A strategy for the selective imaging and inhibition of MT1-MMP to assess its functional roles in breast cancer invasion and metastasis

• Applicant: Dr. Martina Tholen
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2016 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314695690

• Final Report Year: 2020

Final Report Abstract

Proteases play key roles in tumor cell invasion and angiogenesis by mediating degradation of extracellular matrix. Small molecule probes that enable visualization of protease activity are valuable tools in basic research that help us understand function and regulation of proteases. When translated into a clinical setting these chemical tools can be used as contrast agents that visualize the tumor, since protease activity is highly increased in tumor tissue. In the first part of my research fellowship we employed small molecule probes to enlighten the regulation of MT1-MMP, one of the key regulators of protease enhanced cell migration. In previous work, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MT1-MMP activation in the tumor microenvironment. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MT1-MMP activity in the tumor microenvironment. A second focus of my work became the development of imaging probes that can be used as fluorescent intra-surgery contrast agents to help visualize tumor margins. Fluorescence guided surgery has the potential to greatly improve intraoperative decision-making and thereby reduce recurrence rates due to incomplete resections. Fluorescently-quenched probes that are proteolytically cleaved by lysosomal enzymes that are highly abundant in tumor-associated macrophages proved to be a promising strategy to visualize tumor-associated inflammation. For the further development of these probes we followed three different approaches. We vastly improved substrate recognition by incorporation of non-natural amino acids into the protease substrate based on proteolytic profiling of whole tumor tissue using high diversity peptide libraries. Secondly, we optimized the agents for the usage with FDA-approved intra surgery imaging devices in collaboration with Intuitive Surgical the leading developer of robotic surgical systems. In a further approach to increase specificity we developed probes that require sequential processing by multiple tumor-specific proteases to produce a fluorescent signal, so called “AND-Gate” substrates. These developments led to significant improvements of sensitivity and specificity of these agents which will be further advanced into the clinic in collaboration with industry partners. We believe these probes have great potential for application in optical surgical navigation.

Publications

• Chemical Tools for Selective Activity Profiling of Endogenously Expressed MMP-14 in Multicellular Models. ACS Chem Biol. 13(9):2645-2654. (2018)
  Amara N., Tholen M., Bogyo M.
  
• Optimization of a protease activated probe for optical surgical navigation. Mol Pharm. 5;15(3):750-758. (2018)
  Yim J.J.#, Tholen M.#, Klaassen A., Jonathan Sorger J., Bogyo M
  
• Multivariate ‘AND-gate’ substrate probes as enhanced contrast agents for fluorescence-guided surgery
  Widen J.C., Tholen M., Yim J.J., Kerriann M. C., Rogalla S., Bogyo M.
  
• The clinical drug candidate ebselen attenuates inflammation and promotes microbiome recovery after antibiotic treatment for Clostridium difficile infection
  Garland M., Hryckowian A., Tholen M., Loscher S., Van Treuren W, Oresic Bender K., Sonnenburg J.L., Bogyo M.