Interrogating Tumor Microenvironments

Enhance Noninvasive Tumor Monitoring by Exploiting Nanomaterial Properties

 

We first began efforts to interrogate cancer noninvasively through the development of targeted nanoscale imaging probes in 2002. By exploiting both the physics (changes in electronic, magnetic, and optical properties) and the biology (changes in trafficking) of the nanoscale, we demonstrated the ability to sensitively detect solid tumors, and explored the role of the tumor microenvironment in nanoparticle homing to both xenograft and genetically-engineered mouse models of cancer. Subsequently, we sought to develop technologies that would extend beyond detection to interrogate enzymatic activity within the tumor microenvironment, such as that which would occur in tumor infiltration of surrounding ECM. Inspired, in part, by the work of Roger Tsien’s activatable cell-penetrating peptides, we developed MRI contrast agents that could detect activity of ECM-remodeling enzymes MMP2 and MMP9. Further, we sought to achieve monitoring methods that do not require imaging equipment and yet could serve as sensitive and specific reporters of disease state. In order to reach this goal, we developed a new class of ‘synthetic biomarkers’. (Kwong et al., 2012Lin et al., 2013Warren et al., 2014)

 

Our nanoparticle probes are injected systemically, travel through the blood to the disease microenvironment, and are activated by local protease enzymes to release small peptide fragments into the urine for detection by mass spectrometry. The advantage of our system is the amplification of tumor responses by leveraging enzymatic turnover (e.g. a single copy of a protease can cleave hundreds of peptides per hour) and specificity that is introduced by multiplexing. Our study detected colorectal tumors that could not be identified by secreted CEA (a secreted blood biomarker). Based on our knowledge of nanoparticle trafficking in the liver, we also applied this method to study the liver microenvironment as it underwent fibrosis. We found a single synthetic biomarker could be used to monitor fibrosis in response to both xenobiotic and genetic injury, reflecting a conserved tissue response despite distinct underlying etiologies. We have further adapted the synthetic biomarkers to be detectable without the use of mass spectrometry, but rather by antibody-based methods. This adaptation enabled single molecule sensitivities using a bead-based digital ELISA format and a point-of-care lateral-flow assay (i.e. paper diagnostic) for patient monitoring outside of the hospital setting.

 

Synthetic biomarkers for urinary detection of disease.
Synthetic biomarkers for urinary detection of disease. (A) Synthetic biomarkers made up of nanoparticles decorated with mass-barcoded peptide substrates are administered to detect disease. Synthetic biomarkers (i) probe disease sites, (ii) sense the activity of proteases, and (iii) release reporters into the urine as indicators of disease. (B) Thrombin-specific nanoparticles specifically sense cleavage by thrombin (red) but not by non-cognate proteases. (C) In vivo fluorescence image showing cleaved peptide fragments cleared into the bladder in tumor animals. (D) Synthetic biomarkers are more predictive than CEA, a protein biomarker shed into the blood by colorectal tumors. (E) Low-cost paper tests engineered to detect synthetic biomarkers for disease diagnosis in resource-limited settings.