Name: a label-free segmentation approach for intravital imaging of mammary tumor microenvironment
Uploaded: 2022-05-24T13:00:00
Description: Watch this Scientific Journal Video about A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment at JoVE.com

The authors would like to acknowledge NCI R01 CA216248, CA206458, and CA179556 grants for funding this work. We would also like to acknowledge Dr. Kevin Eliceiri and his imaging group for their technical expertise in the early development of our intravital program. We also thank Dr. Ben Cox and other members of the Eliceiri Fabrication Group at the Morgridge Institute for Research for their essential technical design during the early phases of the MIW. Dr. Ellen Dobson assisted with useful conversations about the ImageJ trainable WEKA segmentation tool. In addition, we would like to thank Dr. Melissa Skala and Dr. Alexa Barres-Heaton for the timely use of their microscope. Lastly, we would like to thank Dr. Brigitte Raabe, D.V.M, for all the thoughtful discussions and advice on our mouse handling and care.

All experiments described were approved by the University of Wisconsin-Madison&#39;s Institutional Animal Care and Use Committee. The well-being and pain management in all animal experiments is paramount. Thus, every effort is made to make sure the animal is comfortable and well-cared for at each step of the procedure.
1. Generation of the mammary imaging window (MIW)
<ol>
	<li>To construct the mammary imaging window, fabricate a 14 mm ring from surgical grade stainless stee...

4D intravital imaging is a powerful tool to investigate dynamic physiological interactions within the spatial and temporal context of the native tumor microenvironment. This manuscript provides a very basic and adaptable operational framework to compartmentalize dynamic cell interactions within the tumor mass, the adjacent stroma, or within proximity to the vascular network using only endogenous signals from second harmonic generation or NAD(P)H autofluorescence. This protocol provides a comprehensive, step-by-step metho...

