Experiments

Overview

The APOPTO-SENSE 2.0 system is a dual-cell biosensing platform designed for rapid, quantitative ex vivo drug sensitivity testing in cancers like leukemia, breast cancer, and non-small cell lung cancer. It leverages engineered sensor cells (HEK293T) expressing a synthetic Notch (synNotch) receptor with Annexin V for detecting phosphatidylserine (PS) exposure on apoptotic target cells (e.g., HL-60 leukemia cells). Upon detection, the system activates a Gal4-VP64 transcription factor, driving dose-dependent expression of a TagBFP reporter normalized to a constitutive mCherry internal control.

This wet lab section details the protocols for plasmid construction, cell line engineering, apoptosis induction, co-culture assays, and signal readout. Experiments were optimized for specificity, linearity, and sensitivity, drawing from synthetic biology principles and oncology biomarkers. All steps include controls to ensure rigor (e.g., negative controls for specificity).

Key Objectives:

• Construct and validate synNotch-based sensor cells.
• Induce apoptosis in target cells and detect via co-culture.
• Quantify drug sensitivity through fluorescence ratios.

Plasmid Design and Construction

Overview

Plasmids encode the synNotch receptor (Annexin V extracellular domain fused to Gal4-VP64 intracellular domain) and the reporter cassette (UAS-driven TagBFP with constitutive PGK-mCherry). Designs were synthesized via GeneScript or assembled using Gibson Assembly. After assembly, plasmids are amplified in E. coli for high-yield production, extracted, and verified before transfection into HEK293T cells. This ensures sufficient plasmid quantity (typically 1-5 µg per transfection) and purity (A260/A280 ratio 1.8-2.0) to minimize cytotoxicity and maximize efficiency.

Materials Required

• Synthetic gene fragments: Annexin V (ANXA5), Gal4-VP64, 5x UAS-minimal promoter, TagBFP, mCherry.
• pPiggyBac Transposase vector.
• pPiggyBac-CMV-synNotch-EF1-Blast (synNotch plasmid: CMV promoter, Annexin V ECD, Notch TMD, Gal4-VP64 ICD, EF1-BlastR for selection).
• pPiggyBac-Gal4UAS-TagBFP-PGK-mCherry-EF1-Puro (reporter plasmid: 5x UAS-minimal promoter-TagBFP, PGK-mCherry, EF1-PuroR).
• PCR reagents: High-fidelity polymerase, dNTPs, primers.
• Restriction enzymes (XhoI, HindIII, NEB).
• T4 DNA Ligase (NEB M0202).
• Gibson Assembly Master Mix (NEB E2611).
• TOP10 E. coli competent cells (Thermo Fisher C404010).
• LB broth and agar plates with ampicillin (100 µg/mL).
• QIAprep Spin Miniprep Kit (Qiagen 27106).
• 0.8% agarose gel, GelRed stain, 1 kb DNA ladder.
• NanoDrop spectrophotometer and agarose gel electrophoresis apparatus.

Schedule

1. Gene synthesis or PCR amplification of components (1-2 days).
2. Assembly into PiggyBac vectors (1 day).
3. Transformation into E. coli and colony screening (2 days).
4. Plasmid amplification in E. coli (overnight).
5. Plasmid extraction and verification (1 day).

Procedure

Step 1: Gene Synthesis and PCR Amplification

• Order synthetic genes.
• PCR setup (50 µL reaction): 1 ng template, 0.5 µM each primer, 200 µM dNTPs, 1X Phusion buffer, 1 U Phusion polymerase.
• Cycling: 98°C for 30 sec; 30 cycles of 98°C/10 sec, 55-65°C/30 sec (annealing based on Tm), 72°C/30 sec per kb; final 72°C/5 min.
• Verify products via 0.8% agarose gel electrophoresis: Mix 10 µL PCR product with 2 µL 6X loading dye; run at 100V for 30 min; stain with GelRed; image under UV (use long wavelength to minimize DNA damage).
• Purify bands using QIAquick Gel Extraction Kit (Qiagen 28704): Excise band, dissolve in buffer, bind to column, wash, elute in 30 µL EB.

Step 2: Plasmid Assembly

• Digest backbone vectors: 1 µg vector, 0.5 µL each XhoI/HindIII, 5 µL 10X CutSmart buffer, ddH2O to 50 µL. Incubate at 37°C for 3 hours; inactivate at 65°C for 20 min.
• Ligate inserts: For ligation, mix 50 ng digested vector, insert at 1:3 molar ratio (calculate via NEBioCalculator), 1 µL 10X buffer, 0.5 µL T4 ligase, ddH2O to 10 µL. Incubate at 25°C for 3 hours.

Step 3: Transformation into E. coli

• Thaw TOP10 cells on ice.
• Add 5-10 µL assembly mix to 50 µL cells; incubate on ice for 30 min.
• Heat shock at 42°C for 45 sec; immediately ice for 2 min.
• Add 950 µL SOC medium; incubate at 37°C shaking (225 rpm) for 1 hour.
• Plate 100 µL on LB-ampicillin; incubate overnight at 37°C.
• Include negative control (no DNA) to check for contamination.

