TAMs and cancer cells. Within the TME, TAMs serve as a critical component and act as a “shortcut” that accelerates tumor cell invasion and metastasis. Macrophages have two main phenotypes: (1) The classically activated M1 type; and (2) The alternatively activated M2 type. M1-polarized TAMs exert antitumor effects by releasing pro-inflammatory mediators that recruit and activate immune cells[7,8]. Moreover, M1-type TAMs can exert tumoricidal effects by nitric oxide or reactive oxygen species, triggering DNA damage and apoptosis in tumor cells. Typical biomarkers for M1-polarized TAMs include tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS). By contrast, M2-type TAMs are primarily polarized by T helper 2 cytokines interleukin (IL)-4 and IL-13, along with macrophage colony-stimulating factor. These M2 macrophages secrete anti-inflammatory mediators, including vascular endothelial growth factor, TGF-β, matrix metal loproteinases, and other cytokines, that critically support processes like epithelial-mesenchymal transition, angiogenesis, and immunosuppression. Common M2-type TAM marker markers include arginase 1 (Arg1), TGF-β, and CD206. Unlike M1 macrophages, M2 macrophages can inhibit inflammatory responses, block tumor immune surveillance, and promote tumor proliferation and metastasis. For example, during liver metastasis of CRC, miR-934 promotes M2 macrophage polarization by suppressing PTEN expression, thereby accelerating the activation and transduction of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway[9]. EZH2 inhibitors exert CRC inhibition by acting on the transcription factor signal transducer and activator of transcription 3 (STAT3), converting the M2 phenotype to the M1 phenotype[10]. The tumor immune molecule CD155 in TAMs promotes CRC cell growth by enhancing macrophage polarization towards the M2 phenotype[11].

Fascin actin-bundling protein-1 (FSCN1) is a globular actin-bundling protein in the fascin family. It stabilizes the parallel actin bundles, and it is associated with essential parts of many types of cells with abundant materials, such as microspikes, filopodia, and lamellipodium, within their cytoskeleton. Normal adults have limited expression of FSCN1 at the cellular level. It is primarily associated with few cell types, such as neurons, endothelium, stroma, and dendritic cells, but not epithelial cells. FSCN1 overexpression in transformed epithelial cells drives tumor cell motility and invasion across multiple malignancies[12]. Clinically, increased FSCN1 correlates with poor prognosis, metastasis, and shortened survival in diverse cancers, including CRC, where its expression is significantly associated with advanced clinicopathological stage and adverse outcomes[13-15]. Notably, emerging evidence implicates FSCN1 in immune modulation beyond its canonical cytoskeletal role. In lung adenocarcinoma, FSCN1 activates PI3K-AKT and JAK-STAT signaling to upregulate IL-4 and macrophage colony-stimulating factor, thereby promoting M2 macrophage polarization[16]. Similarly, FSCN1 has been identified as an M2-TAM-associated gene in hepatocellular carcinoma via integrative genomic screening[17]. Although these findings originate from non-CRC contexts, their relevance to CRC is supported by three converging lines of evidence. First, the PI3K-AKT and JAK-STAT pathways are constitutively activated in CRC and well-established drivers of M2-TAM polarization and immune evasion. Second, CRC shares a profoundly immunosuppressive microenvironment characterized by abundant M2-TAM infiltration that correlates with metastasis and therapy resistance. Third, FSCN1 is consistently overexpressed in CRC tissues and linked to aggressive disease, suggesting that it may exert analogous immunomodulatory functions in this malignancy. Collectively, these observations provide a strong biological rationale for investigating whether FSCN1 orchestrates M2 macrophage polarization and metastatic progression in CRC through mechanisms conserved across epithelial cancers.

However, the upstream regulatory mechanism for driving FSCN1 overexpression and its effects on the immune microenvironment in CRC are still not fully clear. The key kinases or signaling molecules orchestrating FSCN1-induced cytoskeletal rearrangement and immune escape are essential targets in treatment development. Under these circumstances, activated Cdc42-associated kinase 1 (ACK1) can be considered an effective onco-motor of tyrosine kinase receptor activity in multiple tumors. ACK1 has been reported to be amplified or overexpressed in prostate, breast, and lung cancer cells. It helps the cell survive, migrate, and invade by phosphorylating several key substrates of cytoskeleton dynamics and growth-factor signal transduction pathways. ACK1 is able to specifically bind with WAVE3, stabilizing it in this role for AR and enhancing filamentation. Previous studies have suggested a direct connection between ACK1 and WAVE3. Moreover, recent research has found that ACK1 signalization may be involved in immune-mediated regulation, and further investigation is needed to elucidate how it affects the tumor-immunity status. ACK1 is the primary controller of Cdc42. Studies have confirmed that Cdc42 is closely associated with actin-bundling proteins such as FSCN1. Therefore, ACK1 could be considered an upstream activator of FSCN1 in CRC. Furthermore, it was thought to be an essential pathway of the FSCN1-ACK1-axis promoting M2-macrophage differentiation and metastatic progression. Activated ACK1 is a tyrosine kinase frequently amplified in multiple cancers, regulating cytoskeletal dynamics and cell migration through phosphorylation of key substrates. As a primary regulator of Cdc42, which interacts directly with actin-bundling proteins, including FSCN1, ACK1 is a plausible upstream activator of FSCN1. Moreover, ACK1 signaling has been implicated in immune regulation, though its role in tumor immunity requires further elucidation. This study hypothesized that ACK1 acts as an upstream activator of FSCN1 in CRC, and that the ACK1/FSCN1 axis promotes M2 macrophage polarization and metastatic progression. This study aimed to delineate this regulatory axis and evaluate its potential as a therapeutic target.

