Project Description

Project Overview

Cancer remains one of the leading causes of mortality worldwide, with treatments like chemotherapy for malignancies such as leukemia, breast cancer, and non-small cell lung cancer often relying on "one-size-fits-all" guidelines derived from population-level statistics. This approach frequently results in suboptimal outcomes, including drug resistance, severe side effects, and missed therapeutic windows. APOPTO-SENSE 2.0 is an innovative synthetic biology platform designed to enable rapid, quantitative ex vivo drug sensitivity testing using a dual-cell biosensing system. By engineering sensor cells to detect apoptosis in patient-like tumor cells, our system provides a proof-of-concept for personalized medicine, transforming cancer treatment from empirical to precise and individualized.

Drawing from advances in synthetic Notch (synNotch) receptors and apoptosis biomarkers, APOPTO-SENSE 2.0 integrates live-cell engineering with co-culture assays to convert phosphatidylserine (PS) exposure—a hallmark of early apoptosis—into measurable fluorescent signals. This not only addresses current limitations in drug screening but also paves the way for broader applications in oncology, including multiplexed detection of diverse cell death pathways.

Problem Statement

Current chemotherapy regimens for aggressive cancers are selected based on large-scale clinical data, often ignoring individual tumor biology. This leads to significant challenges:

  • Treatment Inefficacy and Toxicity: Up to 40-60% of patients show no response to standard therapies, enduring severe side effects (e.g., myelosuppression, cardiotoxicity) while tumors develop resistance.
  • Limitations of Traditional Biopsies: Invasive tissue biopsies fail to capture tumor heterogeneity or dynamic changes during treatment, limiting their utility for real-time monitoring.
  • Gaps in Ex Vivo Testing: Emerging patient-derived cell (PDC) models show promise, with studies correlating ex vivo sensitivity to clinical outcomes (e.g., in leukemia). However, these lack standardization, require long turnaround times (weeks), high costs, and specialized equipment, hindering clinical adoption.

As a result, there is an urgent need for a fast, affordable, and scalable platform to predict drug responses at the individual level, enabling truly personalized oncology.

Project Vision and Solution

APOPTO-SENSE 2.0 aims to develop an intelligent biosensing system that rapidly quantifies chemotherapy-induced apoptosis in tumor cells, providing actionable insights for personalized treatment. By leveraging synthetic biology, we create a "oracle-like" dual-cell system where engineered sensor cells "sense" apoptotic signals from target tumor cells and report them via amplified, dose-dependent outputs.

Core Innovation: Our system detects PS externalization on apoptotic cells—a reliable, early apoptosis marker—using a modular synNotch receptor. This non-enzymatic signal transduction ensures specificity and avoids interference with endogenous pathways. As a minimum viable product (MVP), we focus on linear amplification for dose-dependent responses, with future expansions to feedback loops and multiplexed detection.

This proof-of-concept validates the feasibility of synthetic biology in oncology, potentially reducing treatment failures and improving patient outcomes. For details on experimental validation, see Experiments and Results.

System Design

APOPTO-SENSE 2.0 comprises two complementary cell types in a co-culture setup:

  • Target Cells: Represent tumor cells (e.g., HL-60 leukemia cell line) treated with chemotherapy drugs to induce apoptosis.
  • Sensor Cells: Engineered HEK293T cells (chosen for high transfectability and ease of culture) that detect and report apoptosis.

Workflow:

  1. Treat target cells with a drug (e.g., raphasatin).
  2. Wash and co-culture with sensor cells.
  3. Sensor cells bind apoptotic targets via PS, triggering intracellular signaling and reporter expression.
  4. Quantify output as a fluorescence ratio for drug sensitivity assessment.

Module 1: The Sensor – SynNotch Receptor for Apoptosis Detection

We engineered a synNotch receptor, a highly modular synthetic biology tool previously used in iGEM for antigen sensing, to recognize PS on apoptotic cells.

