Sep 5, 2024

Abstract The study of cooperating genes in cancer can lead to mechanistic understanding and identifying potential therapeutic targets. To facilitate these types of studies, we developed a new dual-inducible system utilizing the tetracycline- and cumate-inducible systems driving HES3 and the PAX3::FOXO1 fusion-oncogene, respectively, as cooperating genes from fusion-positive rhabdomyosarcoma. With this model, we can independently induce expression of either HES3 or PAX3::FOXO1, as well as simultaneously induce expression of both genes. This new model will allow us to further investigate the cooperation between HES3 and PAX3::FOXO1 including the temporal requirements for genetic cooperation. Functionally, we show that dual-induction of PAX3::FOXO1 and HES3 modifies sphere formation in a HEK293T-based system. More broadly, this lentiviral dual-inducible system can be adapted for any cooperating genes (overexpression or knockdown), allowing for independent, simultaneous, or temporally controlled gene expression. Introduction Functional genomics is an important strategy for understanding oncogenesis and therapeutic vulnerabilities. Loss and gain-of-function approaches in a cell culture context can be leveraged to define genetic cooperation and the resulting phenotypic outcomes. To this end, overexpression or knockdown of putative oncogenes and cooperating genes has led to important discoveries regarding how they may be functioning in cancer cells 1 , 2 , 3 , 4 , 5 . However, typically, this type of modeling does not account for critical factors in the tumorigenic process, including timing of expression, the level and duration of expression, or cessation of expression. For these types of questions, an inducible system such as the tetracycline-inducible system or the cumate-inducible system is much better suited. Additionally, combining these systems to have temporal control over multiple genetic events is challenging because of the required number of vectors to introduce into cells, as shown in a previous study in which the cumate and tetracycline expression systems were used in mixed cell populations rather than the same cell line 6 . The tetracycline-inducible system utilizes either tetracycline or a derivative, such as doxycycline, to reversibly induce transcriptional activation or repression 7 . The Tet-On system uses a combination of a tetracycline-controlled transcriptional silencer (tTS), which silences TRE promoters in the absence of tetracycline, and a reverse tetracycline transactivator (rtTA), which is only capable of binding the TRE in the presence of tetracycline 7 . The cumate repressor system utilizes the bacterial cumate repressor (CymR), which binds to the cumate operon (CuO) in the absence of cumate 8 . The tetracycline-inducible and cumate-inducible systems are each meant to be used with a single gene at a time given the packaging limitations for vectors backbones. Thus, these systems are challenging to use for complex studies of gene cooperation. In this study, we combined these two systems in a single cell line via a series of vectors introduced by lentiviral transduction. We engineered the regulatory tTS and rtTA for the Tet-On system and the cumate repressor CymR, all separated by viral T2A sequences, to be in one vector. In a second vector, we designed a tetracycline-inducible HES3 with EGFP separated by a viral T2A sequence. In the third vector, we designed a cumate-inducible PAX3::FOXO1 with mCherry separated by a viral T2A sequence (Fig. 1 A). The rationale for generating this system is motivated by our previous work in PAX3::FOXO1 rhabdomyosarcoma, an aggressive solid tumor that presents in children. Previously, we used a cross-species comparative oncology approach to identify HES3 as a cooperating gene in the disease. We found that HES3 locks cells in an immature cell state, and that its overexpression is predictive of reduced overall survival in patients 3 . Now, we have developed a complementary cell-based system to functionally assess the requirements for HES3 in the disease. By utilizing both the Tet-On and cumate repressor systems in a single cell line, we generated a new system to investigate potential cooperation of PAX3::FOXO1 and HES3, with the capacity to independently control the level and timing of expression of each gene. Fig. 1 Anhydrotetracycline induction of HES3 and cumate induction of PAX3::FOXO1 is titratable. Schematic of tetracycline and cumate induced expression of HES3 and PAX3::FOXO1 (A). Dual inducible HEK293T cells (referred to as 293Tii) were plated on 12-well plates at a density of 250,000 cells per well. After