MATERIALS AND METHODS

Clinical samples and patient characteristics

A total of 20 pairs of freshly frozen colon cancer tissue samples and corresponding adjacent normal tissues were obtained from 20 patients undergoing surgical treatment at Shandong Provincial Hospital Affiliated to Shandong First Medical University.

Inclusion criteria: (1) Patients with histopathologically verified colon adenocarcinoma; (2) Patients who did not receive any preoperative chemoradiotherapy; and (3) Patients with complete medical records and clinical data.

Exclusion criteria: (1) Patients with other concurrent malignant tumors; and (2) Patients with severe co-morbidities such as severe cardiac, hepatic, or renal dysfunction.

The clinicopathological characteristics of the enrolled patients are summarized in Table 1. This study was reviewed and approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University, and all subjects provided informed consent.

Table 1 Clinicopathological characteristics of patients with colon cancer (n = 20), mean ± SD/n (%).

Characteristic	Value
Age (years)	64.5 ± 11.2
Range	42-83
Sex
Male	11 (55.0)
Female	9 (45.0)
TNM stage
I	3 (15.0)
II	7 (35.0)
III	8 (40.0)
IV	2 (10.0)
Lymph node metastasis
Yes	10 (50.0)
No	10 (50.0)

Cells and transfection

CCD-18Co and SW620 cells purchased from ATCC were cultured in DMEM (Gibco, Rockville, MD, United States) supplemented with 10% FBS and 1% penicillin-streptomycin solution under standard conditions (37 °C and 5% CO₂ atmosphere). The full-length coding sequence of human ACK1 (Accession No. NM_001374.2) was synthesized and subcloned into the pcDNA3.1(+) mammalian expression vector containing an empty vector to establish ACK1 overexpression (ACK1-OE) models. The ACK1-OE plasmid and the corresponding empty vector control were constructed by RiboBio Co., Ltd (Guangzhou, Guangdong Province, China). The integrity of the constructed plasmids was confirmed by Sanger sequencing prior to use.

Small interfering RNAs (siRNAs) targeting FSCN1 (si-FSCN1), ACK1 (si-ACK1), and a non-targeting control (si-NC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The cells were seeded in six-well plates and transfected with 2.5 μg of plasmids or 100 nM of siRNAs by using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, United States) in accordance with the manufacturer’s instructions. The transfection efficiency was assessed 48 hours post-transfection via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting. THP-1 human monocytic leukemia cells were differentiated into mature macrophages by using 100 ng/mL of PMA. Subsequently, the differentiated macrophages were co-cultured with conditioned medium (CM) collected from SW620 cells, SW620 cells transfected with vector, and SW620 cells with FSCN1 or ACK1 knockdown.

Xenograft tumor model in nude mice

A total of 10 BALB/c nude mice (weighing 14-18 g, aged 4-6 weeks) were acquired from Beijing Charles River Laboratory Animal Technology Co., Ltd. All experimental animals were housed in pathogen-free animal rooms and given a light/dark cycle every 12 hours, with no restrictions on food and water intake. The mice were randomly divided into two groups (n = 5 per group): (1) The SW620-si-NC group; and (2) The SW620-si-FSCN1 group. SW620 cells transfected with either si-NC or si-FSCN1 were harvested and resuspended at a density of 6 × 10⁷ cells/mL. A volume of 100 μL of the corresponding cell suspension was injected subcutaneously into the right axillary region of each mouse in accordance with its group assignment. Tumor dimensions [long diameter (L) and short diameter (W)] were measured using a digital caliper on days 7, 14, 21, and 28 post-inoculation. Tumor volume was calculated using the following formula: V = (L × W²)/2. On day 28, all mice were euthanized, and tumor tissues were harvested for subsequent analysis. This study was approved by the Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University.

RT-qPCR

Total RNA was extracted using TRIzol reagent and subsequently reverse-transcribed into cDNA by using the PrimeScript RT kit. The reverse transcription reaction volume was 10 μL, and the reaction conditions were set at 37 °C for 15 minutes, with three cycles for the reverse transcription reaction. Subsequently, the prepared PCR reaction mixture was amplified using a real-time quantitative PCR instrument. All primers in this study were provided by Sangon Biotech (Shanghai). Expression data were analyzed using the comparative cycle threshold (2^-ΔΔCt) method, with normalization to housekeeping gene(s).