Step 4: Colony Screening and Amplification in E. coli

• Pick 4-6 well-isolated colonies; inoculate each in 5 mL LB-ampicillin.
• Grow overnight at 37°C shaking (225 rpm).
• Verify inserts via colony PCR or restriction digest: For PCR, boil colony in 20 µL water; use 2 µL as template.
• Scale up positive clones: Inoculate 50-100 mL LB-ampicillin with 1 mL overnight culture; grow overnight.

Step 5: Plasmid Extraction and Verification

• Harvest cells by centrifugation at 4,000g for 10 min.
• Extract using QIAprep kit: Resuspend pellet in 250 µL P1 buffer (with RNase A); lyse with 250 µL P2; neutralize with 350 µL N3; centrifuge at 13,000g for 10 min.
• Bind supernatant to column; wash with 500 µL PB and 750 µL PE buffers; elute in 50 µL EB (pre-warmed to 55°C for higher yield).
• Quantify: Use NanoDrop (expect 100-500 ng/µL; A260/A280 >1.8 for purity).
• Verify: Restriction digest (500 ng plasmid, enzymes as above) and agarose gel; Sanger sequencing of inserts.
• Store at -20°C. Rationale: High-purity plasmids reduce endotoxin in mammalian transfections.

Plasmid Maps:

Figure 1. synNotch receptor (Annexin V extracellular domain fused to Gal4-VP64 intracellular domain)

Figure 2. reporter cassette (UAS-driven TagBFP with constitutive PGK-mCherry).

Sensor Cell Line Construction (HEK293T Engineering)

Overview

Stable integration of synNotch and reporter plasmids into HEK293T cells using PiggyBac transposon system for high-efficiency, selectable expression. This creates a polyclonal stable line with consistent expression, minimizing clonal variability. Post-transfection, verify via fluorescence and Western blot if needed.

Materials Required

• HEK293T cells (ATCC CRL-3216), passage <20 to maintain transfection efficiency.
• DMEM + 10% heat-inactivated FBS + 1% Pen/Strep (complete medium).
• X-tremeGENE HP DNA Transfection Reagent (Roche 06366236001).
• Opti-MEM reduced serum medium (Thermo Fisher 31985062).
• Puromycin (2 µg/mL) and Blasticidin (10 µg/mL) stocks (filter-sterilized).
• Trypsin-EDTA (0.05%, Thermo Fisher 25300054).
• 6-well plates, hemocytometer for counting.
• Fluorescence microscope for initial validation.

Schedule

1. Cell seeding (Day 0).
2. Transfection (Day 1).
3. Selection and expansion (Days 3-10).
4. Verification (Day 11+).

Procedure

Step 1: Cell Seeding

• Thaw or passage HEK293T: Resuspend in complete DMEM; count via hemocytometer (viability >90% with trypan blue).
• Seed 5x10^5 cells/well in 2 mL complete medium in 6-well plates (aim for 60-70% confluence at transfection; rationale: Optimal density prevents overcrowding and ensures high uptake).
• Incubate at 37°C, 5% CO2 for 24 hours. Check confluence microscopically.

Step 2: Transfection

• Prepare plasmids: Use 1 µg PiggyBac Transposase, 1.25 µg synNotch plasmid, 1.25 µg reporter plasmid (total 3.5 µg DNA/well; rationale: Balanced ratio for co-integration).
• In a sterile tube: Add 300 µL Opti-MEM + plasmids; mix gently.
• Add 6 µL (2:1 reagent:DNA ratio) X-tremeGENE HP; incubate at RT for 15-20 min.
• Add dropwise to cells; gently rock plate for even distribution.
• Include mock transfection (no DNA) as negative control.
• Incubate for 48-72 hours without media change (monitor for toxicity: <10% cell death).

Step 3: Selection and Expansion

• Replace media with complete DMEM + puromycin (2 µg/mL) + blasticidin (10 µg/mL).
• Change media every 2-3 days for 7-10 days (monitor cell death; expect 80-90% initial kill in non-transfected controls).
• Once resistant, expand to T-25 flask; cryopreserve aliquots (10% DMSO + 90% FBS, -80°C then LN2).

Step 4: Verification

• Image for constitutive mCherry (red fluorescence) under microscope (excitation 587 nm).
• Confirms stable, functional integration before assays.

Apoptosis Induction and Detection in Target Cells (HL-60)

Overview

Induce apoptosis in HL-60 suspension cells using raphasatin (10 µM) as a model chemotherapeutic agent, mimicking clinical drug sensitivity testing. Validate via Annexin V-FITC/PI flow cytometry for PS exposure and membrane integrity. Include vehicle (DMSO) and untreated controls for specificity.

