Published: September 22nd, 2013
Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.
With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we've added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1
Understanding tissue development and repair is significant to elucidating participating cellular components in wound healing,2,3 regenerative medicine, developmental biology and tumor biology. Under circumstances of repair, numerous cell types infiltrate the surrounding microenvironment to aid in vascularization, ECM deposition, proliferation and tissue restructuring. Cellular factors and phenotypes can be identified based on multiparameter, multiplexed markers that can identify the localization, differentiation status and interaction between cellular components within the investigated microenvironment. Herein, we describe tumor development as a p....
Syngeneic Murine Bone Marrow Transplantation (BMT) as approved by institutional IACUC protocols (Note: Success of this protocol requires utilization of any labeled cell type(s) as the target for multispectral analysis, and to facilitate the downstream analysis, one can exploit any genetic or stable cellular labeling. We suggest that any cell, tissue, or organ-to-be can be applied to a transplant model and that the transplant can contain as many unique labeled populations as the research deems useful. Add.......
Using a multispectral imaging technique to analyze tumors engrafted in our transgenic BMT mouse model, we are able to discern stromal tumor components that are of bone marrow origin. The initial BMT was confirmed three weeks post-transplant by flow cytometry (Figure 1). Orthotopically injected unlabeled ovarian tumors following BMT engraftment confirmations were excised and formalin fixed 6 weeks following initial tumor injection. Paraffin embedded tumor sections were sectioned in <8 μm slic.......
Herein we describe the application of multispectral imaging we describe as MIMicc- multispectral interrogation of Multiplexed cellular compositions, to analyze the bone marrow derived stromal components of the tumor microenvironment, however this methodology and concept can be applied to deciphering other cellular elements that compose a complex tissues such as those seen during wound healing reactions or during a regenerative tissue. In these experiments we utilize transgenic mice to fluorescently distinguish the.......
We are grateful to the discussions, guidance and support from Drs. Michael Andreeff MD, PhD., and Jared Burks PhD. from the MD Anderson Flow Cytometry and Cellular Imaging Core Facility. This work was supported in part by grants from the National Cancer Institute (RC1-CA146381, CA-083639, R01NS06994, CA116199 and CA109451 for FCM. ELS is also supported by the Army Department of Defense (BC083397).....
|Name of the reagent
|.2 μm filter
|10 ml lure lock
|25 G needle
|50 ml conical tube
|Caliper Life Sciences
|Moisture chamber box
|Pen/Strep L Glut
Antigen retrieval buffers:
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