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

Summary

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.

Abstract

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

Introduction

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....

Protocol

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. Additionally, multi-labeled cellular systems, such as those found in transgenic mice containing multiple lineage restricted-fluorescently labeled populat....

Results

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 slices .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
.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