Targeting of FSP1 regulates iron homeostasis in drug-tolerant persister head and neck cancer cells via lipid-metabolism-driven ferroptosis

Patient selection and collection of clinical specimens

Surgical-residual tissue samples of 50 patients with cisplatin-resistant HNSCC were collected from Tri-Service General Hospital (TSGH) to research the expression of FSP1’s upstream and downstream targets. This study was approved by the Institutional Review Board of TSGH and was conducted in accordance with the recommendations of the Declaration of Helsinki for biomedical research (IRB:2-108-05-124). After informed consent was obtained, tissue samples were obtained from the TSGH tissue archive and retrospectively studied. We assessed the expression of FSP1 in a cohort of 50 individuals with head and neck squamous cell carcinoma (HNSCC), comprising 45 males and 5 females, aged between 29 to 75 years, with a median age of 52 years. The tissue specimens for this study were gathered from January 2015 to July 2020. Before treatment commenced, patients underwent comprehensive evaluations that included a thorough clinical history, a physical examination, a barium swallow X-ray, an endoscopy of the upper gastrointestinal tract, and CT scans of both the chest and abdomen. Treatment for all individuals was administered in accordance with the established protocols of TSGH and the NCCN guidelines. Tissue arrays were constructed using tissue samples from 50 participants: 25 normal and 25 recurrent HNSCC tissue samples. FSP1 expression in recurrent HNSCC was determined through immunohistochemical (IHC) staining of the tissue arrays. Antibodies against FSP1 (1:200; 20886-1-AP; Abcam, Waltham, MA USA) were used by following the standard IHC staining protocol and with a similar dilution of mouse immunoglobulin G as that of the negative control. FSP1 expression was analyzed by two independent pathologists. FSP1 immunoreactivity was calculated using the quick score (Q score) method (Q = P × I); the percentage distribution (P) of FSP1-stained tumor cells was scored from 0% to 100%, the intensity (I) of FSP1 expression was scored using a 4-point scale (3, strong staining; 2, moderate staining; 1, weak staining; and 0, no staining), and total scores ranged from 0 to 300. The feed-forward loop of oncogenic activities involved in the regulation of FSP1 and its clinical implications was investigated. This study also analyzed the expression of FSP1 and related genes in head and neck cancer by using datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) databases. Expression Project for Oncology (expO) integrates gene expression data with longitudinal clinical annotations to elucidate human malignancies and provide critical insight into diagnostic markers, prognostic indicators, and therapeutic targets.

Human tongue squamous carcinoma cell lines, drug and persister cell derivation

Cisplatin (99.7% purity, 15663-27-1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). iFSP1 (99.84% purity, HY-136057, MCE, USA) is a potent, selective, and glutathione-independent inhibitor of FSP1 (apoptosis-inducing factor mitochondria-related 2, AIFM2) with an EC50 of 103 nM (drug information from the manufacturer, https://www.medchemexpress.com/ifsp1.html). Two kinds of human tongue squamous carcinoma cell lines were purchased from Merck (HSC-3, SCC 193, USA) and AcceGen Biotechnology (HSC-4, ABC-TC0420, USA). HSC3 and HSC4 cells were seeded in duplicate 24-well culture plates and allowed to reach 70% confluency. Persister cells were derived from treatment of the HNSCC cancer HSC3 and HSC4 cells with 5-μM cisplatin for a minimum of 9 days, with fresh drug administered every 3 days. For cells regrown from persister cells, cisplatin was subsequently removed from the persister cells and fresh cisplatin-free media was replaced every 2 days for 28 days; subsequently, this was performed for both the experiments involving persister and regrown cells.