The extracellular matrix (ECM) in the tumor microenvironment is known to be dynamically deposited and remodeled by multiple cell types to further facilitate disease progression1,2,3. These matrix alterations provide both mechanical and biological cues that alter cell behavior and often result in a continuing cycle of matrix remodeling4. Investigation into the dynamic, reciprocal interplay between tumor cells and the extracellular matrix is often conducted using three-dimensional (3D) in vitro culture or microfluidic systems. While these bottom-up approaches have demonstrated mechanisms of ECM remodeling5,6,7, increased proliferation8, epithelial to mesenchymal transition9,10,11,12, and tumor cell migration and invasion7, 13,14,15,16, their focus has been primarily on a few cell types (e.g., tumor cells or fibroblasts) within a homogeneous 3D matrix compared to the diversity and heterogeneity of interactions present within a physiological tissue. In addition to in vitro systems, ex vivo tumor histology can also provide some insight into these cell-cell and cell-ECM interactions17. Immunohistochemistry has the advantage of being able to analyze multiple cell types with respect to the spatially heterogeneous composition and architecture of the ECM, but the static endpoints of fixed tissue do not capture the dynamic nature of interactions between cells and the microenvironment. Intravital imaging has opened the door to interrogate diverse and dynamic interactions within the physiological&#160;context of the native tumor microenvironment.
The capabilities of intravital tumor imaging are rapidly advancing. Improvements in the design of imaging windows and surgical techniques to implant the windows have enabled long-term longitudinal tumor imaging at a variety of anatomical locations (i.e., primary tumor, lymph nodes, metastatic sites18,19,20). Moreover, the capacity of optical instrumentation to visualize and collect data in multiple dimensions (i.e., spectral, spatial fluorescence intensity, and lifetime), and at high resolution and speed (video rate) is becoming widely accessible. The improved technology provides an opportunity to explore rapid changes in cell signaling and phenotypic dynamics within a physiological environment. Lastly, the expansion of optogenetic tools and the wide array of genetic fluorescent constructs allow for the tagging of specific cell types to capture cell migration in the tumor microenvironment or cell lineage tracing during development or disease progression21,22. The use of these tools in combination with CRISPR/Cas9 technology provides researchers the opportunity to generate unique animal models in a timely manner.
While all these advances make intravital imaging an increasingly powerful method to explore dynamic and physiological cellular interactions, there is still an important need to develop strategies that provide spatial, temporal, and structural context at the tissue level to these biological interactions. Currently, many intravital imaging studies compensate for the lack of visual landmarks such as blood vessels by injecting fluorescent dyes into the vasculature or employing mouse models that exogenously express fluorescent proteins to delineate physical features. Injectable dyes and substrates like fluorescent dextrans are broadly utilized to successfully label the vasculature in intravital collections19, 23, 24. However, this approach is not without limitations. For one, it requires additional mouse manipulations and its utility is limited to short-term experiments. For longitudinal studies, fluorescent dextran can be problematic as we observe the accumulation of dextran in phagocytic cells or diffusion into the surrounding tissue over time25. Exogenous fluorescent protein incorporation into the mouse model has been presented as an alternative to fluorescent dextrans but presents limitations of its own. The availability and diversity of exogenous fluorophores within mouse models are still limited and expensive to create. Additionally, in specific models, such as PDX models, genetic manipulations are not desirable or possible. It has also been shown that the presence of fluorescent or bioluminescent proteins within cells are recognized as foreign within the mouse, and within immunocompetent mouse models, this reduces the amount of metastasis due to the response of the host immune system26,27. Lastly, exogenous fluorescent proteins or fluorescent dyes used for spatial context or to segment subsequent data often occupy prime ranges of the light spectrum that could otherwise be used to investigate the physiological interactions of interest.
The use of the intrinsic signal from the ECM or endogenous fluorescence from cells within the tissue represents a potential universal label-free means to segment intravital data for more in-depth cellular and spatial analysis. Second harmonic generation (SHG) has long been used to visualize the ECM28. With the subsequent development of important tools to aid in the characterization of fiber organization29,30,31, it is possible to characterize cell behavior relative to local ECM structure. In addition, autofluorescence from the endogenous metabolite, NAD(P)H, provides another label-free tool to compartmentalize the tumor microenvironment in vivo. NAD(P)H fluoresces brightly in tumor cells and can be used to discriminate the boundaries of the growing tumor nest from its surrounding stroma21,32. Lastly, the vasculature is an important physiological structure in the tumor microenvironment and the site of key cell-type-specific interactions33,34,35. The excitation of red blood cells (RBC) or blood plasma has been used to visualize the tumor vasculature, and using two- or three-photon excitation (2P; 3P) the measurement of blood flow rates has been shown to be possible36. However, while larger blood vessels are easily identifiable by their endogenous fluorescence signatures, the identification of subtle, variable, and less fluorescent small blood vessels requires more expertise. These inherent difficulties hinder optimal image segmentation. Fortunately, these sources of endogenous fluorescence (i.e., red blood cells and blood plasma) can also be measured by fluorescence lifetime imaging37, which capitalizes on the unique photophysical properties of the vasculature and represents a useful addition to the growing intravital toolbox.
In this protocol, a workflow for the segmentation of four-dimensional (4D) intravital imaging explicitly using intrinsic signals like endogenous fluorescence and SHG is described from acquisition to analysis. This protocol is particularly pertinent for longitudinal studies through a mammary imaging window where exogenous fluorescence may not be practical or possible, as is the case with PDX models. The segmentation principles outlined here, however, are broadly applicable to intravital users investigating tumor biology, tissue development, or even normal tissue physiology. The reported suite of analysis approaches will allow the users to differentiate cellular behavior between regions of aligned or random collagen fiber configurations, compare numbers or behaviors of cells residing in specific regions of the tumor microenvironment, and map the vasculature to the tumor microenvironment using only label-free or intrinsic signal. Together, these methods create an operational framework for maximizing the depth of information gained from 4D intravital imaging of the mammary gland while minimizing the need for additional exogenous labels.

