Mice

hTS [10] and Ink4a/Arf −/− mice ([21]- NCI mouse repository, strain 01XB2) were previously described. To generate hTS transgenic mice in a FVB background, a 2540 bp NruI/NsiI fragment containing hTS cDNA driven by CMV promoter was excised from pcDNA3.1zeo-TS vector [8]. Ectopic hTS was proven to be catalytically active in mice tumor tissues retaining the ability to form a ternary complex with FdUMP [10]. Once hTS mice were crossed with Ink4a/Arf −/−, mice were maintained on a mixed FVB/129/sv background. NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ strain) were generated at University of Florida Animal facility. Mice were maintained within the University of Florida Cancer Genetics Research Center and Communicore barrier facilities in individual ventilated cages. All animal experiments were done in accordance with approved protocols from the Institutional Animal Care and Use Committees (IACUC), according to national and institutional guidelines. Kaplan-Meier survival curves were compared using the log-rank Mantel-Cox test calculated in GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Tumors and organs were systematically collected as frozen for molecular analysis and a piece of tumor/organ was fixed in 10% neutral-buffered formalin for pathology and immunohistochemical analyses.

Genotyping of hTS/Ink4a/Arf −/− mice

Genotype analysis in tail snips was performed by standard PCR analysis using human TS-specific primers [10] as well as a set of primer pairs designed to score for the wild-type, heterozygous, or homozygous Ink4a/Arf genotype. The sequences for TYMS (hTS) primers are: NCI2 5ʹ-ATGCCCTCTGCCAGTTCTATGTGG-3ʹ and H2I 5ʹ-TAGAAGGCACAGTCGAGG-3ʹ; and Ink4a/Arf locus primers are as follows: #I001, 5ʹ-GTGATCCCTCTACTTTTTCTTC-3ʹ, #I002, 5ʹ-CGGAACGCAAATATCGCAC-3ʹ, and #I003, 5ʹ-GAGACTAGTGAGACGTGCTAC-3ʹ. I001/I003 detects a 313 bp band for the knockout and I001/I002 a 278 bp band for the wild type.

Histopathology and immunohistochemistry

For routine histological analysis, specimens were fixed in 10% neutral-buffered formalin for 48 h and transferred to 70% ethanol, processed and embedded in paraffin (Histoserv Inc., Germantown, MD, USA or UF Molecular Pathology Core, Gainesville, FL). Tissues were serially sectioned (5 μm thick) and stained either by conventional H&E or with specific antibodies. H&E-stained slides were interpreted by a certified mouse pathologist (Dr. Victoria Hoffmann, Division of Veterinary Resources, Bethesda, MD; Dr. Mary K Reinhard, ACS Assistant Director at the University of Florida (UF); Dr. Elham Nasri (Bone and Soft Tissue Pathology and Clinical Assistant Professor), Dr. Robert P. Seifert (Hematopathology Program Director and Clinical Assistant Professor) and Michael Feely Gastrointestinal/liver and genitourinary Pathology and Clinical Assistant Professor) at Anatomic Pathology Department at UF, Gainesville, FL. For immunohistochemistry, sections were cut by the NCI-Frederick Pathology Lab (PHL, Frederick, MD, USA) and Molecular Pathology Core (UF, Gainesville, FL).

To prepare HistoGels, 5 × 106 cells were fixed in 10% neutral-buffered formalin for 1 h, then washed twice with PBS by centrifuging at 1000 rpm for 5 min. Then cells were processed using HistoGelTM (Thermofisher) according to manufacturer instructions.

Paraffin-embedded tissues or cells were then deparaffinized and prepared for staining. Antigen retrieval was performed using heated citrate buffer for 15 min, and tissues were then incubated for 30 min with primary antibodies against Mac-2 (1:1000, Cedarlane Laboratories, USA), CD45R (1:200, PharMingen), CD3 (1:100, DAKO), myeloperoxidase (1:750 DAKO). Primary antibodies were visualized with peroxidase ABC kits (Vector Labs) according to manufacturer’s instructions. Tumor cell attribution and diagnosis of tumors was performed by certified pathologist using differential immunophenotyping.

