We would like to thank Ying Yu for ApcMin/+ mice genotyping. This study was supported by funds from INSERM and grants (CA057621 and AI053194) from the National Institutes of Health.

NOTE: All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC), UCSF. All the imaging experiments were non-survival procedures and the animals were euthanized immediately following the end of image acquisition.
1. Generation of Mice
NOTE: ApcMin/+ mice, carrying a mutation on the Apc gene, spontaneously develop 50-100 adenomas in the small intestine.
<ol>
	<...

<ol>
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The authors have nothing to disclose.

In this paper, a detailed protocol is described for spinning disk confocal imaging of myeloid cell dynamics in intestinal tumors for several hours in a live animal, imaged from the serosal side of the gut.
To avoid inflammation and to have optimal physiologic conditions, imaging of the intestine has to be done on the intact organ. However, imaging from the serosal side of the intestine is challenging, since the light has to go through different tissue layers such as smooth muscle before reachi...

Overwhelming evidence now demonstrates that the tumor microenvironment, consisting of heterogeneous cell populations, including fibroblasts, endothelial cells, immune and inflammatory cells, extracellular matrix, and soluble factors, plays a crucial role in the initiation and progression of solid tumors by contributing to almost all hallmarks of cancer1. Indeed, during tumor progression, there are constant dynamic interactions between transformed cancer cells and stromal cells that evolve to generate a microenvironment favorable to malignancy2. Among the immune cells that infiltrate the tumor microenvironment, myeloid cells are the most abundant3. Consisting of tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSCs), dendritic cells (DC) and neutrophils (PMNs), myeloid cells are recruited from bone marrow and progressively infiltrate tumors, releasing cytokines, growth factors and proteases which can promote tumor growth and spread4. The crosstalk between cancer cells and myeloid cells is complex but dynamic. Thus the understanding of the nature of their interactions is crucial for determining why these cells promote cancer progression instead of participating in an anti-tumor immune response, and may help to find new targets to control it.
Direct observation by intravital microscopy provides information on cell dynamics within the tissues of live mice5. A four-color, multi-region, micro-lensed spinning disk confocal system was designed to study stromal cells within mammary tumors6. This approach enables long-term continuous imaging and includes several advantages such as (a) rapid images acquisition to minimize motion artifacts, (b) long-term anesthesia, (c) four color acquisition to follow different cell types, (d) fluorescent labeling of different tumoral components, and (e) observation of different tumor microenvironments within the same mouse to avoid mouse to mouse variability7-9. With this technology, different cell behaviors have been reported in the mammary tumor virus (MMTV) promoter-driven polyoma middle T oncogene (PyMT) model that displays progressive stages of tumorigenesis. Regulatory T-lymphocytes (Tregs, visualized by the Foxp3EGFP transgene) migrate preferentially in proximity to blood vessels whereas DCs (CD11c-DTR-EGFP), carcinoma-associated fibroblasts (Fsp1+/+-EGFP) and myeloid cells (c-fms-EGFP) exhibit higher motility at the tumor periphery than within the tumor mass. In the condition of acute systemic hypoxia, cells migrated differently: Tregs stop migrating in contrast to myeloid cells that continue to move6. In addition, in the same mouse model, it has been shown that doxorubicin sensitivity changes with tumor stage, drug distribution is related to drug response, and doxorubicin treatment leads to CCR2-dependent recruitment of myeloid cells to tumors. Thus, live imaging can also be used to gain insights into drug responses in&#160;situ and the biology of chemoresistance10,11.
Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas12 and mutation of a single copy of the Apc gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer13. The mouse strain ApcMin/+ carries a truncation mutation at codon 850 of the Apc gene and spontaneously develops multiple intestinal adenomas all over the small intestine14-16. Long-term intravital imaging of the intestine is challenging because of the invasiveness of the procedure, since opening the peritoneal cavity is necessary for access to the intestine. Short-term live imaging studies have been previously published on healthy intestine17,18, but long-term direct observation of intestinal tumors has not been reported. A surgical procedure has been designed and refined to visualize tumors through the serosal surface of the intestine, using the intravital spinning disk microscopy system previously used to image breast tumors6,10. In this paper, a protocol is described that allows one to follow the behavior of myeloid cells within the tumors in the small intestine by using ApcMin/+ mice.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;"><a name="RANGE!A1:D30">Name of Material/ Equipment</a></td>
			<td style="width:107px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:227px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ApcMin/+ mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td>2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ACTB-ECFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td style="width:141px;">3773</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cfms-EGFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td align="right">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,000 kDa Dextran, rhodamine-conjugated</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:141px;">D7139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isoflurane</td>
			<td style="width:107px;">Butler Animal Health Supply</td>
			<td style="width:141px;">29450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitrogen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1X PBS</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Saline Buffer</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse Ly-6G (GR1) antibody AF647</td>
			<td style="width:107px;">UCSF Monoclonal antibody core</td>
			<td style="width:141px;"></td>
			<td>Stock 1mg/ml. Use at 7ug/mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atropine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td>Use at 1mg/Kg mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alcohol wipes</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">326895</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">28G1/2 insulin syringe</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">329465</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Remium cover glass</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-548-5M</td>
			<td>24x50-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost plus microscope slides</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-550-15</td>
			<td>25x75x1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Krazy glue</td>
			<td style="width:107px;">Office Max</td>
			<td align="right">7111555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Betadine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heat blanket</td>
			<td style="width:107px;">Gaymar Industries</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hot bead sterilizer</td>
			<td style="width:107px;">Fine Science Tools</td>
			<td style="width:141px;">18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cotton tipped apllicators 6-inch</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:141px;">72310-10</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anesthesia system</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:107px;">Carl Zeiss Inc</td>
			<td style="width:141px;">Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">stage insert</td>
			<td style="width:107px;">Applied Sientific Instrumentation</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse Ox oximeter, software and sensors</td>
			<td style="width:107px;">Starr Life Sciences</td>
			<td style="width:141px;">MouseOx</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nebulizer</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaris</td>
			<td style="width:107px;">Bitplane</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mManager</td>
			<td style="width:107px;">Vale lab, UCSF</td>
			<td style="width:141px;"></td>
			<td>Open-source software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ICCD camera</td>
			<td style="width:107px;">Stanford Photonics</td>
			<td style="width:141px;">XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinning disk confocal sacan-head</td>
			<td style="width:107px;">Yokogawa Corporation</td>
			<td style="width:141px;">CSU-10b</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

By using spinning disk confocal microscopy, non-tumoral and tumoral tissues in the small intestine of ApcMin/+;ACTB-ECFP;c-fms-ECFP mice can be visualized from the serosal surface. After imaging, camera software is used to analyze and adjust the acquisition (Supplementary Figure 1). After intravenous (i.v.) injection of fluorescent 2,000 kDa dextran-rhodamine and a Ly-6G 647 conjugated antibody, blood vessels and PMNs can be detected respect...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

real-time-imaging-myeloid-cells-dynamics-apcmin-intestinal-tumors

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Immunology and Infection

9.5K vues.  INSERM U661, Functional Genomic Institute. By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. 