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 a single cell 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, cancer cells undergo transition and take on characteristics 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 diagnoses.

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.

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The IOD band concept

At Team Czech Republic, we envisioned a tumor marker body atlas. A large number of tumor cell lines as well as healthy tissues of common secondary sites have well-documented surface marker profiles. A dataset of over 700 surface markers comprising of common receptors, transmembrane proteins, and channel families was processed to identify usable members with confirmed antibodies. Each cell line was then linked to a unique combination of up to three markers identified by a simple 6 or 9 digit barcode. We then envisioned The IOD Band, a marker profile probe that locates cells with target marker combinations in samples of peripheral blood.

The fundamental units of the IOD band are intelligent input/output diploid cells called IODs. An IOD is fundamentally simple, comprising two modular inputs and a single modular output. The inputs are locational tags displayed on the surface and pheromones detected by orthogonally expressed receptor and signaling pathway. The inputs combine to yield locally limited cell-cell communication. Pheromones are also the IOD outputs. A rewiring of the signaling pathway transcription factor connects the inputs with the outputs. Hence, a single IOD simply detects a pheromone signal and generates an orthogonal signal in response.

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Design and Functional Prototype

The IOD band is a project built on system level integration. At the outset, it seemed like an impossible goal for a single iGEM project especially as there are no precedents for many of the system parts. So the idea was stripped to its brass tacks to uncover three fundamental modules that prove the diagnostic and engineering principles in a tractable manner.

Module 1 builds synthetic haploid strains with refactored mating loci that are conjugated to make a functional IOD. These strains have the wild-type mating phenotype and differentially express a reprogrammed signalling pathway in their diploid state proving the feasibility of the clone-free assembly concept.

Functional prototypes of both MATa and MATx synthetic haploid strains were constructed and experimentally validated. The successful construction and integration of the refactored loci was confirmed using traditional methods and proves the effectivity of the flanking homologous regions. The retained wild-type mating phenotype of both synthetic haploid strains was validated by direct mating tests where strains were crossed and plated onto minimal medium. This shows that the synthetic strains are easily conjugated. Differential expression of haploid-specific and mating-type specific genes with respect to the wild-type strains was validated by flow cytometry. Using the developed collection of reporter plasmids, it was shown that mating-type specific genes are expressed only in their respective haploid strains and that haploid-specific genes are expressed in both the haploid and diploid strains. This demonstrates that the signaling cascade can be used orthogonally in haploid strains for conjugation purposes and in diploid strains for IOD intercellular signal detection. For the first time in iGEM, the function of a tunable a-specific promoter (a hybrid of the CYC1 and a-specific operon domains) was also validated.

(Top Left) Schematic and theoretical results of synthetic haploid strain mating test. (Bottom Left) Results of the mating test on an SDmin+LEU plate are in exact agreement with the theoretical results above. (Center and Right) Flow cytometry measurements of wildtype and synthetic strains with reporter plasmids. Comparing lower plots with wildtype strains and upper plots with synthetic strains confirms expected levels of expression for all reporters in all strains. Especially significant is the expression of haploid-specific genes in the synthetic diploids.

Module 2 builds a set of orthogonal pheromones and receptors. These pheromone-receptor pairs enable specific localized signalling proving the feasibility of multichannel signal transmission underlying logic operations necessary for reliable diagnosis.

For the first time, orthogonality of five different yeast pheromone-receptor pairs was validated. Alpha factor from various yeast strains was synthetically produced in S. cerevisiae and used to induce the pheromone pathway rewired with the corresponding Ste2 receptors. Fluorescence measurements from the pheromone pathway reporter pFUS1 showed maximal activation in matching pairs that significantly dominated any cross talk between the non-matching pairs. Furthermore, functionality of the synthetic sequences was extensively tested using both physiological and molecular assays. To prove the designed alpha factor, which was truncated to exclude repeats induces mating, a MATa cell synthetically expressing the alpha phermone was shown to schmoo after induction. To prove localization of pheromone based signalling to the immediate neighbourhood of the transmitting cell, a microfluidic experiment was designed that directly measured the signalling range in saturated cultures. An extrapolation of the results indicates that in a culture with OD600 greater than 3.0 only cells in direct contact will transmit signal.

(Top) Response of the tested STE2 receptors to different pheromones, measured relatively to the activation of the corresponding receptor. (Bottom) On the left, activation gradient of the MATa cells with pFUS1 reporter by MATa cells producing synthetic alpha factor in the microfluidics is shown. On the right, MATa cell producing synthetic pheromone to its natural STE2 receptor was captured exhibiting shmooing. This demonstrated successfull production and secretion of the synthetic pheromone.

Module 3 builds a set of location tags that recognize common tumor surface markers and agglutinate cell populations. Location tags displayed in the correct conformation strengthen cell-cell interactions to enable localization of signal transmission.

For the first time, an antibody (anti-EpCam), the most commonly used CTC marker (EpCAM), and the human antigen A antibody (anti-HuA) were displayed on the surface of the cell wall. Successful construction of the display plasmids was confirmed using traditional methods. Successful epitope export and display were then validated by immunofluorescent staining using an upstream hemagglutinin tag. Clumping of cells with complimentary location tags (i.e., anti-EpCAM and Ep-CAM) was shown qualitatively using microscopy and by visual inspection of stained slides with mounted cultures. While all cells have a tendency to aggregate that varies with incubation period and temperature, cells displaying complimentary location tags always generated visibly larger aggregates than their controls. Functioning of location tags in plasma, which is the natural medium for the IOD band diagnostic, was further proven by cell clumping consistently observed in mixed samples of yeast cells with the anti-HuA location tag and red blood cells from a type A donor. The control samples, where anti-HuA location tag display was not induced, showed negligible aggregation. These experiments also prove the compatibility of yeast cells with blood samples as no significant clumping or other abnormalities that would indicate overall concept infeasibility were observed in control samples.

Module 3 - Location tags.

Module 3 - Master experiment results show modest activation in mixed cultures with induced location tag display. Two repeat experiments were performed and compared to pure cultures. Signalling pathway activity in mixed cultures was greater than the linear combination of activities measured in the pure cultures.

A final master experiment integrated the location tag and signalling functions. Signal transmission enabled by location tag display was shown in two strains constitutively displaying complimentary tags (EpCAM and anti-EpCAM epitopes). Strain A represented an IOD bound to a CTC: a transformed MATa strain displaying a fragment of the common epithelial CTC marker EpCAM and carrying a pheromone response reporter (GFP upstream of pFUS1). Strain B represented a second IOD that recognises the EpCAM marker and activates the IOD band response: a transformed MATa strain synthetically producing the S. cerevisiae pheromone. Localised signal transmission is difficult to quantify. Qualitatively fluorescence microscopy revealed greater intensity levels in observed aggregates. Microplate assays suggest modest activation of Strain A in mixed cultures when compared to background activation in pure cultures and controls. Mixed culture controls with strains lacking the location tags exhibited no activation.

It is important to note that modeling and microfluidics were extensively used to support the experimental work. Modeling proved robustness of simple signal transmission networks in a simulation setting with naturally occurring signaling noise. In-house replicated microfluidic chips provided the precise environment necessary to quantify the individual cell-cell interactions. Without these engineering tools, realization and validation of such a complex project would have been as impossible as the

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Module 1 - Synthetic haploids

Module 2 - Orthogonal signals and receptors

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Module 3 - Location tags

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Module 4 - Modeling

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Module 5 - Microfluidics

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