Western blotting

After the concentration of the extracted total protein was determined using BCA assay, equal amounts of protein samples were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Bio-Rad, United States) via wet transfer. The membranes were then blocked for 2 hours at room temperature in TBST containing 5% (w/v) non-fat dry milk. Afterwards, the membranes were incubated overnight at 4 °C with the following appropriately diluted primary antibody working solutions: (1) GAPDH (1:2500, ab9485); (2) FSCN1 (1:10000, ab126772); (3) Arg1 (1:1000, ab124917); (4) CD163 (1:1000, ab182422); (5) CD206 (1:2000, ab125028); (6) ACK1 (1:500, ab74091); and (7) The iNOS (1:1000, ab178945). After the membranes were thoroughly washed, they were incubated with corresponding secondary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system. Band intensities were semi-quantified with ImageJ software.

CCK-8 assay

Cells were detached using 0.25% trypsin, counted, and resuspended in DMEM at a concentration of 3 × 10⁴ cells/mL. Aliquots of 100 μL were then dispensed into each well of a 96-well plate. After a 4 hours adhesion period under standard culture conditions (37 °C, 5% CO₂, humidified), cellular viability was quantified spectrophotometrically at 450 nm with a microplate reader.

Flow cytometric analysis of M1/M2 macrophage polarization

After the culture medium was aspirated and washed two times with PBS, 0.25% trypsin was added to each well, and then the wells were incubated at room temperature for 5 minutes to disperse the cells. After digestion was stopped, full culture medium was added with 10% FBS and transferred to a centrifuge tube for later use. Then, the suspension was dispensed into 100 μL aliquots in each flow cytometry tube, and 0.1 μL of either CD86-APC or CD206-PE fluorescence-labeled antibodies was added to each tube and mixed gently. Next, the suspension was placed in the dark at room temperature for 20 minutes. Afterwards, the cells were centrifuged to separate them from the solution. The supernatant was discarded. Subsequently, the cell pellet was resuspended in 2 mL of PBS and washed several times with PBS to eliminate residual unbound reagents. Approximately 100 μL of residue from the last wash was aspirated using a pipette. Next, 100 μL of 4% paraformaldehyde (PFA) was added to fix the cells for 15 minutes at room temperature to maintain the stability of samples over time during data collection and inactivation of potential biohazards. Previous optimization showed that this moderate fixation approach can preserve the epitopes’ integrity without causing fluorescence loss in CD86 and CD206 expression markers of T cells. A stationary cell suspension was frozen at 4 °C in darkness for no more than 24 hours after collection.

Enzyme-linked immunosorbent assay

The key cytokines TNF-α, IL-1β, IL-10, and TGF-β were quantitatively determined using an enzyme-linked immunosorbent assay kit provided by SolarBio Technology Company Limited in China.

Immunofluorescence

After being fixed in a mixture of 4% PFA for 15 minutes, rinsed three times with PBS and then incubated at room temperature for 20 minutes in a solution containing Triton X-100 at low concentration. The slides were washed three times with PBS, blocked with 5% bovine serum albumin at room temperature for 60 minutes, and placed in a humid box at room temperature (37 °C) for overnight incubation with primary antibodies. After the slides were washed three times with PBS, they were incubated in the dark at room temperature for 1 hour after adding a fluorophore-labeled secondary antibody. Following another three PBS rinses, the slides were covered in a solution of anti-fade mounting agent containing DAPI to observe immunofluorescent signals by using a fluorescent microscope.

Immunohistochemical detection

Paraffin-embedded tissue sections were dehydrated successively as follows: First, they were paraffinized using xylene, followed by rehydration in distilled water. For antigen retrieval, the sections were incubated in 75 °C water bath for 1 hour with gentle agitation, cooled, and washed three times with PBS, two times each time. Afterwards, the sections were incubated with 3% hydrogen peroxide in distilled water for 10 minutes to block endogenous peroxidase and then washed with PBST three times. Primary antibodies at appropriate dilution concentrations were applied directly to the tissue sections to optimize antibody bonding, and then the sections were incubated for 1 hour at room temperature in a moisture-coated container. After being gently washed to remove free primary antibodies, the sections were exposed to corresponding species-specific secondary antibodies at 37 °C for 20 minutes in a water bath to enhance signal detection. Excess secondary antibodies were removed by washing with PBS, they developed according to the DAB method and observed using a microscope. The reaction was terminated, and nuclei were stained by hematoxylin. Afterwards, the sections were dehydrated in 95% (or higher) ethanol solution in accordance with standard protocols. Finally, the sections were scanned using a NanoZoomer S210 Digital Slide Scanner. Positive staining was detected by the presence of brown precipitates and assessed accordingly.