Materials Required

• HL-60 cells (ATCC CCL-240), passage <15.
• RPMI 1640 + 10% FBS + 1% Pen/Strep (complete medium).
• Raphasatin stock (10 mM in DMSO; store at -20°C).
• Annexin V-FITC Apoptosis Detection Kit (BD Biosciences 556547: includes binding buffer, Annexin V-FITC, PI).
• Flow cytometer (e.g., BD FACSCalibur) and FlowJo software.
• PBS (sterile, pH 7.4), 6-well plates, centrifuge tubes.

Schedule

1. Cell seeding and drug treatment (Day 1).
2. Washing and staining (Day 2).
3. Flow cytometry analysis (Day 2).

Procedure

Step 1: Cell Seeding and Apoptosis Induction

• Count HL-60 cells (viability >95%); seed 1x10^6 cells/well in 2 mL complete RPMI in 6-well plates (rationale: Density prevents spontaneous apoptosis).
• Incubate at 37°C, 5% CO2 for 24 hours to acclimate.
• Add raphasatin to 10 µM (dilute from stock; final DMSO <0.1%); include vehicle control (0.1% DMSO) and untreated control.
• Incubate for 24 hours (optimize time via pilot; monitor morphology: Apoptotic cells shrink/bleb).

Step 2: Washing and Collection

• Transfer suspension to 15 mL tubes; centrifuge at 400g for 3 min at RT
• Discard supernatant; wash pellet twice with 5 mL cold PBS (centrifuge each time).

Step 3: Annexin V/PI Staining and Flow Cytometry

• Resuspend in 500 µL binding buffer (from kit).
• Add 5 µL Annexin V-FITC and 5 µL PI per 1x10^5 cells; mix gently.
• Incubate at RT in dark for 15-20 min (Annexin V binds Ca2+-dependent PS; PI stains necrotic cells).
• Analyze immediately: Acquire 10,000 events; gate on FSC/SSC for live cells; plot FITC (FL1) vs. PI (FL2).
• Process with FlowJo: Calculate % early apoptotic (Annexin V+/PI-), late (Annexin V+/PI+), necrotic (Annexin V-/PI+).
• Include unstained and single-stained controls for compensation.

Results:

Figure 3. Flow cytometry analysis of HL-60 apoptosis. Upper: negative control. Below: raphasatin induced apoptosis.

SynNotch Sensor Detection of Apoptotic Cells (Co-Culture Assay)

Overview

Co-culture washed apoptotic/control HL-60 with adherent sensor HEK293T cells to activate synNotch and induce TagBFP. Quantify via fluorescence microscopy and ratio analysis for dose-dependency. Include no-target controls for background.

Materials Required

• Engineered sensor HEK293T cells (from previous section).
• Apoptotic/control HL-60 cells (from previous protocol).
• Complete DMEM, PBS.
• Fluorescence microscope (Zeiss Axio Observer with blue/red filters).
• ImageJ software for quantification.
• 12-well plates.

Schedule

1. Sensor cell seeding (Day 1).
2. Co-culture (Day 2).
3. Imaging and analysis (Day 2-3).

Procedure

Step 1: Sensor Cell Seeding

• Trypsinize sensor HEK293T: Wash with PBS, add 0.5 mL trypsin-EDTA/well, incubate 3-5 min at 37°C, neutralize with 2 mL medium.
• Count cells; seed 5x10^5/well in 1 mL complete DMEM in 12-well plates (aim for 80% confluence; rationale: Adherent layer for co-culture stability).
• Incubate at 37°C, 5% CO2 for 24 hours.

Step 2: Co-Culture

• Wash HL-60 (1x10^6 cells/well) twice in PBS to remove residual drug (centrifuge 400g, 3 min).
• Add to sensor wells; gently mix.
• Co-culture for 15 hours (optimize via time-course; Allows synNotch activation without excessive cell death).

Step 3: Fluorescence Imaging and Quantification

• Wash wells with PBS to remove non-adherent cells.
• Image live: Blue channel (TagBFP, 390/450 nm); Red (mCherry, 587/610 nm); Brightfield.
• Acquire 3-5 fields/well at 20x magnification.
• Analyze in ImageJ: Measure mean intensity (ROI on cells); calculate BFP/mCherry ratio per field; average triplicates.
• Include no-co-culture control for baseline.

Results:

Figure 4. Fluorescence microscopy images demonstrating APOPTO-SENSE 2.0 system activation in co-culture assays. Co-culture of HL-60 target cells with engineered HEK293T sensor cells. Panel A shows the control group (untreated HL-60 cells added to the sensor system), while Panel B shows the experimental group (HL-60 cells treated with raphasatin to induce apoptosis, then added to the sensor system). In each panel: (1) Blue fluorescence channel, representing TagBFP expression; increased brightness indicates detection of apoptotic cells via synNotch activation. (2) Red fluorescence channel, representing constitutive mCherry expression; brightness confirms successful transfection and expression of the reporter plasmid in sensor cells. (3) Brightfield image, illustrating the plated HEK293T sensor cells (bottom layer) with overlaid HL-60 target cells (upper layer). Scale bar: 100 µm. Images were acquired after 15 hours of co-culture using a Zeiss Axio Observer microscope.