Transfection of HNSCC cells with shRNA targeting FSP1 and overexpression

Transient transfection cells were seeded into 24 wells at 2 × 10⁴ cells/well. TurboFect Transfection Reagents (Thermo Fisher Scientific, Waltham, MA, USA) were selected for transfection experiments. Lentivirus containing FSP1 short hairpin (sh) RNA was purchased from Thermo Fisher Scientific and prepared strictly in accordance with the manufacturer’s instructions. Two clones of shRNA were used to effectively silence FSP1 expression: A6 (shRNA1, clone ID: V2LHS-89195) and B10 (shRNA2, V3LHS-639151). shRNA lentivirus infection and construction were conducted in accordance with the standardized practice guidelines of our certified BSL-2 laboratory in the Integrated Laboratories for Translational Medicine, TSGH. Data of the pcDNA3.1 mammalian expression vector (Invitrogen, V79020) were used to design polymerase chain reaction (PCR) primers. The vector map and primer sequences are presented in Supplementary Figure 1. Ten micrograms of empty plasmid (pcDNA3.1 vector control plasmid DNA) or FSP1 expression plasmid (pcDNA3.1-CMV-FSP1) were used. The DNA-lipofectamine reagent complexes were maintained at room temperature for 30 min. The mixture was added to the well, and gentle mixing was achieved by rocking the plate back and forth. Reagent complexes did not need to be removed following transfection. The cells were incubated at 37°C in a CO2 incubator for 48 h. Successfully knocked-down cells were verified either through quantitative reverse transcription–polymerase chain reaction (qRT-PCR) or Western blotting.

RNA preparations

Total RNA was isolated from samples (tumor tissues and cell lines) by using TriZol reagent (Life Technologies, Carlsbad, CA, USA) and quantified using NanoDrop (Thermo Fisher Scientific). RNA from exosomes was extracted using a miRNeasy Micro Kit (QIAGEN, Hilden, Germany). In brief, exosome suspension (20 μL) was mixed with QIAzol lysis buffer (700 μL) and processed in accordance with the vendor’s protocol. RNA samples were subsequently eluted with 25 μL of RNase-free water (Invitrogen™ 10977015, repeated twice with 25 μL of RNase-free water to concentrate the samples). The RNA concentration in the samples was again determined using NanoDrop.

Quantitative real-time PCR (RT-qPCR)

Total RNA was extracted using a TriZol reagent (Invitrogen) in accordance with the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from 1.0 μg of total RNA by using oligo (dT) primers and the PrimeScript II First Strand cDNA Synthesis Kit (Takara, Shiga, Japan). Subsequently, a qRT-PCR kit was used to assess the expression levels of CDK4 and related targets on an MxPro real-time PCR system (Agilent Technologies, Stratagene Mx3005P, USA). GAPDH was used as the internal reference gene. The qRT-PCR reaction was performed as follows: 95°C, 10 min; 95°C, 10 s; 60°C, 20 s; 75°C, 15 s; 40 cycles. A ΔΔCt method was used to determine relative gene expression from qPCR data with GAPDH as an endogenous REF gene. Supplementary Table 1 shows the Q-PCR primer list.

Western blotting

Western blotting was used to determine the quantities of protein and related message transfer proteins. First, 10%, 12.5%, and 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis films were prepared. These were placed in an electrophoresis tank, and electrophoresis buffer (Tris-Glycine-SDS Buffer, Sigma, T7777-1L) was added. Next, 4 μL of loading buffer was added to a 16-μL sample (total protein 20 μg) of the solution. The sample was cooled and denatured (100°C, 10 min) and then placed on the electrophoresis sheet. Electrophoresis separation was performed at 100 V. After approximately 3 h, the gel was removed, and protein transfer was performed. The gel was then placed in ice-cold transfer buffer. The gel was covered with presoaked polyvinylidene difluoride (PVDF) paper and placed in a transfer holder. After the 1-h transfer, the PVDF paper was added to the blocking buffer (CD-110500, CANDOR Bioscience, Wangen im Allgäu, Germany) and shaken for 1 h at room temperature. Primary antibody was added to the TBST buffer (BR510, Biomate, Taipei, Taiwan). The solution was allowed to react overnight at 4°C or at 37°C for 2 h, and it was washed three times with washing buffer (TBS + 0.05% Tween 20) for 10 min each time. Subsequently, the secondary antibody was added to the TBST buffer, and they were allowed to react at room temperature for 1 h and then washed with washing buffer three times for 10 min each time. Finally, the color was developed using an ECL luminescence system, and the result was quantified using a densitometer (Alphalmage 2000, Alpha Innotech, San Leandro, CA, USA). Supplementary Table 2 shows the antibody list.