Colony-formation assay

Low-dose seeding was used to grow single colonies, that is, 1 × 10 cells in each well of a six-well plate. Then, 2 mL of fresh complete medium was added to each well. The plate was rotated in a circular motion so that the cells spread out evenly throughout the wells. For 2 weeks, the cells were cultured in a moist environment and supplemented with all other components of the complete culture medium regularly. On day 14, the culture medium was aspirated, and after being flushed with PBS, it was fixed in a mixture of PFA at room temperature for 30 minutes. The fixative was removed and PBS washes from the wells. Subsequently, 1 mL of crystal violet solution was added to each well, which was then placed in the dark at room temperature for approximately 2 hours. Afterwards, the dye from each well was washed off in PBS multiple times after treatment. The plates were air dried, and colonies were quantified by image capture and manual counting. A colony was considered viable if it contained more than 50 cells, and only such colonies were enumerated.

Wound-healing assay

SW620 cells were seeded into six-well plates and incubated for 12 hours. Afterwards, the cells were washed twice with PBS. Once a confluent monolayer was formed, a straight scratch wound was generated using a sterile pipette tip. The cells were then permitted to migrate into the wounded area for 24 hours under standard culture conditions (37 °C and 5% CO₂). Cell migration was monitored over time by capturing images of the scratch at designated intervals and measuring the gap width by using phase-contrast or bright-field microscopy.

Transwell invasion assay

After the culture medium was added to the lower chamber of the Transwell chamber, the cells were seeded into the upper chamber pre-filled with Matrigel and cultured in the incubator for 48 hours. Next, a cotton swab was used to erase non-invasive cells in the non-upper chamber, and the invaded cells in the lower chamber were fixed and stained with 70% ethanol and 0.1% crystal violet, respectively. The number of invading cells was manually counted using an Olympus optical microscope (Tokyo, Japan).

Statistical analysis

All experimental data were analyzed statistically using SPSS (version 22.0) software. The quantitative results from three independent replicates are expressed as SD. Comparisons between two groups were performed using student’s t-test, and one-way analysis of variance was applied for analysis across multiple groups. A P value of less than 0.05 was considered statistically significant.

RESULTS

High expression levels of FSCN1 and M2 macrophage markers in colon cancer tissues

Figure 1A and B present the RT-qPCR and western blotting data demonstrating that FSCN1 expression was markedly increased in colon cancer tissues compared with their matched adjacent non-tumorous tissues. Further research across colon cancer cell lines showed that compared with the normal colon epithelial cell line CCD-18Co, the SW620 cells had considerably increased FSCN1 expression at the RNA (Figure 1C) and protein levels (Figure 1D). Thus, it is likely involved in colon-cancer genesis or development. To measure the extent to which M2 macrophages infiltrated the TME based on these established markers for determining M2 cells. The Arg1, CD163, and CD206 expression levels in colon cancer tissue were higher than those in adjacent normal tissue (Figure 1E and F). Therefore, patients with CRC have an increased number of M2 type macrophages that can induce an immunosuppressive environment for tumor growth.

Figure 1 Fascin actin-bundling protein-1 is highly expressed in colon cancer tissues and cell lines. A and B: The mRNA and protein expression levels of fascin actin-bundling protein-1 in colon cancer tissues; C and D: The mRNA and protein expression levels of fascin actin-bundling protein-1 in colon cancer cell line SW620 and normal colon epithelial cell line CCD-18Co; E and F: Expression levels of M2 macrophage markers arginase 1, CD163, and CD206 in colon cancer tissues and their paired adjacent tissues. ^cP < 0.001. FSCN1: Fascin actin-bundling protein-1; Arg1: Arginase 1.

Malignant phenotype loss induced by FSCN1 knockdown in CRC cell lines

An si-FSCN1 construct was specifically designed to transfect SW620 cells and explore the effects of FSCN1 on cell phenotypes and functions in colon cancer through siRNA-mediated gene knockout. Meanwhile, a non-specific si-control experiment was established. The results of RT-qPCR and Western blotting showed that the si-FSCN1 group had substantially lower mRNA (Figure 2A) and protein levels (Figure 2B) of FSCN1 than the si-NC group. Therefore, further functional studies using this reagent was deemed feasible. FSCN1 knockdown revealed significantly decreased cell proliferation (Figure 2C). Moreover, relatively fewer transmembrane cells were found in the si-FSCN1 group than in the si-NC group after knocking down FSCN1 (Figure 2D). Thus, suppression of FSCN1 reduced cell migration. The invasive cells in the si-FSCN1 group were significantly fewer than those in the control group, and this difference is also shown by Matrigel invasion tests (Figure 2E). Colony-forming assay showed that the si-FSCN1-transfected cells formed significantly fewer colonies than the cells in the control group (Figure 2F). Therefore, FSCN1 knockdown markedly reduced the proliferation ability and cell division capacity of CRC cells. These findings demonstrated that inhibiting the activity of FSCN1 significantly reduced the proliferation ability, migration capability, invasiveness, and colony-forming efficiency of the colon cancer cell line SW620.

Figure 2 Fascin actin-bundling protein-1 silencing inhibits the malignant phenotype of SW620 cells. A and B: The mRNA and protein expression levels of fascin actin-bundling protein-1; C: Cell proliferation; D and E: Transwell cell migration and cell invasion. Scale bar = 200 μm and 100 μm; F: Clone formation experiment. ^cP < 0.001. FSCN1: Fascin actin-bundling protein-1; NC: Non-targeting control.

