T-cell transduction

The extracellular domain of human FGFR4 (NCBI Gene ID: 2264) fused with mouse Fc was synthesized (Synbio Technologies, Suzhou, China) and the FGFR4-Fc fusion protein was purified from the culture supernatant of the FGFR4-Fc-transduced CHO cells. The anti-FGFR4 antibody was generated from C57BL/6 immunized with the FGFR4-Fc fusion protein. The FGFR4 scFv DNA sequence derived from the anti-FGFR4 antibody was synthesized (Synbio Technologies, Suzhou, China) and cloned into the lentiviral vector pELNS. The 4-1BB and CD3ζ cytoplasmic signaling domains were cloned into the vector to produce the FGFR4 CAR. The Thy1.1 gene was inserted downstream into the FGFR4 CAR as a reporter, together with an SV40 sequence. The lentivirus was multiplied by transfection into 293 T cells and concentrated using ultracentrifugation. Ultraconcentrated FGFR4 CAR lentiviral supernatants were stored at -80 °C instantly for further use.

Peripheral blood mononuclear cells derived from healthy donors with informed consent were activated with anti-CD3 (OKT3) and anti-CD28 (CD28.2) monoclonal antibodies (MoAbs) (5 µg/mL) (eBioscience, Thermo Fisher Scientific; Waltham, MA). For mouse T cell expansion, the anti-CD3 and anti-CD28 antibodies used are 17A2 and 37.51, respectively. Lentiviral supernatants were collected at 48 and 72 h post-transfection and concentrated by centrifugation at 20,000g for 90 min to enhance the infection efficiency of target cells. Activated PBMCs or mouse T cells were subjected to spinoculation with lentivirus in 24-well plates coated with RetroNectin (5 µg/mL) (Takara Bio, Kusatsu, Japan), using a multiplicity of infection (MOI) of 15. Following infection, the cells were cultured in complete RPMI 1640 medium, which consists of RPMI 1640, 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 2 mM l-glutamine, 100 µg/mL streptomycin, and 100 µg/mL penicillin, supplemented with IL-2 (200 IU/mL) (PeproTech, Cranbury, NJ) for human T cells, and IL-2 (200 IU/mL), IL-7 (5 ng/mL), and IL-15 (50 ng/mL) for mouse T cells. The transduced T cells were isolated 72 h later by immunomagnetic selection using biotinylated-Thy1.1 antibody and anti-biotin magnetic beads (BioLegend, San Diego, CA).

Generation of murine CAR-T cells

Murine CD4+ and CD8+ T cells were selected by CD4 and CD8 magnetic Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) from splenocytes obtained from C57BL/6 J mice and stimulated on plates coated with mCD3 and mCD28 MoAb (eBioscience) for 24 h. Activated murine T lymphocytes were transduced with lentivirus supernatants using the same protocol as that used to transduce human T cells. After removal from the RetroNectin plates (Takara Bio), T cells were expanded in the presence of IL-2, IL-7, and IL-15 while changing the medium every 2 days. On day 7, the T cells were collected and used for functional assays in vitro and in vivo.

Cell lines and primary RMS samples

The 293 T and human RMS cell lines RD, RH4, RH18, RH30, and A-204 were purchased from the American Type Culture Collection and Deutsche Sammlung von Mikroorganismen und Zellkulturen. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific) supplemented with penicillin, streptomycin, and 10% fetal calf serum. The cells we used are routinely authenticated and tested for mycoplasma contamination. For in vivo imaging of xenograft models, a lentiviral firefly luciferase construct was transduced into the RH4 and RD cells. Primary human RMS specimens were acquired from the Sun Yat-sen University Cancer Center. The study design was approved by the Sun Yat-sen University Cancer Center Research Ethics Board. Written informed consent for the publication of clinical details was obtained from the patients or their relatives.

