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Abstract

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Protocol

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Materials

References

Medicine

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

Published: September 22nd, 2013

DOI:

10.3791/50385

1Department of Leukemia, MD Anderson Cancer Center, 2Wake Forest Baptist Medical Center, Institute for Regenerative Medicine

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....

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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.......

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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.......

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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.......

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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).

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number
.2 μm filter Fisher 09-740-35A
10 ml lure lock BD 309604
25 G needle Cardinal BF305122
40nm filter BD 352340
50 ml conical tube Fisher 1495949A
Alexafluor 488-ms1 invitrogen A21121
Alexafluor 594-Rb invitrogen A21207
Alexafluor 647-ch Invitrogen A21449
BSA Sigma A7906-500G
Cover slips Corning 2940-243
CRI-Nuance Caliper Life Sciences
Dapi Invitrogen D1306
DMEM CellGro 10-017-CV
Ethanol Dharmacon 4004-DV
FBS invitrogen 16000-044
GFP-ms1 Abcam ab38689
Insulin needle BD 329424
Maleic acid Acros 100-16-7
Moisture chamber box Evergreen 240-9020-Z10
Mounting media dako S3023
Nail hardener Sally Hansen 2103
Pap pen Abcam Ab2601
Pen/Strep L Glut invitrogen 10378016
Steril PBS invitrogen 14040-182
Trypsin invitrogen 252000-56
Blocking Buffer
maleic acid 14.51 g
NaCl 10.95 g
NaOH pellets (to 7.5pH) 9+g
Distilled water 250 ml
*add 0.2% Tween-20 to 1x solution for blocking buffer with 2%BSA/2%FBS. Stir and filter.
Antigen retrieval buffers:
Tris-EDTA Buffer
(10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0)
Tris Base 1.21 g
EDTA 0.37 g
Distilled water 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Tween 20 0.5 ml
*Store at RT for 3 months or at 4 ° C for longer
Sodium Citrate Buffer
(10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
HCl (adjust pH to 6.0) 1N
Tween 20 0.5 ml

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