Effect of FSCN1 knockdown on the polarization of M2 macrophages

THP-1 cells were treated with 100 ng/mL of PMA for 24 hours to induce their differentiation into mature, adherent macrophages and examine the influence of FSCN1 on macrophage polarization. The culture supernatants of SW620, SW620-si-NC, and SW620-si-FSCN1 cells were collected as CM to treat the differentiated macrophages. Compared with macrophages treated with CM from the SW620 and si-NC groups, those exposed to CM from the si-FSCN1 group exhibited an increase in the expression levels of M1 polarization markers NOS2, TNF-α, and IL-1β (Figure 3A-C), suggesting a shift toward the antitumor M1 macrophage phenotype. On the contrary, the mRNA expression levels of M2 polarization-related markers Arg1, CD206, and IL-10 were downregulated in the si-FSCN1 group (Figure 3D-F), indicating that FSCN1 silencing can inhibit macrophage polarization toward pro tumor M2 polarization. In addition, the immunofluorescence staining results showed that the positive signal of CD86 (M1 marker) was enhanced in macrophages treated with CM in the si-FSCN1 group (Figure 3G), whereas the fluorescence intensity of CD163 (M2 marker) was weakened (Figure 3H). These results indicated that downregulation of FSCN1 in colon cancer cells can alter the TME through secretion of factors, thereby reprogramming macrophage polarization direction, promoting its transformation to M1 type, and inhibiting M2 type polarization. These findings were validated in another CRC cell line, SW480. The results demonstrated that FSCN1 expression was similarly upregulated in SW480 cells. Knockdown of FSCN1 inhibited SW480 cell proliferation. Moreover, macrophages co-cultured with CM from si-FSCN1-treated cells (si-FSCN1-CM) exhibited increased expression of NOS2 and decreased expression of Arg1. These findings indicated that FSCN1 silencing suppressed macrophage polarization toward the pro-tumor M2 phenotype (Supplementary Figure 1).

Figure 3 Knockdown of Fascin actin-bundling protein-1 inhibits M2 phenotype of macrophages. A-C: The mRNA expression levels of M1 markers NOS2, tumor necrosis factor-α, and interleukin-1β; D-F: The mRNA expression levels of M2 markers arginase 1, CD206, and interleukin-10; G and H: Immunofluorescence staining of M1 marker CD86 and M2 marker CD163. Scale bar = 50 μm. ^cP < 0.001. THP-1 cells were induced to differentiate into macrophages by PMA and treated with conditioned medium containing SW620, SW620-si-NC, and SW620-si-FSCN1 cells, respectively. TNF-α: Tumor necrosis factor-α; FSCN1: Fascin actin-bundling protein-1; CM: Conditioned medium; Arg1: Arginase 1; IL: Interleukin; NC: Non-targeting control.

Effect of ACK1 knockdown on the malignant phenotype of colon cancer cells

The effect of FSCN1 silencing on ACK1 expression was examined to investigate the potential molecular mechanisms by which FSCN1 regulates the biological behavior of colon cancer cells. The results demonstrated that compared with the SW620 and si-NC groups, transfection with si-FSCN1 markedly reduced the mRNA (Figure 4A) and protein expression levels (Figure 4B) of ACK1, indicating that FSCN1 may act as a positive regulator of ACK1 expression. An ACK1-OE plasmid and a control vector were constructed and si-NC and si-ACK1 groups were set up to further validate the function of ACK1 in colon cancer. The results demonstrated that the ACK1 mRNA and protein expression levels in the si-ACK1 group decreased, whereas the ACK1-OE group successfully achieved high ACK1 expression (Figure 4C and D), indicating ideal transfection efficiency. The functional experiment results showed that knocking down ACK1 significantly inhibited the proliferation (Figure 4E), migration (Figure 4F), and invasion abilities (Figure 4G) of SW620 cells, and the number of clones formed was significantly reduced (Figure 4H). These findings indicated that ACK1 played a pro cancer role in colon cancer cells, and its expression was regulated by FSCN1, suggesting that FSCN1 may affect the progression of colon cancer by regulating ACK1 expression.

Figure 4 Fascin actin-bundling protein-1 positively regulates activated Cdc42-associated kinase 1 expression and activated Cdc42-associated kinase 1 promotes malignant phenotype of colon cancer cells. A and B: The mRNA and protein expression levels of activated Cdc42-associated kinase 1 (ACK1) after fascin actin-bundling protein-1 knockdown; C and D: Detection of ACK1 overexpression and knockdown (si-ACK1) efficiency; E: Cell proliferation; F and G: Transwell cell migration and invasion. Scale bar = 200 μm and 100 μm; H: Clone formation experiment. ^cP < 0.001. ACK1: Activated Cdc42-associated kinase 1; FSCN1: Fascin actin-bundling protein-1; ACK1-OE: Activated Cdc42-associated kinase 1 overexpression; NC: Non-targeting control.