Flow cytometry

To detect surface markers, approximately 1 × 106 cells were washed once in 100 µL phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA) and then labeled with 5 µL of antibodies in accordance with the manufacturer’s instructions for 30 min at 4 °C in the dark. FGFR4 expression in the RMS cell lines was detected using an FGFR4-PE antibody (BioLegend, 4FR6D3). A mouse Thy1.1 MoAb (BioLegend, OX-7) was used to evaluate the FGFR4 CAR transduction rate in the T cells. We performed flow cytometry using MoAbs specific to human CD3 (OKT3), CD4 (GK1.5), CD8 (SK1), CD19 (HIB19), CD27 (M-T271), CD28 (CD28.2), CD45 (HI30), CD45RA (HI100), CD62L (DREG-56), CCR7 (G043H7), PD-1 (29 F.1A12), PD-L1 (29E.2A3) and TIM3 (A18087E), and murine CD3 (17A2), CD4 (GK1.5), CD8 (53-5.8), CD11b (M1/70), CD11c (N418), CD19 (6D5), and PD-1 (RMP1-14) (All from BioLegend) conjugated with BV421, BV510, BV605, BV711, FITC, AF488, PerCP-cy5.5, PE, PE-cy7, APC, and APC-cy7 fluorochromes. A titration was performed to determine the optimal concentration of the FGFR4 scFv-Fc for FACS. We tested a range of concentrations from 1 µg/mL to 20 µg/mL and found that 10 µg/mL provided the best signal-to-noise ratio based on the preliminary experiments. A BD Fortessa flow cytometer (BD Biosciences) was used for flow cytometric analysis, and data were analyzed using FlowJo version 7.6.5 (BD Biosciences).

Immunohistochemistry and tissue histopathology

Frozen RMS specimens were sectioned at the Sun Yat-sen University Cancer Center with consent from the Research Ethics Board. Inclusion/exclusion criteria is pre-established before the experiments. The slides were dried for 30 min at 18–26 °C, fixed in 4% paraformaldehyde in PBS for 15 min, blocked with 3% H2O2 (Sigma-Aldrich, St. Louis, MO) in distilled water for 20 min, then blocked with 1% BSA and 10% normal goat serum (Sigma-Aldrich) in PBS for 1 h at room temperature. Slides were stained with the primary antibody at 4 °C overnight. Normal human tissue microarray and tumor tissues were stained with FGFR4 MoAb (abcam, ab151444, clone 19H3, 1:1000 dilution). Horseradish peroxidase polymer-conjugated goat anti-mouse secondary antibody (Dako, Glostrup, Denmark) was used. Slides were developed using DAB chromogen (Cell Signaling Technology, Danvers, MA), counterstained with CAT hematoxylin (Biocare Medical, Pacheco, CA), dehydrated in ethanol, and cleared in xylene (Fisher Chemical, Thermo Fisher Scientific). Cover slips were added using a histological mounting medium (Fisher Chemical, toluene solution). Stained tumor microarray slides were digitally imaged at ×200 magnification using an Aperio ScanScope XT (Leica Microsystems, Wetzlar, Germany). Tumor microarray slides were de-arrayed to visualize the individual cores, and each core was visually inspected. Folded tissues were excluded from analysis using a negative pen, and all other artifacts were automatically excluded using Aperio Genie software (Leica Microsystems). Stained tissues were blindly evaluated with respect to clinical patient data and intensity was scored as 0-Negative, 1-Low antibody staining, 2-High membrane staining by two pathologists.

In vitro cytotoxicity assay

RMS cells were washed and 1 × 106 cells/mL in PBS were labeled with 1 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) at 37 °C for 15 min. 20,000 CFSE-labeled RMS cells were pre-plated in the 96-well plate for 4-6 hr and then co-cultured with FGFR4 or CD19 CAR-T cells at effector-to-target (E:T) ratios of 0.5:1, 1:1, 2:1, 4:1, and 8:1 for 24 h. Subsequently, all cells were harvested and stained with CD45 and 7-AAD, along with annexin V to assess T cell apoptosis.

T-cell proliferation assay

A suspension of 1 × 107 T cells was incubated with 4 ml of pre-warmed PBS containing 5 μM of Carboxyfluorescein Succinimidyl Ester (CFSE) for 15 minutes at 37 °C. Subsequently, the cells were pelleted by centrifugation, resuspended in fresh, pre-warmed RPMI medium, and incubated for an additional 30 minutes at 37 °C to ensure complete labeling of the cells with the fluorescent probe. After a final wash, the cells were resuspended in culture medium suitable for growth. Aliquots of 500 μl of cell suspension, each containing approximately 5 × 105 labeled T cells, were co-cultured with RMS cells in the presence of exogenous IL-2 for different time points. The cells were harvested for analysis via flow cytometry, where proliferating T cells of various generations were identified based on the decreasing intensity of CFSE fluorescence as a result of successive cell divisions.