  • Extracellular Domain: Annexin V, a calcium-dependent protein with high affinity for PS (Kd ~ nM range). This "bait" domain enables direct binding to apoptotic cell surfaces, validated in prior studies for detecting neuronal apoptosis.
  • Transmembrane and Intracellular Domains: Derived from Notch, with the intracellular payload as a Gal4-VP64 fusion (yeast-derived transcription factor for orthogonal signaling in mammalian cells, minimizing off-target effects).
  • Mechanism: Binding induces proteolytic cleavage (e.g., by γ-secretase), releasing Gal4-VP64 to the nucleus.

Rationale: SynNotch's modularity allows customization, ensuring specificity to apoptosis without cross-reactivity to healthy cells.

Module 2: The Reporter – Dose-Dependent Signal Amplification

To convert sensor activation into a quantifiable output, we designed a linear amplification circuit as our MVP, avoiding "all-or-nothing" responses from positive feedback loops.

  • Transcriptional Driver: Released Gal4-VP64 binds upstream activating sequences (UAS; 5-9 tandem repeats) upstream of a minimal promoter.
  • Reporter Gene: TagBFP (blue fluorescent protein) for signal readout, chosen for brightness and spectral separation.
  • Internal Reference: Constitutive mCherry (red fluorescent protein) driven by PGK promoter, normalizing for cell number and transfection efficiency.
  • Output Metric: TagBFP/mCherry fluorescence ratio, providing a robust, quantitative measure of apoptosis intensity.

Rationale: This design ensures linearity (proportional to apoptotic cell input), critical for dose-response curves in drug testing. Future iterations could incorporate orthogonal systems (e.g., TetR) for multiplexing.

For engineering details and iterations, see Engineering.

Experimental Blueprint

Our wet lab validation focused on HL-60 (leukemia model) and HEK293T cells:

  • Plasmid construction, amplification in E. coli, and transfection.
  • Apoptosis induction and flow cytometry confirmation.
  • Co-culture assays with fluorescence readout.

Results demonstrated specific, dose-dependent activation (see Results for figures showing elevated TagBFP in apoptotic co-cultures).

Significance and Broad Applications

APOPTO-SENSE 2.0 represents a paradigm shift in cancer diagnostics, bridging synthetic biology with clinical oncology. Its significance lies in enabling rapid (hours to days) prediction of drug responses, potentially reducing chemotherapy failures by 20-30% based on PDC correlation studies.

Clinical Impact

  • Hematological Cancers (e.g., Leukemia): Test peripheral blood or bone marrow blasts ex vivo for sensitivity to drugs like cytarabine, guiding personalized regimens.
  • Solid Tumors (e.g., Breast, Lung): Use biopsy-derived organoids for testing targeted therapies, overcoming heterogeneity.
  • Advantages Over Traditional Methods: Faster, more sensitive, cost-effective, and scalable compared to NGS or animal models.

Future Expansions and Prospects

  • Multiplexed Detection: Integrate sensors for other death modes (necrosis, pyroptosis, ferroptosis, autophagy) using orthogonal transcription factors, enabling comprehensive "cell death profiling" in one assay.
  • Patient-Derived Models: Transition to PDCs or organoids for true personalization, with ethical frameworks discussed in Human Practices.
  • High-Throughput Screening: Adapt for 96-well formats, integrating with robotics for drug libraries.
  • Broader Oncology Applications: Extend to immunotherapy monitoring or resistance prediction, potentially impacting global cancer care (e.g., in low-resource settings via portable fluorescence readers).
  • Societal Benefits: By reducing ineffective treatments, it could lower healthcare costs and improve quality of life, aligning with UN Sustainable Development Goal 3 (Good Health and Well-Being).

This project not only contributes standardized BioBricks to the iGEM registry but also inspires future teams through our "Mammalian Synthetic Receptor Design Toolkit" (see Contributions).