Flow cytometry

106 cells obtained from spleen or lymph nodes were blocked using rat anti–mouse CD16/CD32 Fc Block (BD Biosciences) and then stained in PBS with 2% FBS containing the BD Bioscience antibody CD11b-FITC (1.2 μg/ml). Cells were incubated on ice for 45 min. Following incubation, cell suspensions were subjected to two wash cycles involving resuspension of cell pellets in 3 mL PBS with 2% FBS, followed by centrifuging at 1000 rpm at 4 °C for 5 min, followed by supernatant removal. Finally, samples were resuspended in 300 mL of PBS with 2% FBS and analyzed by flow cytometry using a BD LSRII FACScan flow cytometer and analyzed using the BD FACSDiva software.

shRNA knockdown in hTS/Ink4a/Arf −/− mice

For shRNA treatment, 2-month-old hTS/Ink4a/Arf −/− animals were injected IP with 10 MOI TYMS shRNA#61, #64 or with NS shRNA. Animals were monitored until survival endpoint. Kaplan-Meier survival curves were compared using the log-rank Mantel-Cox test calculated in GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Tumors and organs were systematically collected and frozen for molecular analysis and a section of tumor/organ was fixed in 10% neutral-buffered formalin for pathology and immunohistochemical analyses.

Mouse spleen and lymph node single cell suspensions

Spleens were extracted and divided longitudinally in two halves. One piece was fixed in 10% buffered formalin for histological analysis and the other piece was homogenized in PBS using the plunger of an insulin syringe, disrupting the tissue against a 100-μm nylon cell strainer. Cells were then centrifuged at 1000 rpm for 5 min to discard supernatant. Cells were washed again with PBS and counted in a Nexcelom cell counter (Perking Elmer, MA, USA).

BrdU detection in splenic mouse cells

For BrdU labeling, 5.9-month-old hTS/Ink4a/Arf −/− animals were injected IP with 1 mg of BrdU (BD Pharmigen) and let them rest for 4 h before sacrifice to harvest spleens. Spleens were harvested and processed to obtain single cell suspensions as described above. Then, cells were stained with BrdU for flow cytometry detection. Briefly, 106 cells were fixed with BD Cytofix/Cytoperm buffer for 30 min on ice, washed with BD Perm/Wash buffer and incubated in Cytoperm Plus Buffer for 10 min on ice. Cells were then re-fixed with BD Cytofix/Cytoperm buffer for 5 min at room temperature and after washed with BD Perm/Wash buffer, cells were treated with 30 μg of DNase for 1 h at 37 °C to expose incorporated BrdU. After washing thoroughly with BD Perm/Wash buffer, cells were incubated with anti-BrdU for 20 min at room temperature and prepared in staining buffer to be analyzed by flow cytometry in a BD Accuri C6 Plus cytometer (BD Biosciences, NJ, USA). Analysis was performed using FlowJo v9 (BD Biosciences, NJ, USA).

Establishment of cell lines

Single-cell suspensions from a hTS/Ink4a/Arf −/− spleen (hTS5278 – number refers to mouse ID) and an Ink4a/Arf −/− lymph node (5318 – number refers to mouse ID) were made in RPMI 1640 (Sigma) supplemented with 10% FBS (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 50 μm ß–Mercaptoethanol (Sigma) and 1% Penicillin/Streptomycin (Gibco). A piece of the tissue was homogenized in PBS using the plunger of an insulin syringe, disrupting the tissue against a 100-μm nylon cell strainer. Then, 50 cells were seeded per well in a 96-well plate. After four weeks in culture, growing tumor cells were expanded to verify their genotype and negativity for Mycoplasma (Sigma). hTS5278 was further infected with lentiviral luciferase (Addgene #19785) and maintained in puromycin 8 ug/ml; the new cell line was designated Luc-hTS5278.

Reverse-transcription PCR

Total RNA was prepared using Trizol reagent (Invitrogen). cDNA was synthesized using SuperScript reverse transcriptase (Invitrogen) following the manufacturer’s instructions. Briefly, 5 μg of total RNA was converted to cDNA, and further used for PCR analysis. Primers used for PCR were TS-3F, 5ʹ-GGGGCAGATCCAACACAT-3ʹ and TS-4R, 5ʹ-CTCCCTTGGAAGACAGCTCTTTA-3ʹ. Actin primers, included with the cDNA synthesis kit (Invitrogen), were used to normalize the PCR products. All reactions were done for 35 cycles: (95 °C for 30 s, 60 °C for 30 s and 72 °C for 60 s).