overnight adherence, cells were treated with titrating amounts of either: anhydrotetracycline at 0, 1, 5, 10, 25, or 50 ng/ml; or cumate at 0, 1, 5, 10, 25, or 50 µg/ml. Shown are representative images of cells imaged at 24 h post-induction (hpi) on a Leica DMI microscope with a 10 × objective for GFP fluorescence (HES3) or mCherry fluorescence (PAX3::FOXO1) and then harvested for western blotting (B,C). Brightfield images were exposed for 5 ms. GFP and mCherry images were exposed for 2 s. Scale bar is 500 µm. (D,E) Cells were lysed, and then 20 µg of protein was loaded into each well of a 4–15% gradient gel. After transferring to a PVDF membrane, the membrane pieces were blotted with either a HES3 primary antibody (D), FOXO1 primary antibody that recognizes the PAX3::FOXO1 fusion and endogenous FOXO1 (E), or TUBULIN primary antibody (D,E). (F,G) Quantification of western blot data for HES3 (F) or PAX3::FOXO1 (G) protein expression. Shown is the ratio of HES3 or PAX3::FOXO1 expression normalized to TUBULIN, and presented as the fold-change to no drug treatment (far left on both graphs). Each point represents a biological replicate (n = 3 for each condition). The error bars represent the mean ± standard deviation. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated three times. Anhydrotetracycline p-values: 0 ng vs 25 ng, p = 0.00081; 0 ng vs 50 ng, p = 0.00008; 1 ng vs 25 ng, p = 0.00112; 1 ng vs 50 ng, p = 0.0001; 5 ng vs 25 ng, p = 0.0108; 5 ng vs 50 ng, p = 0.00075; 10 ng vs 50 ng, 0.00348. Cumate p-values: 0 µg vs 5 µg, p = 0.0005; 0 µg vs 10 µg, p = 0.00004; 0 µg vs 25 µg, p = 0.00001; 0 µg vs 50 µg, p = 0.000002; 1 µg vs 5 µg, p = 0.0055; 1 µg vs 10 µg, p = 0.0003; 1 µg vs 25 µg, p = 0.00004; 1 µg vs 50 µg, p = 0.00001; 5 µg vs 50 µg, p = 0.008. HES3 and associated TUBULIN blot were cut from the same membrane prior to primary antibody hybridization. PAX3::FOXO1 and associated TUBULIN blot were cut from the same membrane prior to primary antibody hybridization. Western blots have been cropped from original and the uncropped blots are presented in Supplemental Fig. 7. Acronyms: tTS, tetracycline-controlled transcriptional silencer; rtTA, reverse tetracycline transactivator; TRE, tetracycline response element promoter; cymR, cumate repressor; cuO, cumate operator site; P3F, PAX3::FOXO1; mCh, mCherry. Cell culture HEK293T cells (CRL-3216, ATCC, RRID: CVCL_0063) were grown in Dulbecco’s modified Eagle’s media with GlutaMAX (DMEM, 10569044, Gibco) supplemented with 10% FBS, 1 × Penicillin/Streptomycin, and 10 mM glutamine. Cells were passaged every 3–4 days with TrypLE (12604013, Gibco). Cells were authenticated by STR and tested for mycoplasma annually through Genetica Inc a subdivision of LabCorp. Lentiviral plasmids To design the plasmid containing the Tet-On regulatory proteins and the cumate repressor, we used VectorBuilder’s Tet Regulatory Protein Expression Lentiviral Vector as a base, which contains both the tTS and rtTA proteins linked via a T2A sequence. We then added the CymR sequence directly downstream of this linked by a second T2A sequence. Additionally, this plasmid contains a blasticidin resistance gene for antibiotic selection. The plasmid map is available in Supplemental Figure S1 . To design the plasmid containing the tetracycline-inducible gene, we used VectorBuilder’s Mammalian Tet Inducible Gene Expression Lentiviral Vector as a base, adding the coding sequence for either a multicloning site (MCS) or human HES3 with no stop codon directly downstream of the TRE sequence, linked to EGFP with a T2A sequence. Additionally, this plasmid contains a puromycin resistance gene for antibiotic selection. The plasmid maps are available in Supplemental Figure S2 for MCS version and Supplemental Figure S3 for HES3 version. To design the plasmid containing the cumate-inducible gene, we used VectorBuilder’s Mammalian Tet Inducible Gene Expression Lentiviral Vector as a base. Then we replaced the TRE sequence with a CMV promoter and CuO sequence, adding the coding sequence for either a multicloning site (MCS) or human PAX3::FOXO1 with no stop codon directly downstream of the TRE sequence, linked to mCherry with a T2A sequence. Additionally, this plasmid contains a hygromycin resistance gene for antibiotic selection. The plasmid maps are available in Supplemental Figure S4 for MCS version and Supplemental Figure S5 for PAX3::FOXO1 version. These plasmids were generated and packaged into lentivirus by VectorBuilder with a minimum virus titer of 1 × 108 TU/mL. Lentiviral transduction HEK293T cells were transduced with each vector sequentially to prevent potential cytotoxicity from PAX3::FOXO1 expression. The multiplicity of infection (MOI) used for each vector was 5. For transduction, 10,000 cells were plated on a 6-well plate with 2 mL of culturing media and allowed to adhere for 16 h overnight. Then, transduction media was made with 80% culturing media, 20% TransDux MAX Lentivirus Transduction Enhancer (LV860A-1, System Biosciences), and 0.5% TransDux (LV860A-1, System Biosciences). 