Tumor spheroid formation assay

HNSCC persister cells were transferred to serum-free low-adhesion culture plates containing Dulbecco’s modified Eagle medium/F-12 containing N2 supplement (Invitrogen), 20 ng/mL EGF, and 20 ng/mL basic fibroblast growth factor (stem-cell medium; PeproTech, Rocky Hill, NJ, USA) for 2 weeks to allow tumor sphere formation. The spheres were counted under a microscope. The tumor ball formation efficiency was calculated as the ratio of the number of balls to the number of implanted cells.

Cell migration assays

HNSCC persister cells were seeded and cultured in six-well plates for 24 h. The cells were incubated with mitomycin (10 μg/mL) for 1 h. A linear scratch was created by moving the tip of a 200-μL pipette through the cell monolayer. Cellular debris was removed, and the cells were allowed to migrate for 24–48 h. Gap healing was determined using a microscope (Nikon, Japan) from micrographs taken before and after the wound was created. Migration distance was measured from images (three random fields) obtained at indicated time points. The gap size was subsequently analyzed using ImageJ software (Wayne Rasband National Institutes of Health, Bethesda, MD, USA).

Cell invasion assays

An invasion assay was performed in accordance with a previously established protocol. In brief, 3 × 10⁵ HNSCC persister cells were seeded onto matrigel (BD Biosciences, San Jose, CA, USA) in culture plate inserts (pore size of 8 μm, Corning Inc., Corning, NY, USA) in serum-free medium. Three independent and random fields per well were photographed and the number of cells per field was counted. An average of the three determinations was obtained for each chamber. Each invasion assay was performed a minimum of three times.

Fatty acid metabolism assay

The experiment described involves using the Lipid Extraction Kit (Chloroform Free, ab211044, Abcam) for analyzing cell lysates to study the interaction of FSP1 with lipid metabolism. Pellet 5 × 10⁵ cells by centrifugation at 1000 × g for 5 minutes. The cell pellet was washed once with PBS (Phosphate-Buffered Saline). The washed pellet was then resuspended in 25–50 μL of PBS. For the lipid extraction, 25 μL of the cell suspension was transferred to a clean 1.5 mL microcentrifuge tube 500 μL of Lipid Extraction Buffer which was added to the cells. This mixture was vortexed immediately for 1–2 minutes. The homogenate was then centrifuged at 10,000 × g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was agitated on an orbital shaker at room temperature for 15 minutes. A subsequent centrifugation step at 10,000 × g for 5 minutes was performed, and the lipid-containing supernatant was carefully transferred to a new tube. The volume of the supernatant was recorded, and then it was dried in a vacuum concentrator or a 37°C incubator overnight until a thin film was visible, indicating complete drying. The dried lipid sample was then ready for further processing. These steps are conducted as per the instructions provided in the kit’s manual, and they are crucial for the successful extraction and analysis of lipids from cell samples. Subsequent quantitative determination was performed using Human Fatty Acid Oxidation In-Cell ELISA Kit (ab118182, Abcam) and Cholesterol/Cholesteryl Ester Assay Kit (ab65359, Abcam).

Patient-derived xenograft mouse models

Xenografting was performed with mice homozygous for the severe combined immune deficient (SCID) mutation. Twenty-five 8-week-old female nonobese diabetic (NOD) and SCID mice obtained from BioLASCO Taiwan (Taipei, Taiwan) were bred under standard experimental pathogen-free conditions in accordance with the protocols of the Animal Care and Use Committee of Taipei Medical University (Approval number: LAC-2021-0657 and LAC-2021-0377). Briefly, primary HNSCC tumors were placed in RPMI 1640 in an ice bath in the surgical site. Thin slices of tumor were cut into 2–3 mm3 pieces and washed three times with RPMI 1640. The samples were minced into fine fragments that would pass through an 18-gauge needle. They were then mixed 1:1 (v/v) with Matrigel (Collaborative Research, Bedford, MA, USA), achieving a total volume of 0.2 mL per injection. The tissue mixture was injected subcutaneously in both flanks of the 8-week-old male SCID mice. Twenty 8-week-old female NOD and SCID mice obtained from BioLASCO Taiwan were bred under standard experimental pathogen-free conditions. The mice were divided into four groups (untreated, cisplatin, dapagliflozin, or combination therapy; five per group). The mice received different treatments: vehicle (PBS orally five times per week) or dapagliflozin (10 mg/kg, orally three times per week). Tumor volume was measured using a standard caliper every other week with the following formula:

$$\text{V}\text{=}\text{(L}\text{×}\text{W2)/2}$$

where L is the long axis and W is the width of the tumor. Animals were humanely sacrificed following the experiments, and tumor and tissue samples were collected for further analyses.