qRT-PCR

0.2 μg of RNA was converted to cDNA and subjected to quantitative PCR using SYBR Green TaqMan Master Mix (Applied Biosystems) according to the manufacturer’s instructions. qRT-PCR products and their dissociation curves were detected using a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA) with Taqman Probes for TYMS (Hs00426591_m1) and R18S (Hs99999901_s1) (Applied Biosystems). TS mRNA expression was normalized relative to that of GAPDH mRNA in the same sample.

shRNA knockdown assays

Luc-hTS5278 cells were infected with lentiviral supernatants expressing shRNAs against TYMS (TS shRNA#61, TRCN0000045664; TS shRNA#64, TRCN0000045667; both from Sigma). A non-specific (NS) shRNA control vector (Sigma SHC002) was used as a negative control. Infected cells were seeded in 96 well plates at a density of 5 × 104 cells per well and proliferation was assessed using the MTS viability assay. For in vivo studies, 4 × 106 cells were infected with 10 MOI lentiviral particles for 48 h, trypsinized, and dissolved in 200 ul of PBS to be injected IP into the abdominal cavity of SCID mice. Tumor growth was measured every week using Xenogen IVIS from Perkin Elmer for 7 weeks. Animal experiments complied with University of Florida IACUC and international regulations and ethical guidelines.

Plasmid preparation

All plasmid constructs were propagated in DH5α (Invitrogen) on LB plates or in liquid media 100 μg/ml Ampicillin at 37 °C. Liquid cultures were grown in orbital shaking at >200 RPM. Plasmid extraction was performed using maxi-prep (Qiagen) following the manufacturer’s recommendations. All constructs were verified by analytical digest and/or Sanger sequencing.

Lentiviral production

Of, 12 × 106 HEK293T were seeded per T175 flask and incubated overnight at 37 °C and 5% CO2. 10–20 h after seeding, HEK293T were co-transfected with lentiviral construct TS shRNA #61, TS shRNA#64 or non-specific (NS) shRNA (32 μg), viral packaging plasmid (psPAX, 16 μg) and viral envelope plasmid (pMD2.G, 16 μg) using a mixture of 1 mg/ml polyethyleneimine (Polysciences) and 1.5 M NaCl. Viral supernatant was collected, filtered through a 0.45 μm filter 40 h post-transfection and pulled together. Pooled supernatants were concentrated using an Amicon Ultra-15 100 K cutoff filter device (EMD Millipore, Burlington, MA, USA) according to manufacturer’s instructions. Final concentrated virus was aliquoted and stored at −80 °C till used. Viral titer of the concentrated supernatant was determined by a Lenti-X qRT-PCR Titration Kit (Clontech).

Protein isolation

Protein lysates were generated using RIPA buffer (Santa Cruz) for 20 min on ice, followed by centrifugation for 15 min at 13000 rpm at 4 °C. Protein-containing supernatant was transferred to fresh microcentrifuge tubes and stored at −20 or −80 °C until further use. Protein was quantified using Bradford Assay (BioRad) following manufacturer’s recommendations, with standard curves generated with bovine serum albumin (Sigma Aldrich).

Western blotting

In total, 20 μg of total protein lysate was loaded per lane of 10% Novex Tris Glycine Gel (Invitrogen). SDS-PAGE was run at 150 V for 1.5 to 2 hr. Proteins were transferred to nitrocellulose membranes using iBlot (Invitrogen, Waltham, MA, USA). Membranes were blocked with 5% non-fat dry milk (LabScientific) in Tris-buffered saline supplemented with Tween20 (0.1%) (TBS-T) for 45–60 min at RT. Membranes were incubated on a plate shaker overnight at 4 °C with TS-106 antibody (1:300 as previously described [8]), H2AX (Ser139) (1:1000, Cell Signaling #9718) or GAPDH (1:1000, Millipore ABS16) diluted in blocking buffer. Membranes were washed extensively with TBS-T (minimum 4X for 5 min), followed by incubation with horseradish peroxidase-conjugated secondary antibody goat anti mouse IgG (BioRad) or goat anti rabbit (BioRad) in blocking buffer 30–60 min at RT on a plate shaker. Membranes were washed extensively with TBS-T (minimum 4X for 5 min). Signal was detected using Super Signal West Pico Plus Chemiluminescent substrate (ThermoScientific) following manufacturer’s recommendations. Membranes were either developed using multiple film (Genesee Scientific, San Diego, CA) processed in a Kodak X-Omat 2000A processor with exposures ranging from 2 s to 2 min or by scanning membranes in Amersham Imager 680 for different times for optimal image analysis. For quantification of protein levels relative to loading control, densitometric analysis was performed by ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland, USA).