72 h after transduction, transduction media was removed and fresh culturing media was added with selection antibiotic. For the tTS/rtTA/CymR vector (developed in this study, Supplemental Figure S1 ), 3 µg/mL of blasticidin (A1113903, Fisher Scientific) was used for selection. For the TRE:HES3-T2A-EGFP vector (developed in this study, Supplemental Figure S3 ), 1 µg/mL of puromycin (A1113803, Fisher Scientific) was used for selection. For the CMV-CuO:PAX3::FOXO1-T2A-mCherry vector (developed in this study, Supplemental Figure S5 ), 300 µg/mL of hygromycin (10-687-010, Fisher Scientific) was used for selection. Each antibiotic was used for selection of its respective construct for two weeks post transduction. Then the next construct and drug selection were sequentially added. The only time a combination of drugs was included (all three) was in the two weeks after FAC sorting. After the two week selection periods for vector constructs, the drugs (puromycin, hygromycin, blasticidin) were not included in the media during experiments. These concentrations were determined by performing an antibiotic selection kill-curve. Briefly, 10,000 cells were plated in each well of a 96-well plate in growth media. In triplicate, titrated concentrations of blasticidin, puromycin, or hygromycin were added to each well every two days for six days total. After six days of exposure to antibiotic, crystal violet staining was done. After aspirating out all growth media, the cells were fixed with 4% paraformaldehyde (50-276-31, Fisher Scientific) for 15 min at room temperature and then washed with 1 × PBS. After aspirating out the PBS, crystal violet stain was added for 5 min (250 mg crystal violet (C0775, Sigma-Aldrich) added to 100 mL of 20% methanol). The crystal violet stain was then removed, and the plate was gently washed by submerging in a container of still tap water, and repeated once with fresh tap water. The plate was then allowed to air dry. To extract and quantify, 10% glacial acetic acid in water was added to each well and vigorously agitated for 30 s. The plate was then read on a plate reader at 590 nm. Tetracycline and cumate induction Dual-inducible 293 T cells that were successfully transduced with all three vectors were plated on either 6-well or 12-well plates and allowed to adhere for 16 h overnight. Anhydrotetracycline (C4291, ApexBio Technology) was used as an effector for the Tet-On system and binds the rtTA protein at a much higher affinity than tetracycline 9 . Anhydrotetracycline was dissolved in dimethyl sulfoxide (DMSO, D2650, Sigma-Aldrich) and added in concentrations ranging from 1 ng/mL to 50 ng/mL. The resulting DMSO concentrations were kept to a maximum of 0.1% total volume. A water-soluble cumate solution (QM150A-1, System Biosciences) was used to induce PAX3::FOXO1-T2A-mCherry, and was added in concentrations ranging from 1 µg/mL to 50 µg/mL. Western blotting Cells used for western blotting were collected with TrypLE, spun down at 8000xg for 5 min, aspirated of supernatant media, and snap frozen to be stored at − 80 °C. Cells were then lysed with RIPA buffer and 1 × protease inhibitor (PI78442, Fisher Scientific) for 2 h on ice. Protein concentration was determined by BCA assay (PI23227, Fisher Scientific). For each sample, 20 µg of protein was loaded into a 4–15% gradient mini-PROTEAN TGX precast protein gel (4561086, Bio-Rad). Gels were run at 150 V in 1 × Tris/Glycine/SDS buffer (1610772, Bio-Rad) until the dye front has just run off the gel, approximately 45 min. The samples were then transferred to a PVDF membrane at 400 mA for 2 h at 4 °C in 1 × Tris/Glycine buffer (1610771, Bio-Rad). Membranes were then cut at approximately 70 kDA and 37 kDa markers prior to antibody hybridization to prevent potential cross-reaction between HES3, TUBULIN, and FOXO1 antibodies. Membranes were then blocked in casein blocking buffer (PI37528, Fisher Scientific) with 0.05% Tween-20 for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies in fresh casein blocking buffer with 0.05% Tween-20. Primary antibodies used were mouse anti-alpha Tubulin (3873S, Cell Signaling Technologies, RRID: AB_1904178) at 1:1000, mouse anti-HES3 (PCRP-HES3-1A10, Developmental