Immunohistochemical staining

Tissues collected from the experimental animal sacrifice were fixed in 10% (vol/vol) formalin for 24 h and embedded in paraffin. Bones were decalcified prior to paraffin embedding. Paraffin-embedded 4-μm tissue sections were dewaxed through incubation with xylene for 2 min twice and were rehydrated with 100% ethanol twice for 2 min, 95% ethanol for 2 min, 75% ethanol for 2 min, and ddH₂O for 2 min. The tissue sections were stained with hematoxylin for 2 min, washed with tap water for 10 min, counterstained with eosin for 30 s, and then dehydrated with 75% ethanol for 30 s twice, 95% ethanol for 30 s, and 100% ethanol and xylene for 30 s. After being mounted using mounting medium, the stained tissue sections were examined under a light microscope, and the tumor areas were digitally photographed. Tumor areas in the images were calculated using ImageJ software, version 1.5.

Statistical analysis

SPSS (IBM, Armonk, NY, USA) was used to perform all statistical analyses. Each experiment was performed three times. All data in the figures are expressed as mean ± standard deviation. Comparisons between groups were performed using the t-test. All statistical tests were two-sided, and p < 0.05 was considered significant. Continuous data were analyzed using the paired t-test or Wilcoxon rank test. Categorical data were analyzed using χ2 or the Fisher exact test. Survival analysis was estimated using the Kaplan–Meier method with the log-rank test to calculate differences between curves.

Availability of data and materials

The datasets that are used and analyzed by the current investigation will be provided by the corresponding author in reply to the reasonable demands. Experimental procedures, characterization of new compounds, and all other data supporting the findings are available in the supplementary materials.

FSP1 is highly expressed in tissues procured from cisplatin-resistant patients and is correlated with FSP1 expression

Our study focused on examining the genes that become active following cisplatin treatment in HNSCC cells that are resistant to chemotherapy. We used data from the Gene Expression Omnibus (GEO) database (GSE72384) to conduct our investigation. Through a Volcano Plot analysis, we identified two key indicators where red dots signify genes with increased expression levels in the TS samples compared to the control samples, and blue dots indicate genes with decreased expression levels. Our results showed that the gene ALDH3A1, associated with Ferroptosis biomarkers, and the gene SOX2, involved in cancer stemness, were both upregulated (Figure 1A). Related clinical tables of FSP1 expression and correlation analysis are presented in Table 1. Subsequently, we examined the gene expression profiles associated with HNSC tissue samples listed in Table 1. A gene expression heat map is displayed in Figure 1B. Gene sequencing from the cohort (provided in Table 1) indicated that FSP1 and related regulatory genes were highly expressed in patients’ tissues. In our preliminary clinical observations, the following results were obtained from the patient-derived primary cells that were subjected to tissue staining and separation. As presented in Figure 1C, FSP1 was more highly expressed in tissues procured from cisplatin-resistant patients compared with those from non-cisplatin-resistant patients, and cisplatin resistance was correlated with FSP1 expression. The IHC results showed that the expression level of FSP1 in HNSCC tumor tissues was significantly increased (p < 0.05). The analysis of FSP1 expression in patient tissues, both at the mRNA and protein level, revealed that samples subjected to chemotherapy displayed elevated levels of FSP1 compared to untreated samples. This suggests that the deviant FSP1 expression subsequent to chemotherapy may have a potential regulatory role in cancer cells. Consistent results were obtained for the tissues isolated from the specimens of patients with relapsed HNSCC. Regardless of whether mRNA or protein was considered, the cisplatin-treated recurrence samples exhibited higher FSP1 expression than the nonrecurrence samples (Figure 1D). To further clarify the effect of abnormal FSP1 expression in head and neck cancer on patient survival, we used online open databases to analyze and support our hypothesis. The TCGA database revealed that poorer survival of patients with higher expression of FSP1 (AIFM2) (Figure 1E) and that FSP1 is highly abnormally expressed in head and neck cancer tissues and expression increases with tumor progression (Figure 1F). Finally, Figure 1G illustrates the high expression of FSP1 in head and neck cancer. Significant positive correlations with GPX4 (ferroptosis), ACSL4 (fatty acid metabolism), SOD2 (antioxidant), HIF1A (tumor growth and metastasis), FAT1 (fatty acid metabolism) and IREB2 (Iron-responsive element). The outcomes of our investigation were in line with those of the wider TCGA HNSC cohort. These preliminary findings indicate that there may exist a regulatory association between FSP1 and cancer cell metabolism with regards to various aspects such as fatty acids, antioxidation, and iron ions. It suggests that FSP1 expression could potentially have a role in regulating these biological processes in cancer cells. Further research is necessary to establish the precise nature of this relationship and to explore the underlying molecular mechanisms involved.