Cytokine secretion assay

To assess cytokine secretion, supernatants from co-cultures of FGFR4 or CD19 CAR-T cells with RMS cells were collected at 24 h post-incubation. These samples were then analyzed using a 30-plex Luminex assay (MilliporeSigma, Burlington, MA). For the experiment, cells were plated at a density of 1 × 105 cells per well in a 48-well plate, maintaining an effector-to-target (E:T) ratio of 1:1 for both CAR-T and RMS cells. Cultures were established in 250 μl of medium, and after 24 h of incubation, the supernatant was harvested for subsequent multiplex Luminex analysis.

Xenograft animal model

Animal studies were approved by the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animal Monitoring Institute, and the animal experiment facility was accredited by the American Association for Accreditation of Laboratory Animal Care. Six-week-old female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were maintained under pathogen-free conditions. Five mice were randomly assigned to each group, with no investigator blinding. RD or RH4 cells were inoculated subcutaneously into NOD/SCID mice. On days 10 and 15 after tumor cell inoculation, CD19 or FGFR4 CAR-T cells were injected intravenously into tumor-bearing mice. The mice were sacrificed when the tumor volume reached 1000 mm3. For bioluminescent imaging, xenografted mice were injected intraperitoneally with D-luciferin (150 mg/kg) and imaged using a Xenogen-IVIS imaging system with Living Image software (PerkinElmer, Waltham, MA, exposure time = 1 min) under isoflurane anesthesia after ten minutes post-injection.

Murine STS xenograft model in C57BL/6 J mice

The murine MCA-205 cell line was engineered to overexpress mFGFR4 (NCBI Gene ID: 14186) using retrovirus and was inoculated subcutaneously into six-week-old C57BL/6 J female mice. Fourteen days after tumor cell implantation, the mice were irradiated with 400 cGy to create a lymphodepleted environment. Two and seven days post-irradiation, mice were infused intravenously with syngeneic control non-transduced (NT) cells, mCD19 CAR-T cells, or mFGFR4 CAR-T cells. Tumor growth was monitored using ultrasound imaging. The mice were euthanized 30 days after T-cell infusion. Immune cell compositions of the blood, spleen, bone marrow, and draining lymph nodes were assessed by flow cytometry, and counting beads and tissues were analyzed by immunohistochemistry using hematoxylin and eosin staining.

Quantitative real-time PCR

The Paraffin-Embedded Tissue RNA Extraction Kit (Invitrogen, Carlsbad, CA, USA) was employed to extract RNA from FFPE samples, following the manufacturer’s instructions. Subsequently, the cDNA synthesis kit (Promega, Madison, Wisconsin, USA) was utilized to generate cDNAs through reverse transcription. Taq Pro Universal SYBR qPCR Master Mix (Invitrogen, Carlsbad, CA, USA) was utilized for quantitative PCR, with β-ACTIN serving as the internal control. The PCR cycle was initiated at 95 °C for 30 s and then continued with 40 cycles of 10 s at 95 °C and 30 s at 60 °C, with three biological replicates included. Three replicates of each sample were conducted, and the 2−∆∆Ct method was used to calculate the relative fold change in expression when compared to the control group. The PCR primers used were as follows:

4-1BB-CD3ζ Forward Primer: 5′-AAGAGAGGCAGAAAGAAGCTG-3′;

4-1BB-CD3ζ Reverse Primer: 5′-CCGTTCCCTCTACCCATGTGA-3′;

β-ACTIN Forward Primer: 5′-GATTGCGGGTTTGATCTCCAG-3′;

β-ACTIN Reverse Primer: 5′-GATTGCGGGTTTGATCTCCAG-3′.

Statistical analysis

The sample size to ensure adequate statistical power was based on prior experience in the laboratory. Data are reported as mean ± standard deviation. Student’s t-test was used to evaluate the statistical significance of the differences. Kaplan–Meier analysis was used to compare survival between groups. P values < 0.05 were considered significant. The data were analyzed using SPSS version 19 (IBM, Armonk, NY).