Abstract

The IOD band is a general diagnostic test enabling early detection and mapping of tumor mobility. Over a billion unique tests are made accessible to field experts outside of synthetic biology with a unique clone-free assembly feature. Tumor mobility is incredibly difficult to diagnose due to the rarity of circulating tumor cells (CTCs) and the complexity of surface marker combinations. The IOD band strives to make it easy. The central players are processing units called Input Output Diploids or IODs. IODs use antigen recognition and intercellular communication to create a logical network by which even single cells carrying the desired marker profile can be identified in a background of millions. Affirmative CTC localisation triggers a global response manifested by IOD initiated clumping at levels visible to the naked eye. As such, IOD bands do in a test tube what normally requires days to do in the lab.

Motivation

Tumor mobility is likely the most significant prognostic factor for all types of cancer. Contained primary tumors often present no symptoms and if discovered early can be safely removed without needing subsequent chemotherapy. If left untreated, however, primary tumors spread through the lymphatic or blood circulatory systems to other parts of the body. Given enough time, the cancer cells transition and take on the forms of cells from other organs. At this stage, the cells invade compatible organs and secondary tumors called Mets develop. Early mets are less diverse and still present hope for treatment. Later mets, however, are too diverse and are usually associated with terminal diagnosis.

The difference between early stage and late stage diagnosis can be staggering. The table below lists the survival rate differential between early and late diagnosis for common cancer types.

Early detection of cancer and its localization is very difficult. General early detection tests look for specific molecular traces in samples of blood or urine. Such tests usually carry a high rate of false positives and are difficult to calibrate for each individual. In addition, these test provide limited information regarding the primary and secondary tumor sites. Indeed, the localization of tumors is a real issue. Total body scans are impractical at the necessary resolution level and carcinogenic if applied regularly.

Circulating tumor cells (CTCs) provide an alternate path for tumor detection. These stray cells originate from the tumor site and enter the blood stream after begin pushed out from the forefront of the primary tumor. These stray cells are also the first to invade other organs and seed secondary tumors. During the process of detachment and invasion CTCs undergo several transitions downregulating local adhesian molecules and upregulating distal adhesian molecules and stem cell markers. Deciphering CTC surface markers holds the key to understanding the tumor's ability to invade the host system.

Methods and tools for detecting circulating tumor cells (CTCs) are very limited. CELLSEARCH circulating tumor cell test is the only FDA approved diagnostic method. CTCs are magnetically separated from samples of peripheral blood using the common epithelial marker EpCAM.Subsequently, the cells are stained and individually scanned using an automated positioning and scanning system. Final results are submitted to an expert for review. In clinical practice, however, simple enumeration of CTCs is most commonly used. Another diagnostic waiting for FDA approval is Adnatest, which goes a step further with broad spectrum separation of cells and RT PCR analysis. Multiple antibodies are used to capture not only EpCAM+ cells but also CA15-3+, Her2new+ and others. RT PCR kits are targeted at common primary tumors. Customised CTC screens are possible for research purposes only through immunostaining in combination with microdisection.

The takeaway message is current CTC diagnostic tests are time consuming, require advanced training and equipment, are impractical for early diagnosis, and are too broad to yield localization or mobility information.

The IOD band concept

At team Czech Republic, we envision a body surface marker atlas. Each cell type is unique in its interaction with the system. A kidney cell is ... Hence marker profiles serve as unique area codes linking a cell to an organ.

The IOD band is a system that reads marker profiles and generates a signal visible to the eye when an interesting profile is found.

The IOD band is based on intelligent synthetic organisms called IODs that work together to solve complex problems such as the deciphering of marker profiles.

Probably the most important IOD feature is the way they are assembled. A unique IOD band can be assembled clone-free in approximately a day.

Functional prototype

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More detailed description

Module 1 - Synthetic mating types

This module develops synthetic haploids of both mating types that preserve the ability to process an extracellular signal via pheromone response pathway even after mating - in diploid state. Naturally in diploid cell, all components of the pathway are switched-off, therefore we designed synthetic haploid strains that mate into a diploid with functional pheromone pathway. As a result, the IODs use robust signaling pathway for the signal processing.

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Module 2 - Synthetic signals and receptors

In order to achieve more complex behaviour of our IODs, each IOD needs to be able to communicate with others. Since the pheromone response pathway was used for signal transduction, we prepared several synthetic signals and receptors that couple to this pathway and allow our IODs to communicate without a cross-talk. Read more...

Module 3 - Reversible cellular adhesion

To enable our modified yeast cells to bind extracellular markers, we expressed single-chain variable fragments of antibodies (scFvs) on their surface. These proteins are fused with the adhesion subunit of a-agglutinin, which is a protein naturally used by S. cerevisiae for aggregation during mating. Binding of the antibody to its specific marker triggers release of a pheromone and relays the signal to other yeast cells. We used a modified Yeast Display technique to enable our cells to present the antibody fragments on their surface at all times. We were able to detect and measure the binding of several oncogenic, epithelial, and blood markers. Read more...

Module 4 - Modelling

IOD systems function at both the intracellular and intercellular level. Intracellular biochemical reactions determine signalling activities and gene expression. Extracellular reactions include signalling molecule diffusion and cell movement including cell-cell interactions. At each level models of various complexity are available. We selected minimalistic models that capture only the key design elements and integrated these models in a single simulator CeCe. Subsequently simulation was used to study the robustness and efficiency of different signal transmission patters in identifying cells with specific marker profiles while minimising false positives. Read more...

Module 5 - Microfluidics

The diffusion processes are slow, and the inertial effects are negligible on micro-scale with low Reynolds number ^{[Angelescu2011] [Nam2002]}. Hence microfluidics enables complex control of the cellular microenvironment. Microfluidic experiments in conjunction with live fluorescence microscopy were designed and performed to verify and characterise the developed IOD system.

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