Figure 1. Ferroptotic biomarker FSP1 is aberrantly expressed in human HNSCC tissues. (A) Volcano Plot analysis from the Gene Expression Omnibus (GEO) database (GSE72384). (B) Heat map of expression of FSP1 and related genes in TSGH HNSCC cohort (n = 50). (C) IHC staining of FSP1 expression in cisplatin-treatment HNSCC recurrence tissues (left) and nonrecurrence tissues (right) and Q score of FSP1 expression in patients with HNSCC. (D) Graphical representation of FSP1 expression in patients with HNSCC (top image: FSP1 expression in patient tissue; bottom image: FSP1 mRNA expression in patient tissue). (E) Kaplan–Meier curves indicating the effects of low and high BTK expression on the overall survival of patients with HNSCC (n = 259). (F) Ferroptotic biomarkers of FSP1 were aberrantly expressed in box and stage plot of the TCGA HNSCC cohort. (G) Analysis of correlations of FSP1 with iron metabolism, fatty acid metabolism, Iron-responsive element and antioxidant related genes. ^*p < 0.05, ^**p < 0.01.

Cisplatin DTP HNSCC cancer cell line generation and validation

We first established human tongue squamous cell carcinoma cell lines HSC3 and HSC4 DTP cancer cells that could tolerate cisplatin. Two kinds of human tongue squamous carcinoma cell lines were purchased from Merck (HSC-3, SCC193, USA) and AcceGen Biotechnology (HSC-4, ABC-TC0420, USA). Figure 2A presents the analysis of the expression of FSP1 and ACSL4 in the HNSCC cell line. We used the depmap online tool to investigate the vulnerabilities of cancer and identify targets for therapeutic development (https://depmap.org/). The findings indicate that HSC3 and HSC4 cell lines are relevant to the expression of AIFM2 and ACSL4 and can be utilized in this study. Out of the two cell lines, HSC3 exhibits a greater expression of AIFM2 compared to HSC4. It is hypothesized that following Cisplatin treatment, more pronounced differences could be observed, and HSC3 will serve as a research sample for subsequent ferroptosis studies. HSC3 was used as the experimental cell line in this study, and HSC4 was used as the control cell line. Subsequently, we generated drug-tolerant persister cells from these two cell lines. HNSCC cancer cell lines were treated for approximately 9 days with a cytotoxic concentration of cisplatin (5 μM; Figure 2B). The results revealed that a small fraction of HNSCC cancer cells (3%–5%) entered a quiescent (persister) state to evade the strong selective pressure of high-concentration cisplatin (Figure 2B, upper right panel). We assessed whether these persister cells were consistent with the reported observation of a reversible state of drug resistance. Removal of cisplatin (the cells were cultured in cisplatin-free media for >28 days) allowed the persister cells to regrow (Figure 2B, lower right panel). Subsequent cisplatin treatment led to persister cells being derived again and indicated that these cells had reacquired sensitivity to cisplatin (Figure 2B, lower left panel). The reversibility of drug resistance in HNSCC cancer persister cells, which has also been reported in HSC3 persister cell models, is indicative of a resistance mechanism. Relative gene expression assay findings indicated higher expression of FSP1, GPX4, and ACSL4 in cisplatin-DTP cell lines (presented in Figure 2C). The rederived persister cells were discovered to be sensitive to cisplatin. These cells are named cisplatin-DTP cells and further annotated as HSC3-P and HSC4-P. The results indicated that the persister cells were considerably more resistant to cisplatin than were their parental cells (Figure 2D). We compared DTP and non-DTP cell line functional phenotypes. In cell immunostaining, we found abnormal expression of FSP1 and ACSL4 in both DTP cell types (Figure 2E). The DTP cell phenotypes were characterized by poor migration and high drug resistance. As previously reported, DTP HNSCC cell lines have strong stemness properties, indicated by greater diameters of formed spheres (Figure 2F). The relative gene expression survey of FSP1, GPX4, and ACSL4 with and without shFSP1 transfection is presented in Figure 2G. In an effort to provide more concrete evidence of the crucial role played by FSP1 in the regulation of lipid metabolism in head and neck squamous cell carcinoma (HNSCC) drug-tolerant persisters (DTP) cells, we conducted a detailed analysis focused on the effects of FSP1 knockdown on the metabolic pathways within these cells. Specifically, we targeted the FSP1 gene for silencing and subsequently monitored the resultant changes in lipid metabolism. The data obtained from this investigation was quite telling; it demonstrated a marked reduction in the cellular concentrations of key lipid constituents, namely fatty acids and cholesterol. These findings are critical as they suggest that FSP1 is a significant regulator of lipid metabolism in these cancer cells. The observed downregulation of lipid components following FSP1 suppression could have profound implications for the metabolic state and viability of the HNSCC DTP cells. This pivotal data is comprehensively illustrated in Figure 2H, where a clear visual representation of the reduced lipid levels post-FSP1 silencing is presented.