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	<tbody>
		<tr>
			<td>#1.5 12mm round cover glass</td>
			<td>Warner Instruments</td>
			<td># 64-0712</td>
			<td>MIW construction</td>
		</tr>
		<tr>
			<td>1.0 mL syringe for SQ injection</td>
			<td>BD</td>
			<td>309659</td>
			<td>Syringe</td>
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		<tr>
			<td>20x objective</td>
			<td>Zeiss</td>
			<td>421452-988</td>
			<td>Water immersion</td>
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		<tr>
			<td>27G needle for SQ injection</td>
			<td>Covidien</td>
			<td>1188827012</td>
			<td>Needle</td>
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		<tr>
			<td>40x objective</td>
			<td>Nikon</td>
			<td>MRD77410</td>
			<td>Water immersion</td>
		</tr>
		<tr>
			<td>5-0 silk braided suture</td>
			<td>Ethicon</td>
			<td>K870</td>
			<td>Suture for MIW implantation</td>
		</tr>
		<tr>
			<td>Artificial tears gel</td>
			<td>Akorn</td>
			<td>NDC 59399-162-35</td>
			<td>Eye gel</td>
		</tr>
		<tr>
			<td>Betadine solution, 5%</td>
			<td>Fisher Scientific</td>
			<td>NC1558063</td>
			<td>Surgery antiseptic</td>
		</tr>
		<tr>
			<td>cotton-tipped applicator</td>
			<td>Fisher Scientific</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Loctite</td>
			<td>1365882</td>
			<td>MIW construction</td>
		</tr>
		<tr>
			<td>fluorescent dextran</td>
			<td>Sigma</td>
			<td>T1287-50mg</td>
			<td>intravenous labelling of vasculature</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Mckesson.com</td>
			<td>Miltex&#160;#18-782</td>
			<td>stainless, 4 inch, curved</td>
		</tr>
		<tr>
			<td>GaAsP photomultiplier tube</td>
			<td>Hamamatsu&#160;</td>
			<td></td>
			<td></td>
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			<td>heating blanket</td>
			<td>CARA 72 heating pad</td>
			<td>&#160;038056000729</td>
			<td>Temperature selectable</td>
		</tr>
		<tr>
			<td>heating chamber</td>
			<td>home built</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent lifetime handbook</td>
			<td>Becker and Hickl</td>
			<td></td>
			<td>https://www.becker-hickl.com/literature/handbooks</td>
		</tr>
		<tr>
			<td>inverted microscope base</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Akorn</td>
			<td>NDC 59399-106-01</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Liqui-Nox</td>
			<td>Fisher Scientific</td>
			<td>16-000-125</td>
			<td>MIW cleaning</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Norbrook</td>
			<td>NDC 55529-040-10</td>
			<td>Analgesic</td>
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		<tr>
			<td>Micro Hose</td>
			<td>Scientific Commodities INC.</td>
			<td>&#160;BB31695-PE/1</td>
			<td></td>
		</tr>
		<tr>
			<td>multiphoton scan head</td>
			<td>Bruker Ultima II</td>
			<td></td>
			<td>Multiphoton scanhead and imaging platform</td>
		</tr>
		<tr>
			<td>NADH FLIM filter</td>
			<td>Chroma</td>
			<td>284994</td>
			<td>ET 440/80 m-2P</td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>CVS</td>
			<td>339826</td>
			<td>Depilatory cream</td>
		</tr>
		<tr>
			<td>objective heater</td>
			<td>Tokai Hit</td>
			<td>STRG-WELSX-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>SHG/FAD filter</td>
			<td>Chroma</td>
			<td>320740</td>
			<td>ET450/40m-2P</td>
		</tr>
		<tr>
			<td>Sparkle glass cleaner</td>
			<td>Amazon.com</td>
			<td>B00814ME24</td>
			<td>Glass Cleaner for implanted MIW</td>
		</tr>
		<tr>
			<td>SPC-150 photon counting board</td>
			<td>Becker and Hickl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>surgical light</td>
			<td>FAJ</td>
			<td>B06XV1VQVZ</td>
			<td>Magnetic LED gooseneck light</td>
		</tr>
		<tr>
			<td>surgical micro-scissors</td>
			<td>Excelta</td>
			<td>366</td>
			<td>stainless, 3 inch</td>
		</tr>
		<tr>
			<td>Triple antibiotic ointment</td>
			<td>Actavis Pharma</td>
			<td>NDC 0472-0179-34</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>TV catheter</td>
			<td>Custom</td>
			<td>BD 30G needle: 305106</td>
			<td>Catheter for TV injection</td>
		</tr>
		<tr>
			<td>Two photon filter</td>
			<td>Chroma</td>
			<td>320282</td>
			<td>ET585/65m-2P</td>
		</tr>
		<tr>
			<td>two-photon laser</td>
			<td>Coherent charmeleon</td>
			<td></td>
			<td>Tunable multiphoton laser</td>
		</tr>
		<tr>
			<td>ultrasound gel</td>
			<td>Parker</td>
			<td>PKR-03-02</td>
			<td>Water immersion gel</td>
		</tr>
		<tr>
			<td>Urea crystals</td>
			<td>Sigma</td>
			<td>U5128-5G</td>
			<td>Optional: FLIM IRF</td>
		</tr>
	</tbody>
</table>

