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).

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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	<li>Kidd, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+the+tumor+microenvironment:+quantitative+assessment+of+adipose-derived+and+bone+marrow-derived+stroma.">Origins of the tumor microenvironment: quantitative assessment of adipose-derived and bone marrow-derived stroma.</a> PLoS ONE. 7, e30563 (2012).</li><li>Quintavalla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+labeled+mesenchymal+stem+cells+(MSCs)+maintain+multilineage+potential+and+can+be+detected+following+implantation+into+articular+cartilage+defects.">Fluorescently labeled mesenchymal stem cells (MSCs) maintain multilineage potential and can be detected following implantation into articular cartilage defects.</a> Biomaterials. 23, 109-119 (2002).</li><li>Sasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+are+recruited+into+wounded+skin+and+contribute+to+wound+repair+by+transdifferentiation+into+multiple+skin+cell+type.">Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type.</a> Journal of immunology. 180, 2581-2587 (2008).</li><li>Spaeth, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+transition+to+tumor-associated+fibroblasts+contributes+to+fibrovascular+network+expansion+and+tumor+progression.">Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression.</a> PLoS ONE. 4, e4992 (2009).</li><li>Wels, J., Kaplan, R. N., Rafii, S., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migratory+neighbors+and+distant+invaders:+Tumor-associated+niche+cells.">Migratory neighbors and distant invaders: Tumor-associated niche cells.</a> Genes and Development. 22, 559-574 (2008).</li><li>Weis, S. M., Cheresh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+molecular+pathways+and+therapeutic+targets.">Tumor angiogenesis: molecular pathways and therapeutic targets.</a> Nat. Med. 17, 1359-1370 (2011).</li><li>Bataille, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+immunofluorescence+on+paraffin-embedded+tissue+sections.">Multiparameter immunofluorescence on paraffin-embedded tissue sections.</a> Applied immunohistochemistry &amp; molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. 14, 225-228 (1097).</li><li>van Vlierberghe, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-color+staining+combining+fluorescence+and+brightfield+microscopy+for+simultaneous+immune+cell+phenotyping+and+localization+in+tumor+tissue+sections.">Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections.</a> Microscopy research and technique. 67, 15-21 (2005).</li><li>Robertson, D., Savage, K., Reis-Filho, J. S., Isacke, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+immunofluorescence+labelling+of+formalin-fixed+paraffin-embedded+(FFPE)+tissue.">Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue.</a> BMC cell biology. 9, 13 (2008).</li><li>Qiu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracting+a+cellular+hierarchy+from+high-dimensional+cytometry+data+with+SPADE.">Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE.</a> Nat Biotechnol. 29, 886-891 (2011).</li><li>Belizario, J. E., Akamini, P., Wolf, P., Strauss, B., Xavier-Neto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+routes+for+transgenesis+of+the+mouse.">New routes for transgenesis of the mouse.</a> Journal of applied. 53, 295-315 (2012).</li></ol>

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...

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 prototypical example for this multicolor-multicellular transplant model followed by multispectral imaging and spectral unmixing methodology.	
	Tumor progression is a multistep process that is marked by several acquired capabilities that include enhanced proliferation, antiapoptotic, invasive and angiogenic properties.4 Tumor development is facilitated by non-neoplastic cells that are recruited into the surrounding environment to provide growth factors, structural matrices, vascular networks and immune modulation.1,5,6 This microhabitat consists of cells derived from local, neighboring tissues such as adipose, and blood vessels and distant sources such as bone marrow derived cells 1. The extent of non-neoplastic cell incorporation depends on the demand from the tumor, which often corresponds with the stage/grade of the tumor. To comprehend the role of the tumor supportive microenvironment, one must understand the origin and the differentiation potential of the non-neoplastic cell populations.	
	This protocol has been designed to aid in the interpretation of tumor progression through the visualization of both the bone marrow derived cellular components, and the local tissue derived cells. Utilizing fluorescent reporter gene-expressing transgenic mice, we transplanted GFP (green-fluorescent protein) bone marrow into a lethally irradiated RFP (red-fluorescent protein) mouse. Following successful bone marrow engraftment, a syngeneic tumor cell line is injected orthotopically and allowed to engraft for 4-8 weeks. The resulting tumor is excised from the mouse and processed for immunofluorescent (IF) staining to visualize the stromal components. 	Multiplexing IF markers is a commonly used technique that involves significant optimization7-9, however using a multispectral imaging/unmixing platform improves the potential for fluorescent marker combinations that possess spectral overlap. Herein we present a technique we call MIMicc- multispectral interrogation of Multiplexed cellular compositions to stain and analyze up to eight markers within a tumor section on a single slide in order to analyze the cellular origins, cellular differentiation status and cell-cell interactions of components within the tumor microenvironment. This simplified example has the potential to be expanded upon in order to analyze five, six, or more markers utilizing antibodies or intracellular promoter-driven fluorescent expression. Table 1 lists potential fluorescent antibody staining combinations with appropriate species taken into account.

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

quantitative multispectral analysis following fluorescent tissue transplant for visualization of cell origins, types, and interactions

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&#39;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

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 &lt;8 &mu;m slic...

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

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 ...

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9.3K Views.  MD Anderson Cancer Center. 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.