Effect of ACK1 knockdown on the M2 polarization of macrophages

we collected SW620 cells and their stable culture supernatants transfected with si-NC or si-ACK1 as CM and treated THP-1 cells pretreated with PMA. Flow cytometry analysis revealed that compared with macrophages treated with CM from the SW620 and si-NC groups, those exposed to CM from the si-ACK1 group exhibited a significantly increased proportion of CD86⁺ (M1-type) macrophages and a reduced proportion of CD206⁺ (M2-type) macrophages (Figure 5A), suggesting that ACK1 knockdown promoted polarization toward the M1 phenotype while suppressing M2 differentiation. Furthermore, the macrophages treated with si-ACK1 CM secreted increased the levels of M1-associated cytokines TNF-α (Figure 5B) and IL-1β (Figure 5C), whereas the production of M2-related anti-inflammatory factors IL-10 (Figure 5D) and TGF-β (Figure 5E) was markedly decreased. Moreover, iNOS expression was upregulated (Figure 5F and G) and Arg1 expression was downregulated (Figure 5F and H) in the macrophages of the si-ACK1 group, thus validating their phenotypic transition at the protein level. In summary, ACK1 silencing in colon cancer cells shifted macrophage polarization toward the antitumor M1 phenotype by modulating secreted factors and suppressing immunosuppressive M2 polarization, thereby highlighting a critical role for ACK1 in shaping the tumor-immune microenvironment.

Figure 5 Activated Cdc42-associated kinase 1 silencing inhibits M2 phenotype of macrophages. A: Flow cytometry analysis of the ratio of CD86⁺ (M1) and CD206⁺ (M2) in macrophages; B-E: Enzyme-linked immunosorbent assay was used to detect the secretion levels of M1 and M2 macrophage cytokines; F-H: Protein expression of inducible nitric oxide synthase and arginase 1. ^cP < 0.001. PMA induced differentiation of THP-1 macrophages was treated with conditioned medium containing SW620, SW620-si-NC, and SW620-si-CK1 cells, respectively. TNF-α: Tumor necrosis factor-α; TGF-β1: Transforming growth factor-β1; iNOS: Inducible nitric oxide synthase; Arg1: Arginase 1; ACK1: Activated Cdc42-associated kinase 1; NC: Non-targeting control; CM: Conditioned medium; IL: Interleukin.

FSCN1 mediating M2 polarization of macrophages via ACK1 signaling pathway

A rescue experiment was performed to investigate whether FSCN1 regulates macrophage polarization through the ACK1 signaling pathway. The ACK1 protein expression was markedly decreased in the si-FSCN1 group compared with that in the control group. However, co-transfection with an ACK1-OE plasmid effectively restored the ACK1 protein levels (Figure 6A). The RT-qPCR results revealed that the CM from the si-FSCN1 group significantly upregulated the mRNA expression levels of M1-associated cytokines IL-1β (Figure 6B) and TNF-α (Figure 6C) while downregulating the mRNA levels of M2-associated cytokines IL-10 (Figure 6D) and TGF-β (Figure 6E). Notably, ACK1-OE reversed these effects. The upregulation of IL-1β and TNF-α was attenuated, whereas the downregulation of IL-10 and TGF-β was partially rescued, indicating that restoring ACK1 expression counteracted the modulatory effect of FSCN1 knockdown on macrophage polarization. Furthermore, the Western blotting analysis demonstrated that in macrophages treated with CM from the si-FSCN1 group, the protein levels of iNOS (an M1 marker) increased (Figure 6F and G) and Arg1 (an M2 marker) expression decreased (Figure 6F and H). Bu contrast, in the si-FSCN1 + ACK1-OE group, the upregulation of iNOS was attenuated, and Arg1 expression was partially recovered. Collectively, these findings indicated that the pro-M1 polarization effect induced by FSCN1 silencing was reversed upon ACK1 re-expression, demonstrating that FSCN1 mediated macrophage polarization by regulating ACK1 expression. This functional dependency between FSCN1 and ACK1 suggests that the FSCN1/ACK1 axis plays a critical role in modulating the immune microenvironment in colon cancer. As shown in Supplementary Figure 2, ACK1 knockdown promoted the secretion of the M1 macrophage marker TNF-α and suppressed the secretion of the M2 marker IL-10. Furthermore, ACK1-OE reversed the inhibitory effect of FSCN1 silencing on M2 macrophage polarization, consistent with the observations in SW620 cells.

Figure 6 Fascin actin-bundling protein-1 regulates macrophage polarization through activated Cdc42-associated kinase 1. A: Activated Cdc42-associated kinase 1 protein expression; B-E: The mRNA expression levels of M1 and M2 macrophage cytokines; F-H: Protein expression of Inducible nitric oxide synthase and arginase 1. ^bP < 0.01. ^cP < 0.001. FSCN1: Fascin actin-bundling protein-1; TNF-α: Tumor necrosis factor-α; TGF-β1: Transforming growth factor-β1; iNOS: Inducible nitric oxide synthase; Arg1: Arginase 1; ACK1: Activated Cdc42-associated kinase 1; NC: Non-targeting control; ACK1-OE: Activated Cdc42-associated kinase 1 overexpression; CM: Conditioned medium; IL: Interleukin.

