This was our original plan for in vitro, to combine both bacteriotherapy and current immunotherapy techniques. Following the synthesis of our bacterial genome, we would insert the DNA sequence that selectively secretes immunotherapeutic molecules into the synthesized genome to leverage the power of both. The following experimental procedure also describes how we planned on validating the tumor suppressing abilities of our individual microbes.
Disease modeling using 3D Organoid
Organoids are three-dimensional (3D) structures that are comprised of multiple cell types, self-organized to recapitulate embryonic and tissue development in vitro. This model has been shown to be superior to conventional two-dimensional (2D) cell culture methods in mirroring functionality, architecture, and geometric features of tissues seen in vivo. As a result, the traditional 2D monolayer often becomes irrelevant to human physiology, possibly producing inaccurate results to treatment candidates.
After an in-depth look at the results of the computational analyses, we will ultimately select 2-4 microbes deemed cancer-promoting as well as cancer-suppressing. These selected microbes will be characterized in vitro using the 3D organoid of HNSCC. These organoids will first be cultured as stem cells, and later differentiated into various cell types using specific media conditions. Firstly, an Enzyme-Linked Immunosorbent Assay (ELISA) will be performed on the media of the organoid co-cultured with the microbe of interest vs the control. Following the attachment of a colorgenic enzyme substrate in the 96-well plate, the wells will be quantified with an electric plate reader. This test will validate the correlation of a certain microbe to a specific immune or cancer pathway from the computationally determined pathway analysis results. Next, in order to validate for immune infiltration, immune cells will be co-cultured with the microbe and the organoid, and counted every 24 hours to quantify their proliferation using a hemocytometer.
After validating the mechanisms and interactions of these key microbes, we will select a tumor-suppressing microbe to develop a combination immunotherapy approach. We will engineer this clinically relevant microbe to express the peptide CLP002 that specifically binds to PD-L1 to blockPD-1/PD-L1 interaction synchronously at a threshold population density. The amino-terminal end of CLP002 will be fused to a twin-arginine translocation or Tat pathway-specific signal peptide for the secretion of CLP002 across the cytoplasmic membrane. This approach takes advantage of natural bacterial quorum sensing and only when they reach critical density within tumors, will they express and secrete the therapeutic agent. As a result, this approach avoids releasing therapeutics in unintended parts of the body, and the localized delivery of therapeutic agents greatly improves the potency. This synchronized circuit will couple positive and negative feedback loops, specifically utilizing the luxl promoter that drives expression of both its own activator (positive feedback) and the gene producing CLP002 (negative feedback). This process has been proved to be an effective method to successfully localize the tumor and combat human cancer in a study by M Omar Din Et. al.. However, in M Omar Din et. al.’s study, their negative feedback of tumor density detection resulted in the lysis of the cell releasing the therapeutic payload. For our microbe, not only will the microbe secrete CLP002, the tumor-suppressing pathways the microbe originally participated in will also continue to be active to potentially regress the tumor. Recently, various combination immunotherapies to treat HNSCC patients have yielded unprecedented results.
Using the 3D Organoid, we will validate the effectiveness of this engineered microbe by injecting them inside a coculture of immune cells and the HNSCC organoid. A MTT assay will be performed to compare cell proliferation to a control, as well as the injection of the unengineered, tumor-suppressing microbe. This sequence of the circuit will be identified and submitted to the BioBrick Registry for iGEM. While we hope this engineered bacteria may prove to be an effective therapeutic agent, low survival rates associated with HNSCC are also partly due to failure in early diagnosis. Only one third of HNSCC patients are diagnosed at an early stage and can mainly be attributed to the lack of appropriate screening and diagnostic biomarkers. Recent advances in genome sequencing technologies and metagenomic analysis, however, provide us with a broader understanding of these commensal microbes, and emerges a plethora of potential biomarkers. The microbes identified and characterized previously as tumor-promoting may be useful as a biomarker for HNSCC and for developing diagnostic tools for early diagnosis in the future. Ultimately, in this project, we hope to engineer a therapeutic bacteria that combines already established and novel immunotherapies to combat HNSCC. Furthermore, we hope to present a panel tumor-promoting microbes as potential biomarkers as well as the mechanisms by which they operate.
-  Economopoulou P., Kotsantis I., Psyrri A. “The promise of immunotherapy in head and neck squamous cell carcinoma: combinatorial immunotherapy approaches” ESMO Open , 2016
-  Ho, Beatrice Xuan et al. “Disease Modeling Using 3D Organoids Derived from Human Induced Pluripotent Stem Cells.” International journal of molecular sciences, 2018
-  Freudl, R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact, 2018
-  Din, M., Danino, T., Prindle, A. et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature, 2016