Cell viability assays

For proliferation assays, 5 × 104 Luc-hTS5278 cells were plated in 96 well plates and 16–20 h after seeding, cells were infected with concentrated lentiviral TS shRNAs at different multiplicities of infection (MOI) (2–12 MOI). For GI50 determination, Luc-hTS5278 cells were seeded at 4000 cells/well in 96-well plates. 16–20 h after seeding, cells were treated with the indicated treatment: pemetrexed (LC Labs #P-7177; 1 nM to 20 nM) or gemcitabine (LC Labs #G-4177; 1 nM to 30 nM). Cells were grown in the presence of TYMS shRNA with or without gemcitabine or pemetrexed for 72 h. Cell viability was assessed by MTS using Cell Titer 96 R Aqueous One Solution Cell Proliferation Assay Kit (Promega), following manufacturer’s recommendations. Chemiluminescent output (integration time 1000 ms) was measured on a SpectraMax M3 (Molecular devices, San Jose, CA, USA). Data were normalized to max/min and plotted in GraphPad Prism GI50 were estimated.

Cytogenetic analysis

hTS5278 and 5318 cells were incubated in a T25 flask with 10 μg/ml Colcemid (Karyomax, Invitrogen) 1.5 h prior to harvest. Cells were then dissociated with 0.8 ml 0.05% trypsin/EDTA and treated with 0.075 M KCl hypotonic solution for 15 min at 37 °C to be then fixed with methanol: acetic acid (3:1). Metaphase slides were prepared from the cell harvest and let dry at 42 °C overnight. The metaphases were hybridized with a 20-color mouse SKY paint kit (ASI) according to the manufacturer’s protocol [43]. Spectral images of the hybridized metaphases were acquired using a SD301 SpectraCubeTM system (Applied Spectral Imaging Inc., Carlsbad, CA, USA) mounted on top of an epifluorescence microscope Axioplan 2 (Zeiss, Dublin, CA, USA). Images were analyzed using Spectral Imaging 4.0 acquisition software (Applied Spectral Imaging Inc., Carlsbad, CA, USA). G banding was simulated by an electronic inversion of DAPI-counterstaining.

Comet assay

Neutral comet assay was used to detect DNA double-strand breaks and was performed based on the manufacturer’s instructions (Trevigen, Gaithersburg, MD). Briefly, hTS5278 and 5318 cells were cultured for 48 h before being collected and resuspended in low-melting agarose (R&D Systems) at a final concentration of 0.9% before spreading on 20-well Comet Slides (R&D Systems). Slides were placed at 4 °C in the dark for 10 min and then immersed in prechilled lysis solution at 4 °C for 60 min to lyse cells before washing twice with deionized H2O and submerging Comet Slides in prechilled 1X Neutral Electrophoresis Buffer (deionized H2O containing Tris Base and Sodium Acetate, pH = 9) for 30 min, at 4 °C. Electrophoresis was run for 45 min with applied voltage at 21 V and a current of approximately 300 mA for 45 min using the Comet Assay Electrophoresis System II (R&D Systems, Minneapolis, MN, USA). The electrophoresis unit and buffer were chilled to 4 °C. After excess of Neutral Electrophoresis Buffer was drained, slides were gently immersed in DNA Precipitation Solution for 30 min at room temperature. Slides were rinsed twice in deionized H2O, fixed in 70% ethanol for 30 min at room temperature and dried at 37 °C using a standard incubator for 15 min. DNA was stained with 40 μl 1× SYBR Gold nucleic acid gel stain (S-11494, Life Technologies) per well. Comets were visualized using Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan). Double strand breaks were quantified using the OpenComet Image-J plugin [44].

Analysis of TYMS in the TCGA soft tissue sarcoma (STS) cohort

Anonymized STS patient level data (overall survival in days, live/dead status, and RSEMv2 expression) were downloaded, and plotted using R 2.15.0 statistical environment.

TYMS expression (TPM + 1) data for STS was extracted from RNA-seq data from TCGA Research Network https://www.cancer.gov/tcga and analyzed in UALCAN portal [25].

Statistics

Statistical analyses were performed using the GraphPad Prism 9 (GraphPad Software, USA). Values are represented as data ± SD. Data were analyzed by 2-tailed Student’s test or 2-way ANOVA for comparison between groups. Kaplan-Meier survival curves were analyzed using the log-rank Mantel-Cox test to determine significance. For all studies, P values <0.05 were considered statistically significant.