a label-free segmentation approach for intravital imaging of mammary tumor microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The installation of the MIW and basic experimental planning are the first steps in this process. This particular MIW design and protocol are more amenable to longitudinal studies19 and has been successfully utilized with both upright and inverted microscopes. In this case, an inverted microscope was used as it has resulted in greater image stability of the mammary gland with fewer breathing artifacts. In Figure 1A, we provide the dimensions of the rigid MIW and a grap...

Watch this Scientific Journal Video about A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment at JoVE.com

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Intravital imaging allows us to observe physiological interactions within the tumor microenvironment. And by using this method, specific dynamic behaviors that are observed can be compartmentalized using local tissue features. This protocol uses only an ubiquitous label-free signal from collagen fibers or autofluorescent metabolites to segment the TME, thereby minimizing the amount of manipulations to the mouse.This method is broadly applicable to any intravital system where understanding dynamic interactions relative to extracellular matrix structures or vascular structures is required. Demonstrating this procedure will be Dave Inman, senior research specialist, and Erica Hoffmann, a graduate student from the laboratory. Start preparing a mammary imaging window cover glass by soaking a 1.5 12 millimeter round cover glass in 100%ethanol for 10 minutes, then dry the cover glass under a heat lamp and secure the glass to the metal mammary imaging window frame using cyanoacrylate adhesive.Cure the adhesive overnight. The next day, use an acetone soaked swab to clean the assembled mammary imaging window of the excess adhesive before submerging the mammary imaging window in 70%ethanol for at least 10 minutes to assist with the cleansing. After drying, store the cleaned mammary imaging window in a sterile Petri dish.Before beginning the surgery for the window implantation, autoclave surgical tools and sanitize surfaces with 70%ethanol. Prepare a sanitized tabletop for the surgery with a warming blanket covered with a sterile field. Set the warming blanket such that the temperature measured on top of the sterile field is 40 degrees Celsius.Use auxiliary cold lighting and magnifying glasses for the surgical procedure. Wear PPE consisting of a sterile single-use lab coat, surgical sleeves, gloves, eye protection, and face mask. Next, remove the fur of the anesthetized mouse at the fourth inguinal mammary gland with a depilatory cream and rinse the surgical site with a sterile water soaked gauze.Then prepare the depilated surgical site for surgery by sanitizing the skin surface with three alternating Betadine and ethanol scrubs. When done, gently lift the skin over mammary gland number four using forceps. Once the skin is pulled away from the body wall, remove a one millimeter section of the dermal layer at the tip of the forceps with surgical microscissors.Without cutting the underlying gland, create a 10 millimeter incision and then release the mammary gland from the dermal layer with the gentle movement of the forceps. Add PBS to cover the exposed gland. Use a 5-0 silk braided suture to create a purse string suture along the periphery of the opening, then insert an edge of the mammary imaging window so that the dermal layer engages into the receiving notch of the mammary imaging window.While stretching the epithelium at the opposite end of the mammary imaging window, push the metal mammary imaging window into place such that the dermal layer fully engages the receiving notch around the entire mammary imaging window circumference. Then cinch the purse string to draw the dermal layer into the notch and tie off the layer to secure the window. Apply a topical antibiotic to the dermal layer at the mammary imaging window and continuously monitor the mouse.After regaining sufficient consciousness to maintain sternal recumbency, house the mammary imaging window implanted mouse separately on soft bedding with an igloo placed in the cage and allow the mouse to recover for 48 hours before imaging. For the imaging, use a forced air system set to 30 degrees Celsius. Use an additional objective heater to avoid drift in Z focus and allow the system to come to equilibrium at 30 degrees Celsius for at least one hour before imaging.Then set up a heating chamber on the microscope stage. After confirming the