Figure 2. Cisplatin DTP HNSCC cancer cell line generation and validation. (A) Cell line expression analysis to optimize the experimental parameters used HSC3; HSC4 was the control. (B) HSC3 parental cells were treated with 5 μg/mL cisplatin for 9 days, removed from cisplatin for 28 days, and then reincubated with cisplatin for 9 days to generate a stable DTP cell line. (C) Relative gene expression assay indicated higher expression of FSP1, GPX4, and ACSL4 in cisplatin-DTP cell lines. (D) HSC3-P was compared with parental counterpart in terms of sensitivity to cisplatin treatment for validation. (E) Immunofluorescence staining analysis of the expression of FSP1 and ACSL4 in the DTP HNSCC cancer cell line. (F) Diameter of tumor spheres and (G) relative gene expression of FSP1, GPX4, and ACSL4 with and without shFSP1 transfection. (H) Downregulation of lipid components following FSP1 suppression. (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Scale bar: 5 μm.

Overexpression of FSP1 enhances cellular function in HSC cell lines and is reversed by FSP1 inhibitors

To elucidate the role of FSP1 in conferring drug resistance, we performed a thorough investigation on two HSC cell lines. Using pCDNA 3.1 as our expression vector, we successfully overexpressed FSP1 in these cells, with confirmation of this overexpression demonstrated in Figure 3A. A subsequent cell migration assay provided evidence that FSP1 overexpression enhances the motility of HSC cells, an effect that was not mitigated by treatment with cisplatin. Interestingly, the introduction of an FSP1 inhibitor not only reduced cell migration but also allowed cisplatin to exhibit a more potent inhibitory effect on the migration rate, as shown in Figure 3B. Furthermore, RNA analysis of the HSC DTP cell lines, which show persistent cisplatin resistance, revealed atypical expression patterns of genes implicated in ferroptosis (FSP1), fatty acid metabolism (ACSL4), and cellular antioxidation (SOD2). This dysregulated gene expression profile was normalized upon the application of an FSP1 inhibitor, suggesting a reversal of the resistant phenotype, as depicted in Figure 3C. The alteration in gene expression correlating with cellular responses was further supported by cell viability assays, confirming these observations, as seen in Figure 3D. Delving deeper into the effects of FSP1 inhibition on the biological characteristics of HNSCC-DTP cells, including their stemness, invasive capacity, and metastatic potential, we employed immunoblotting techniques. This allowed us to dissect the downstream protein expression related to ferroptosis regulation. The findings presented in Figure 3E shed light on the potential molecular mechanisms by which FSP1 modulates these characteristics in DTP cells. To affirm FSP1’s role in lipid metabolism in HNSCC DTP cells, we scrutinized the consequences of FSP1 suppression on lipid-related cellular activities. The results unambiguously indicated that silencing FSP1 leads to a significant reduction in the intracellular concentration of fatty acids and cholesterol, as illustrated in Figure 3F. This supports the hypothesis that FSP1 is integral to the proper regulation of lipid metabolism. Lastly, Figure 3G presents a comprehensive overview of the putative regulatory network of ferroptosis within the cell, highlighting three intertwined pathways: ferroptosis itself, fatty acid metabolism, and cellular antioxidation. This diagram serves to visualize the complex interactions and regulatory mechanisms that may be affected by the perturbation of FSP1 activity within these cells, further emphasizing its pivotal role in cellular metabolism and drug resistance phenomena.