Studies Hybridoma Bank, RRID: AB_2618684) at 0.5 µg/mL, and rabbit anti-FOXO1 (2880S, Cell Signaling Technologies, RRID: AB_2106495) at 1:1000. Then membranes were washed three times for 10 min each in 1 × phosphate buffered saline (PBS) with 0.05% Tween-20. Membranes were then incubated at room temperature for 2 h with secondary antibodies in fresh casein blocking buffer with 0.05% Tween-20. Secondary antibodies used were goat anti-mouse horse radish peroxidase (HRP)-conjugated (1706516, Bio-Rad, RRID: AB_11125547) at 1:10,000 and goat anti-rabbit HRP-conjugated (1721019, Bio-Rad, RRID: AB_11125143) at 1:10,000. Membranes were then washed three more times for 10 min each in 1 × PBS with 0.05% Tween-20. Membranes were then imaged on a C-DiGit Chemiluminescent Western Blot Scanner (103375-240, VWR) using SuperSignal West Pico PLUS Chemiluminescent Substrate (PI34577, Fisher Scientific) to image TUBULIN and PAX3::FOXO1/FOXO1, and SuperSignal West Atto Ultimate Sensitivity Substrate (38554, Fisher Scientific) to image HES3. While Pico PLUS Chemiluminescent Substrate is not sensitive enough to show membrane edges on a C-DiGit Chemiluminescent Western Blot Scanner, Atto Ultimate Sensitivity Substrate is sensitive enough to show membrane edges on this imager. Sphere formation Sphere formation assay was adapted from a previous study 10 . Double inducible cells were seeded in 6-well ultralow attachment plates (07-200-601, Fisher Scientific) at 10,000 cells per well in serum free DMEM with GlutaMAX (10569044, Gibco) supplemented with 20 ng/ml bFGF (3718-FB-100, R&D Systems), 20 ng/ml EGF (236-EG-200, R&D Systems), and 1X B27 (17-504-044, Fisher Scientific). Cells were continuously induced with either diluent control, anhydrotetracycline, cumate, or both at time of seeding and for every 4 days for 12 days total. Spheres were imaged and counted manually using a Leica DMIL LED inverted microscope at 12 days after seeding. Imaging Images were taken with a Leica DMIL LED microscope with a 10 × objective for 2D adherent growth or a 5 × objective for 3D sphere growth. Brightfield images had a 5 ms exposure time, GFP a 2 s exposure time, and RFP/mCherry a 2 s exposure time. For Fig. 1 , the displayed pixel value range is 300–5000 for GFP and 200–5000 for mCherry. For Fig. 2 , the displayed pixel value range is 300–20,000 for GFP and mCherry. For Fig. 3 , the displayed pixel value range is 2000–16,000 for GFP and 2000–7000 for mCherry. For Fig. 4 , the displayed pixel value range is 1300–65,000 for GFP and 3500–10,000 for mCherry. Fig. 2 HES3 and PAX3::FOXO1 induction are both reversible. 293Tii cells were plated on 6-well plates at a density of 250,000 cells per well. After overnight adherence, cells were treated with either 50 ng/ml anhydrotetracycline (HES3 induction) or 50 µg/ml cumate (PAX3::FOXO1 induction). Shown are representative images of uninduced cells (A), anhydrotetracycline-induced cells at 24 h post-induction (hpi) and 120hpi (B), and cumate-induced cells at 24hpi and 192hpi (C). Cells were imaged on a Leica DMI microscope with a 10 × objective. Brightfield images were exposed for 5 ms. GFP and mCherry images were exposed for 2 s. Scale bar is 500 µm. Cells were then harvested for western blotting. Cells were lysed, and 20 µg of protein was loaded into each well of a 4–15% gradient gel. After transferring to a PVDF membrane, the membrane pieces were blotted with either a HES3 primary antibody (D), FOXO1 primary antibody that recognizes the PAX3::FOXO1 fusion and endogenous FOXO1 (E), or TUBULIN primary antibody (D,E). Relative expression of either HES3 (D) or PAX3::FOXO1 (E) was then quantified using ImageJ (relative to TUBULIN, and presented as the ratio compared to uninduced cells). Each point represents a biological replicate (n = 3 for each condition). The error bars represent the mean ± standard deviation. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated three times. HES3 reversibility p-values: uninduced vs 24hpi, p = 0.00006; 24hpi vs 120hpi, p = 0.00006; uninduced vs 120hpi, p = 0.99. PAX3::FOXO1 reversibility p-values: uninduced vs 24hpi, p = 0.00001; uninduced vs 120hpi, p = 0.0003; uninduced vs 192hpi, p = 0.11; 24hpi vs 120hpi, p = 0.019; 24hpi s 192hpi, p = 0.00009; 120hpi vs 192hpi, p = 0.005. Blots have been cropped from original (D,E). HES3 and associated TUBULIN blot were cut from the same membrane prior to primary antibody hybridization. PAX3::FOXO1 and associated TUBULIN blot were cut from the same membrane prior to primary antibody hybridization. Western blots have been cropped from original and the uncropped blots are presented in Supplemental Fig. 8.