In vivo antitumor effects of FSCN1 knockdown

A subcutaneous xenograft tumor model was established using nude mice with SW620 cells to examine the role of FSCN1 in promoting colon tumor proliferation and altering the composition and function of TME in vivo. The tumors in the si-FSCN1 group were much smaller than those of the control group (Figure 7A). Tumor growth was longitudinally observed, and the results showed that the tumor grew more slowly in the si-FSCN1 group than in the control groups (Figure 7B). Additionally, the tumor weight at death in the si-FSCN1 group showed a significant reduction compared with that in the control group (Figure 7C). Overall, silencing of FSCN1 inhibited CRC growth in vivo. The FSCN1 and ACK1 in the tumor tissue of the si-FSCN1 group showed lower expression levels than those in the control group. The positive rate of the cell proliferation marker Ki-67 was more pronouncedly suppressed (Figure 7D). Therefore, FSCN1 is involved in facilitating tumor progression. The flow cytometry analysis of tumor-infiltrating immune cells indicated a considerable increase in the percentage of CD86⁺ M1-type macrophages within the TME of the si-FSCN1 group and a noticeable decrease in CD206⁺ M2-type macrophages (Figures 7E). These results indicated that FSCN1 knockdown can return the immune environment in TME to promote macrophage polarization toward an antitumor direction. The immunofluorescence double-staining results showed that the iNOS-positive signal increased in the si-FSCN1 group of tumor tissues (Figure 7F), whereas the Arg-1-positive signal was reduced (Figure 7G), suggesting an increase in M1 polarization and a decrease in M2 polarization. In addition, the mRNA expression of IL-1β in the si-FSCN1 group increased, whereas the expression of IL-10 showed a decrease (Figure 7H and I). Therefore, FSCN1 siRNA-mediated knockdown not only inhibited tumor growth but also modified the antitumor immune landscape through the regulation of ACK1 expression and macrophage FSCN1 states, indicating anticancer property.

Figure 7 Fascin actin-bundling protein-1 silencing inhibits colon cancer growth and reshapes the tumor immune microenvironment in vivo. A: Representative tumor photos; B: Tumor growth curve; C: End point tumor weight; D: Immunohistochemistry staining of fascin actin-bundling protein-1, activated Cdc42-associated kinase 1, and Ki67. Scale bar = 200 μm; E: Flow cytometry analysis of polarization status of macrophages in tumors; F and G: Immunofluorescence staining images of Inducible nitric oxide synthase and arginase 1, with DAPI counterstaining of the cell nucleus. Scale bar = 50 μm; H and I: The mRNA levels of interleukin-1β and interleukin-10. ^bP < 0.01. ^cP < 0.001. FSCN1: Fascin actin-bundling protein-1; iNOS: Inducible nitric oxide synthase; Arg1: Arginase 1; ACK1: Activated Cdc42-associated kinase 1; NC: Non-targeting control; IL: Interleukin.

DISCUSSION

CRC is a highly prevalent and lethal malignancy, ranking among the leading causes of cancer-related incidence and mortality worldwide. Metastasis remains the primary contributor to therapeutic failure and poor clinical outcomes. Remodeling of TME plays a pivotal role in this metastatic process, with TAMs, being the most abundant immune cell population within TME, exerting a profound influence on tumor behavior through their polarization state. M2 macrophages, as “tumor-promoting” macrophages, can create favorable conditions for tumor cell invasion and metastasis by secreting various cytokines, promoting angiogenesis, and extracellular matrix remodeling[18]. Therefore, finding key molecules that regulate M2 polarization of macrophages has become an important breakthrough in blocking colon cancer metastasis.

Research has confirmed that the invasiveness and metastatic potential of various malignant tumor cells are associated with the upregulation of FSCN1 expression[19,20]. The present study focuses on the relationship between FSCN1 and macrophage polarization in TME, revealing that FSCN1 was abnormally upregulated in colon cancer tumor tissues, accompanied with a considerable infiltration of tumor-associated M2 macrophages. The in vitro experiments and in vivo xenograft tumor models in nude mice demonstrated that silencing FSCN1 suppressed the malignant invasiveness of tumor cells, reduced tumorigenicity in vivo, and inhibited M2 polarization of macrophages. These results suggest that FSCN1 may participate in colon cancer metastasis by regulating macrophage M2 polarization, providing a new perspective for understanding the complex mechanisms of colon cancer metastasis.