Figure 3. Overexpression of FSP1 enhanced cellular function in HSC cells and was reversed by FSP1 inhibitor. (A) Overexpression of FSP1 in the transfected HSC cell line. (B) Diameter of tumor spheres. (C) RNA analysis of related genes of HSC DTP cells through FSP1 inhibitor treatment. (D) HSC DTP cells were compared with parental counterparts in terms of their sensitivity to cisplatin, FSP1 inhibitor, and combination treatment. (E) Western blotting indicated the expression levels of iron-metabolism-related markers FSP1, IREB2, FBXL5, and GPX4; fatty acid metabolism marker ACSL4; and antioxidant marker SOD2 in HSC DTP cells. Actin was used as a loading control. (F) Downregulation of lipid components following FSP1 inhibition. (G) Potential regulatory network of ferroptosis in cells, involving three pathways: ferroptosis, fatty acid metabolism, and cellular antioxidation. (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Scale bar: 5 μm.

Inhibition of FSP1 significantly suppresses metastasis in patient-derived xenograft mouse models in vivo

We evaluated the therapeutic effects of iFSP1 by creating a patient-derived xenograft mouse model through orthotopic inoculation with patient-derived tumor cells. Patient-derived tumor cells were injected into the right flank of NOD-SCID female mice for in vivo validation of the findings in the in vitro study. The mice were divided into four groups: vehicle control, cisplatin alone (orally five times per week), iFSP1 alone (orally five times per week), and the combination of both drugs (combining both regimens), respectively. A flowchart showing the in vivo experimental design and treatment schedule is presented in Figure 4A. The tumors that developed in the mice receiving the combination treatment were markedly smaller at the indicated time points than those that developed in the control mice, with a 1.6-fold difference in tumor size by week 8 (p < 0.01). However, no significant effect on mouse bodyweight at week 6 was observed (Figure 4B). Furthermore, the mice in the combination treatment group had a considerably higher survival rate than those in the other groups (Figure 4C). Using tumor samples derived from the tumor xenograft mouse model, we demonstrated that the expression of FSP1, ki67, SOD2, and ACSL4 proteins was significantly suppressed in the FSP1 inhibition and combined treatment groups compared with that in the control group. The Q score of tissue staining was also calculated. The findings indicated that FSP1 plays a crucial role in the malignant progression of HNSCC and in the modulation of markers (Figure 4D, 4E).

Figure 4. Inhibition FSP1 significantly suppressed metastasis in the patient-derived xenograft mouse model. (A) Flowchart showing the in vivo experimental design and treatment schedule. (B) Tumor size and body weight curve over time indicated that the combination of cisplatin and FSP1 inhibitor suppressed tumor growth and caused no apparent systematic toxicity. (C) The Kaplan–Meier survival curve indicated that the group of mice receiving combined treatment had a higher survival ratio than did the other groups. (D) The Q-score of tissue staining. (E) Immunostaining analysis of tumor sections indicated that the combined treatment prominently suppressed FSP1, ACSL4, SOD2, and ki67 expression compared with other sections. (^**p < 0.01; ^***p < 0.001).