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The prognosis for high-grade glioma (HGG) remains dismal and the extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients was scored by pathologists and expression levels were compared to that of mouse xenografts. Intratumoral distribution of panitumumab-IRDye800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 h post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Tumor specific uptake of panitumumab-IRDye800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

Background: The prognosis for high-grade glioma (HGG) remains dismal and extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Methods: Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients during was scored by pathologists and expression levels were compared to that of mouse xenografts. Results: Intratumoral distribution of pan800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 hours post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Conclusion: Tumor specific uptake of pan800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

The tumor microenvironment (TME) can be defined as non-transformed stromal elements within the tumor, effecting their growth, invasion, and metastatic ability. Cancer cells leave the primary tumor during metastasis, then spread and colonize in distant organs. In breast cancer, metastasis mechanisms are not yet fully understood within the TME. However, epithelial to mesenchymal transition (EMT) is a known essential step in metastasis, in which patterned epithelial cells transform into migratory cells with mesenchymal phenotypes. Developing state-of-the-art multiparametric in vivo imaging devices increases the opportunities to observe dynamic cellular properties and their interactions with the surrounding stroma and TME during the critical steps of invasion and metastasis. Currently available commercial imaging technologies only enable in vivo imaging under anesthesia in a fixed posture. In contrast, real-time confocal imaging of the EMT and TME is not possible in animals. We have adapted our miniature dual-axis confocal (DAC) fluorescent microscope to track breast cancer cells having fluorescent reporters indicating key molecular splicing events during EMT to overcome these limitations. This project aims to study EMT more thoroughly using rat breast cancer MTLn3 cell lines by mimicking in vivo scenarios through 3D in vitro mono/co-culture spheroids, 3D multi-spheroid matrix overlay models, and 3D sandwich. We have engineered MTLn3 cells with fluorescent reporters correlating with different EMT stages, depending on their invasiveness levels. The ultra-low attachment method is used to engineer 3D MTLn3 spheroids, embed within TME matrices, and monitor advanced in vitro live-cell imaging techniques for up to 10 days. Another goal is to use miniature DAC microscopes, implanted in animal models, to study the fundamental changes occurring during carcinoma progression. This transdisciplinary project's final goal is to optimize the DAC microscope as an implantable and insertable device to investigate EMT-associated splicing events in the TME of orthotopic rat breast carcinoma models. This will be achieved by growing engineered MTLn3 cells and spheroids in their mammary fat pads and following microscopic changes for a certain period. This work will contribute to further developing the miniature implantable DAC microscopes for 3D high resolution, multiparametric imaging to reveal dynamic changes within the TME during carcinoma progression. Citation Format: Ehsanul Hoque Apu, Seock-Jin Chung, Michael J. Mandella, Frank Schonig, Frank B. Gertler, Zhen Qiu, Christopher H. Contag. Implantable and insertable miniature dual-axis confocal (DAC) microscope to detect epithelial-mesenchymal transition (EMT) in the breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB243.

Wide-field fluorescent imaging for fluorescence molecular guidance has become a promising technique for use in imaging guided surgical navigation, but quick and intuitive microscopic inspection of fluorescent hot spots is still needed to confirm local disease states of tissues. To address this unmet need, we have developed a clinically translatable dual-modality handheld surgical microscope that incorporates both, wide-field (mesoscopic) fluorescence imaging and high-resolution (microscopic) horizontal optical-sectioning. This is accomplished by integrating a commercially available wide-field fiberscope, modified for two-color (660nm and 785nm) fluorescent detection, into a compact package (5.5 mm dia.) which also contains a dual-axis confocal (DAC) microscope. DAC microscopy is a high-sensitivity, high-resolution fluorescent imaging technology that benefits from the specificity of molecular probes, and enables interrogation of deeper regions of tissue by performing optical-sectioning of tissue. The DAC microscope has been designed with custom catadioptric micro-lenses to provide broadband multispectral capability for fluorescence imaging of multiple fluorophores over a broad spectral range (VIS to NIR), and also uses a novel MEMS-based scanning system for horizontal sectioning, and thus enables access to deeper regions of tissue at resolutions comparable to histological analysis. Large field-of-view (mm scale) is further provided by image mosaicing. The instrument thus provides simultaneous mesoscopic and microscopic fluorescence imaging over a broad spectral range for intuitively performing fast in-vivo search and microscopic confirmation of optical molecular markers in tissue, which is a capability that will become increasingly important for precise tumor resection in oncology as more optical molecular markers become approved for human use.

MEMS based microendoscopes have become important imaging tools for early cancer diagnosis and precise tumor resection. Due to various technical challenges, few microendoscopes have been translated to clinics or applied to human patients. Through synergistic collaborations, we have developed novel MEMS scanner enabled microendoscopic multispectral (640nm to 780nm) three- dimensional dual-axis confocal fluorescent imaging system for translational applications, including early cancer detection and staging on colorectal cancer, molecular imaging guided surgical navigation on head and neck cancer. Based on dual-axis confocal microscopic architecture, we have miniaturized the imaging system with compact form-factor by integrating micro-optics and a patterned gold coated MEMS scanners, which have been custom-made and mass-produced in the nanofabrication foundry. The metal coating of the scanning mirror provide over 80% high reflectivity over near infra-red range. Both axes of the MEMS scanner could perform large tilting angle (> 6 degree mechanical scan angle) at DC and resonant mode. By advanced computational imaging approach, we have achieved real-time cross-sectional imaging in either raster or lissajous pattern scanning with fast frame rate (> 10 Hz) with large field-of-view (> 600 microns). Advanced real-time mosaicing algorithm has been developed to achieve broader view in millimeter scale. By utilizing molecular contrast probes conjugated with fluorescence dye, we have successfully demonstrated multi-spectral ex-vivo and in-vivo imaging on small animal tumor models and human tissue specimens, aimed for both early cancer detection and molecular imaging guided surgical navigation.