Protein immunoblotting experiments were conducted to gain in-depth insights into how FSCN1 modulates M2 macrophage polarization at the molecular level. The results showed that FSCN1 regulated the expression of ACK1, a non-receptor tyrosine kinase that plays a pivotal role in regulating multiple essential biological processes, including, but not limited to, cell proliferation, migration, survival, and cytoskeletal remodeling[21]. In recent years, the pro-tumorigenic role of ACK1 in the progression of various cancers has been widely recognized and reported. In CRC tissues, increased ACK1 expression is commonly observed in clinical specimens and strongly correlated with higher tumor stage, lymph node involvement, and unfavorable patient outcomes[22]. Mechanism studies have shown that ACK1 can activate various key signaling pathway node molecules, such as AKT, androgen receptor, and β-catenin, through phosphorylation, thereby promoting tumor cell proliferation, anti-apoptosis, and invasion ability[23,24]. ACK1, as an upstream regulator of epithelial-mesenchymal transition, enhances the migratory and invasive potential of CRC cells. Small-molecule inhibitors targeting ACK1 have shown good antitumor activity in preclinical models, not only inhibiting the growth of CRC cells but also enhancing the sensitivity of traditional chemotherapy drugs. This study showed that in SW620 cells, FSCN1 silencing can reduce ACK1 expression and M2 polarization-related cytokine secretion in macrophages, leading to a weakened invasion ability of colon cancer cells. In previous studies, ACK1 was mainly regarded as a key molecule for autonomous regulation of tumor cells. However, the present study revealed that ACK1 can participate in macrophage polarization by regulating FSCN1, thereby expanding the understanding of its biological functions.

FSCN1 has also been reported to be aberrantly overexpressed in various malignancies, including breast cancer and gastric cancer, where its expression positively correlates with lymph node metastasis, distant metastasis, and poor prognosis. This is consistent with the positive correlation between FSCN1 overexpression and metastatic potential observed in CRC tissues in the present study, further strengthening the evidence that FSCN1 functions as a tumor-promoting factor. To date, most studies have primarily focused on the role of FSCN1 in regulating tumor cell biological behavior, such as promoting cell migration and invasion through cytoskeletal remodeling. Previous studies revealed that ACK1 drives M2 polarization of macrophages and modulates inflammatory cytokine production by activating downstream signaling cascades such as STAT3, AKT, and nuclear factor kappa B[25,26]. While earlier studies have predominantly focused on ACK1’s functions within tumor cells, the present findings revealed a novel mechanism whereby FSCN1 interacts with ACK1 to modulate its activity, thereby influencing macrophage polarization toward the M2 phenotype. This finding links FSCN1 with the ACK1 signaling axis.

Several limitations of this study warrant consideration. First, the clinical cohort comprised 20 paired tissue samples only, thus limiting the statistical power for subgroup analyses and possibly affecting the generalizability of the clinical findings. Complementary in vivo xenograft experiments that independently validated the functional role of the ACK1/FSCN1 axis in promoting tumor growth and M2 macrophage polarization were conducted to address this limitation. The consistency between the clinical observations and the animal model results strengthened the reliability of the conclusions despite the modest sample size. However, future studies with larger, multicenter cohorts are needed to further confirm the clinical relevance of the findings and evaluate the potential of ACK1 and FSCN1 as prognostic biomarkers or therapeutic targets in CRC.

Beyond the FSCN1/ACK1 axis identified in this study, it is also embedded within a broader network of immune regulatory pathways controlling CRC. Several previous studies have shown that M2 macrophage polarity formation involves the participation of the STAT3/nuclear factor kappa B/PI3K/AKT pathway complex. In the present study, ACK1-mediated activation of AKT indicated that the FSCN1/ACK1 axis acts together with canonical pathways in promoting the immunosuppressive environment. Additionally, cytoskeletal transformation exhibits cross-talk with changes in immune checkpoint genes. Actin reorganization protein can affect the movement and survival stability of programmed death ligand-1 attached to the outside of tumor cells. Given that ACK1 has been shown to be a strong kinase that regulates cell morphology, the FSCN1/ACK1 pathway may affect programmed death ligand-1 levels or distribution and participate in T-cell exhaustion together with macrophage-rewired phenotype. A recent report showed that ACK1 activity promotes metabolic reprogramming through enhanced glycolysis. This process also reshapes the metabolic micro-environment in favor of M2 polarization. The cross-talk between the FSCN1/ACK1 pathway and other concurrent immune-metabolic networks needs to be explored to establish integrated therapy strategies combining ACK1 inhibitors with programmed cell death-1 blockade or metabolic drugs.

CONCLUSION

Silencing of FSCN1 expression can effectively inhibit colon cancer tumor growth, reorganize TME, and inhibit ACK1-mediated M2 polarization. The in-vitro experiments revealed decreased invasiveness. Intervening in the FSCN1/ACK1 axis may inhibit colon cancer metastasis through disorganized cell-automotive movement and promote the formation of pro-metastatic micro-environments. Although the in vivo data primarily support the role of FSCN1 in tumor growth and regulation of the microenvironment, future studies using specific metastasis models, such as tail-vein injection or orthotropic implantation, are needed to verify whether FSCN1 has anti-metastatic effects systemically. This paper puts forward some new ideas on the molecular mechanisms underlying colon cancer and ACK1/FSCN1 as an alternative therapy.