anesthesia with the toe pinch method, add an eye ointment to the mouse. To maintain proper hydration, inject 0.5 milliliters of PBS subcutaneously every two hours for the duration of the imaging session.Apply a water-based gel instead of water to the objective and clean the outside of the mammary imaging window glass with a cotton applicator and glass cleaner before transferring the mouse to the pre-warmed microscope stage. After laying the mouse on the microscope stage, press the collar of the mammary imaging window into a 14 millimeter receiving hole in the stage insert to stabilize the images and fit the isoflurane hose. Then bring the imaging field into focus using the microscope oculars and Brightfield illumination, observing vasculature with blood flow.Check the stability of the field of view. If breathing movement artifacts are present, apply gentle compression to the backside of the gland with a small foam block and a cincture-like piece of adhesive tape. After compression is applied, verify that blood flow is maintained throughout the field of view.Once the mouse is sedated and securely positioned on the inverted microscope stage, start locating regions of interest. Using a light source directed at the mammary imaging window, use the oculars of the microscope to identify potential areas for investigation. The focus should be on seeing vasculature and blood flow.Add and save the XY positions in the software. When the appropriate power levels are set, set up the Z stack and observe the appearance of abundant collagen fibers at 20 to 50 micrometers beneath the glass surface of the mammary imaging window. Collagen will become less prevalent as the microscope sections deeper into the tumor.The voids in the second-harmonic generation or SHG reveal the location of tumor masses. Set the top Z slice beneath the layer of solitary cells with the first collagen fibers appear at 50 to 100 microns. Set the bottom Z slice at 250 micrometers where the fibers fade out and the poor signal dominates.Then repeat the procedure for all XY positions saved. Once the Z stack range is set, increase the dwell time to eight microseconds and optimize the power and detector settings. Optimize the power levels needed to excite the tissue for each experiment and use powers up to 90 milliwatts at 750 nanometers or 70 milliwatts at 890 nanometers at the back aperture of the objective.Next, start with 10-minute intervals between collection points for most intravital migration movies and adjust the time intervals according to experimental goals. If signs of phototoxicity like cell blebbing or rapidly increasing autofluorescence and excessive photobleaching are observed, reduce laser power or increase timelapse intervals as the conditions indicate. For fluorescence lifetime imaging or FLIM of NADPH, start preview scanning and adjust the laser power until the readout of the constant fraction discriminator or CFD is between 10 to the fifth and 10 to the sixth.Exceeding CFD beyond 10 to the sixth result in photon pileup and poor overall results. Once the power level is set, set the integration time between 90 to 120 seconds and start the FLIM collection to acquire photons from the field of view for the allotted time. A typical field of view within a label-free mammary tumor demonstrated abundant collagen fibers near the window and a decrease in abundance deeper into the tumor.The use of fluorescence lifetime or NADPH aided in the identification of the vasculature. Some dark regions in the images were tumor folds or the butting up of adjacent tumor lobes. To identify vasculature, a composite image using NDPH FLIM was validated by comparing maximum intensity projections of the intravital stack before and after tail vein injection of fluorescent dextran.The representative analysis shows the quantification of four mice and the fields of view. To delineate the boundaries of the label-free tumor masses, NADPH autofluorescence was used. The images reported the metabolic signatures of the cells and the location of blood vessels.The AFD autofluorescence also identified the macrophages. With the segmentation scheme, the label-free tumor was segmented into compartments for the tumor nest, stroma, and vasculature using only SHG and NADPH autofluorescence. Additionally, the stroma and collagen fibers can also be classified into local regions of the aligned fibers.The most important thing to remember for this protocol is to make sure that the mouse is properly sedated and restrained so that the field of view remains stable for imaging.

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