Team:British Columbia/Implementation
PROJECT
Implementation
TARGET USERS
Our tool will mainly be used by cancer researchers, and further downstream by physicians and cancer patients undergoing treatment. As such, we broke down the user cases into research scenarios and patients.
Given our end goal is to apply in patients, we first consider the use of VNP3000, the specific strain of Salmonella that is used in clinical trials.
VNP3000 is the strain of Salmonella used in clinical trials (Clairmont et al, 2000; Broadway et al, 2014). Genetic modifications harbored by VNP3000 include purI, msbB, and 108-kb Suwwan deletion that involves 128 genes, purM inversion, 50 nonsynonymous SNPs, and a derivative ∆htrA (Broadway et al, 2014). We envision our biosensor will eventually be integrated onto this strain of Salmonella to align with current practices.
IMPLEMENTING DETECTME AS A RESEARCH TOOL IN MICE
Benchmarking Step
Beyond our in-vitro lab work, the next step is to implement our project as an in vivo setting in mouse tumour models to provide a research tool to better characterize tumours in-vivo. Before that, we need to first characterize and validate the function of the biosensor in mouse models. Specifically, cancer researchers can inject tumour-bearing mice with Salmonella containing our biosensor, and perform immunohistochemistry (IHC) staining for immune markers the biosensor detects, and benchmark with the results of our reporter tool.
We will also consider testing different routes of administration. We chose intravenous (IV) injection as Dr. Saltzman suggested injection would result in a higher colonization rate. Oral administration and intratumoral injection are other options as well.
Using our system, we could detect light production from the luciferase-luciferin reaction via in vivo imaging. The comparison between IHC tumour immune analysis and our immune biosensor would allow us to characterize and validate our tool. We will also seek to determine the most physiologically relevant luciferin concentration threshold to decipher positive versus negative immune infiltration of the tumour(s) in question.
Safety Considerations
A critical safety concern is how the biosensor will interact with the host system, and how that interaction might affect our detection. In implementing our system in mice, we will be aiming to address questions such as, at what concentration would the biosensor become potentially toxic to the body? Although it is highly attenuated, how immunogenic is our Salmonella? THe mouse model will also help us to assess the stability of luciferin in-vivo, possible interactions between luciferin and other compounds in the body, and ultimately, how this will affect our detection readout.
In-vivo Tumour Characterization
Our modular system may have profound implications for research. First, we can apply this reporter system to gain insight on the TME by detecting the expression of other relevant markers in various types of tumours. For example, in the case of bladder cancer, it could be used to aid in tumour subtyping and characterization by detecting different biomarkers for subtypes such as neuroendocrine, urothelial, and more. This information would help inform prognosis and treatment.
Another application of our system is to rapidly screen for predictive biomarkers. For example, with the rise of immunotherapy, there is an increasing need for biomarkers that indicate tumour response to immunotherapy. Our modular platform can be used to screen for various potential biomarkers in tumours under different treatment conditions, to help improve understanding of the correlation between predictive markers and treatment response.
IMPLEMENTING DETECTME IN CLINICAL SETTINGS
To bring the tool to a clinical setting, we will adopt the split protein reporter system as outlined on the Design page for more convenient and cost-effective reporter detection in urine, which opens the door to greater clinical applications. The prior split-lux operon system will inform our choice of promoters which respond to relevant biomarkers, which we can then substitute into the split-galactosidase AND-gate reporter system. This will allowing for excretion of luciferin to be detected in urine.
Benchmarking Step
To this end, we will first characterize the system in vitro, to assess the efficiency of the split-galactosidase protein as a reporter. The proposed animal experiments for this system will be similar to that proposed for the split operon system, in addressing the safety and efficacy of the system in vivo. Some additional questions that we will want to address are the ways that beta-galactosidase and Lu-Gal react in the whole animal, and how luciferin reacts within the body and in urine. We will also want to assess dose dependency of the system, meaning the change in beta-galactosidase production and consequent luciferin excretion as a function of immune biomarker concentration. The final benchmark would be determining the limit of detection for luciferin in urine?
Clinical Trials
Once we are able to ensure safety and efficacy of our beta-galactosidase reporter system in vivo, we bring our project to the clinical trials stage. In this stage, we will continue to assess the safety and efficacy of our tool in healthy controls and in patients. To further characterize and validate our tool, Dr. Mathieu Roumiguie suggested that we compare the tumour immune marker IHC analysis of each individual with the output provided by our tool. Based on the output of our reporter, physicians can decide the best treatment scheme for the patient.
How would this fit into current options for biopsy and sensing?
From our discussion with Dr. Mathieu Roumiguié, he described the importance of biopsies in making diagnoses (various aspects: staging, subtyping, immune infiltration). However, he also shared that there is a need for non-invasive methods to learn more about tumours and a need for biomarkers to indicate immunotherapy response. Moreover, he indicated that to continue monitoring and check for recurrence, patients are sometimes required to perform further imaging and potentially additional biopsies. Thus, our tool would fit in as a non-invasive method of informing tumour immune state and providing a less invasive way to monitor disease.
In the case of bladder cancer, the immunotherapy option would be BCG (Bacillus Calmette-Guerin). Dr. Roumiguie suggested that our tool could be used to monitor tumour immunity while patients are receiving BCG, and potentially be used to investigate differences between tumours that do and do not respond to immunotherapy.
Throughout our project, we formed a partnership with the Humboldt Berlin iGEM team given our shared ideas of using tumour-targeting bacteria for cancer care. Learn more about our initiatives at Partnership.