Femur Window Chamber Model for <em>In Vivo</em> Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Princess Margaret Cancer Centre

Radiotherapy is a widely used cancer treatment. However, understanding how ionizing radiation affects tumor cells and their vasculature, particularly at cellular, subcellular, genetic, and protein levels, has been limited by an inability to visualize the response of these interdependent components within solid tumors over time and in vivo. Here we describe a new preclinical experimental platform combining intravital multimodal optical microscopy for cellular-level longitudinal imaging, a small animal x-ray microirradiator for reproducible spatially-localized millimeter-scale irradiations, and laser-capture microdissection of ex vivo tissues for transcriptomic profiling. Using this platform, we have developed new methods that exploit the power of optically-enabled microscopic imaging techniques to reveal the important role of the tumor microvasculature in radiation response of tumors. Furthermore, we demonstrate the potential of this preclinical platform to study quantitatively--with cellular and sub-cellular details--the spatio-temporal dynamics of the biological response of solid tumors to ionizing radiation in vivo.

In vivo optical imaging of tumor and microvascular response to ionizing radiation.

La radioth&eacute;rapie est le traitement du cancer largement utilis&eacute;. Toutefois, comprendre comment les rayonnements ionisants affecte des cellules tumorales et leur syst&egrave;me vasculaire, en particulier au cellulaire, cellulaire, g&eacute;n&eacute;tique et le taux de prot&eacute;ines, a &eacute;t&eacute; limit&eacute;e par une incapacit&eacute; &agrave; visualiser la r&eacute;ponse de ces composants interd&eacute;pendants dans les tumeurs solides au fil du temps et in vivo. Nous d&eacute;crivons ici une nouvelle plate-forme exp&eacute;rimentale pr&eacute;clinique combinant la microscopie intravitale optique multimodale pour l&#39;imagerie longitudinal au niveau cellulaire, une microirradiator de petits animaux aux rayons x pour les irradiations de l&#39;&eacute;chelle millim&eacute;trique reproductible localis&eacute;e dans l&#39;espace et microdissection laser-capture d&#39;ex vivo des tissus pour le profilage de transcriptomique. &Agrave; l&#39;aide de cette plate-forme, nous avons d&eacute;velopp&eacute; de nouvelles m&eacute;thodes qui exploitent la puissance des techniques d&#39;imagerie microscopiques optiquement compatible pour r&eacute;v&eacute;ler le r&ocirc;le important de la microcirculation de la tumeur &agrave; la suite de la radiation des tumeurs. En outre, nous avons d&eacute;montr&eacute; le potentiel de cette plate-forme pr&eacute;clinique d&#39;&eacute;tudier quantitativement--avec les d&eacute;tails cellulaires et subcellulaires--la dynamique spatio-temporelle de la r&eacute;action biologique des tumeurs solides &agrave; un rayonnement ionisant in vivo.

Imagerie optique in vivo de la tumeur et microvasculaire r&eacute;action aux rayonnements ionisants.

Radioterapia &egrave; un trattamento del cancro diffuso. Tuttavia, capire come le radiazioni ionizzanti colpisce le cellule tumorali e loro vascolarizzazione, specialmente al cellulare, subcellulare, genetica e livelli di proteine, &egrave; stata limitata da un&#39;incapacit&agrave; di visualizzare la risposta di questi componenti interdipendenti all&#39;interno dei tumori solidi nel tempo e in vivo. Qui descriviamo una nuova piattaforma sperimentale preclinica combinando intravital microscopia ottica multimodale per cellulare-livello longitudinale imaging, un microirradiator piccolo animale a raggi x per irradianza riproducibile spazialmente localizzate-scala millimetrata e laser capture microdissection di ex vivo di tessuti per l&#39;analisi di approfondimento. Usando questa piattaforma, abbiamo sviluppato nuovi metodi che sfruttano la potenza delle tecniche di imaging microscopiche otticamente abilitato a rivelare il ruolo importante del microvasculature tumore in risposta di radiazioni di tumori. Inoltre, noi dimostrare le potenzialit&agrave; di questa piattaforma preclinica per studiare quantitativamente - con dettagli cellulari e subcellulare - la dinamica spazio-temporale della risposta biologica dei tumori solidi a radiazioni ionizzanti in vivo.

In vivo imaging ottico del tumore e microvascolare risposta alle radiazioni ionizzanti.

Bacterial infection significantly impedes wound healing. Clinical diagnosis of wound infections is subjective and suboptimal, in part because bacteria are invisible to the naked eye during clinical examination. Moreover, bacterial infection can be present in asymptomatic patients, leading to missed opportunities for diagnosis and treatment. We developed a prototype handheld autofluorescence (AF) imaging device (Portable Real-time Optical Detection, Identification and Guidance for Intervention - PRODIGI) to noninvasively visualize and measure bacterial load in wounds in real time. We conducted preclinical pilot studies in an established nude mouse skin wound model inoculated with bioluminescent Staphylococcus aureus bacteria. We tested the feasibility of longitudinal AF imaging for in vivo visualization of bacterial load in skin wounds, validated by bioluminescence imaging. We showed that bacteria (S. aureus), occult to standard examination, can be visualized in wounds using PRODIGI. We also detected quantitative changes in wound bacterial load over time based on the antibiotic treatment and the correlation of bacterial AF intensity with bacterial load. AF imaging of wounds offers a safe, noninvasive method for visualizing the presence, location, and extent of bacteria as well as measuring relative changes in bacterial load in wounds in real time.

Autofluorescence imaging device for real-time detection and tracking of pathogenic bacteria in a mouse skin wound model: preclinical feasibility studies.

Biomarker-specific imaging probes offer ways to improve molecular diagnosis, intraoperative margin assessment, and tumor resection. Fluorescence and photoacoustic imaging probes are of particular interest for clinical applications because the combination enables deeper tissue penetration for tumor detection while maintaining imaging sensitivity compared to a single optical imaging modality. Here we describe the development of a human epidermal growth factor receptor 2 (HER2)-targeting imaging probe to visualize differential levels of HER2 expression in a breast cancer model. Specifically, we labeled trastuzumab with Black Hole Quencher 3 (BHQ3) and fluorescein for photoacoustic and fluorescence imaging of HER2 overexpression, respectively. The dual-labeled trastuzumab was tested for its ability to detect HER2 overexpression in vitro and in vivo. We demonstrated an over twofold increase in the signal intensity for HER2-overexpressing tumors in vivo, compared to low-HER2-expressing tumors, using photoacoustic imaging. Furthermore, we demonstrated the feasibility of detecting tumors and positive surgical margins by fluorescence imaging. These results suggest that multimodal HER2-specific imaging of breast cancer using the BHQ3-fluorescein trastuzumab enables molecular-level detection and surgical margin assessment of breast tumors in vivo. This technique may have future clinical impact for primary lesion detection, as well as intraoperative molecular-level surgical guidance in breast cancer.

Jiachuan Bu

Princess Margaret Cancer Centre

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow