A special thank you is given to Kwami Ketosugbo and Emily Cowen for being volunteers for imaging.This research is funded by a grant from the National Cancer Institute /National Institutes of Health (P30-CA008748) made to the Memorial Sloan Kettering Cancer Center.

All the protocols described below follow the guidelines of the institutional human research ethics committee.
1. RCM device and imaging protocol
NOTE: There are two commercially available in vivo RCM devices: wide-probe RCM (WP-RCM) and handheld RCM (HH-RCM). The WP-RCM comes integrated with a digital dermatoscope. These two devices are available separately or as a combined unit. Below are the image acquisition protocols using the latest generation (Generation 4) of the WP-RCM and HH-RCM devices along with their clinical indications.
<ol>
	<li>Lesion selection and clinical indications
	<ol>
		<li>Look for the following types of lesions: dermoscopically equivocal pink (BCC, squamous cell carcinoma [SCC], actinic keratosis [AK], other benign lesions) or pigmented lesion (nevi and melanoma, pigmented keratinocytic lesions); a nevus that has recently changed on clinical or dermoscopy examination; inflammatory lesions to determine inflammatory patterns.</li>
		<li>Perform mapping for lentigo maligna (LM) margins to determine the extent of the lesion and mapping and selection of biopsy sites for disease with subclinical extension such as extramammary Paget&#39;s disease (EMPD) and LM.</li>
		<li>Carry out noninvasive monitoring of nonsurgical treatment such as topical drugs (imiquimod), radiation, photodynamic therapy, and laser ablation.</li>
	</ol>
	</li>
	<li>For device selection, use the WP-RCM device for lesions located on relatively flat surfaces of skin (the trunk and extremities) and the HH-RCM device for lesions on curved surfaces (the nose, earlobes, eyelids, and genitalia). 
	NOTE: Selection of the imaging device will mainly depend on the location of the lesion.</li>
	<li>For imaging, position the patient on a fully reclining chair or a flat examination table with pillows or an armrest for support and to achieve a flat imaging surface. 
	NOTE: Older generation (Generation 3) WP-RCM devices took ~30 min per lesion. Imaging a single lesion may require ~15 min with the newer generation (Generation 4) WP-RCM device currently being used in clinics. Despite the improved acquisition time, positioning the patient comfortably will ensure minimum motion artifacts and aid the acquisition of superior quality images. The following steps may help with correctly positioning the patient:</li>
	<li>To prepare for imaging, clean the lesion and surrounding skin with an alcohol wipe to eliminate any dirt, lotion, or make-up. Shave hairy skin surfaces before attaching the tissue window to avoid air bubbles that can hinder the visualization of tissue microstructures. 
	NOTE: For removing heavy cosmetics or sunscreens, clean the site with a gentle soap and water prior to cleaning with alcohol.</li>
	<li>Image acquisition using the WP-RCM device (Figure 1, Figure 2, Supplemental Figure S1, Supplemental Figure S2, and Supplemental Figure S3) 
	&#8203;NOTE: WP-RCM devices are capable of capturing stacks, mosaic, live single-framed videos, and single-framed images.
	<ol>
		<li>To attach a disposable plastic window cap to the lesion (Figure 1), position the probe perpendicular to the lesion for the best images. Refer to Figure 1A-F for an example of attachment. Add a drop of mineral oil on the center of the plastic window, carefully spreading it across the window width (Figure 1A). Remove the paper backing from the adhesive side of the plastic window. Stretch the skin gently to avoid wrinkling and attach the window. 
		NOTE: Use food-grade mineral oil that is safe and has a high viscosity. Ensure that the lesion is centered and covered in its entirety. For lesions larger than 8 mm x 8 mm, either image areas of concern based on dermoscopy or perform separate imaging sessions to cover the entire lesion.</li>
		<li>Acquiring dermoscopy images (Figure 1C,D) 
		NOTE: A dermoscopy image is acquired to serve as a guide for navigating within the lesion. The following steps should be used to ensure perfect registration between the dermoscopy image and the confocal image.
		<ol>
			<li>Hover the WP-RCM probe over the plastic window cap and approximate the best angle of insertion for the probe (Figure 1C). Locate the small, white arrow located on the side of the probe (Figure 1C) and align it with the arrow on the side of the dermoscopy camera (Figure 1C).</li>
			<li>Insert the dermoscopy camera into the plastic window cap (Figure 1D). Press the trigger on the camera to acquire an image. Remove the dermatoscope. Before starting the imaging session, ensure that the dermatoscope image covers the entire lesion surface.</li>
		</ol>
		</li>
		<li>To attach the RCM probe to the plastic disposable cap (Figure 1E,F), place a pea-sized amount of ultrasound gel inside the disposable plastic window cap (Figure 1E). Insert the probe within the cap until a sharp click is heard (Figure 1F). 
		NOTE: For the best images, insert the probe perpendicular (at a 90&#176; angle) to the plastic window. The height of the examination chair can be raised to achieve a flatter surface, reduce motion artifacts, expel air bubbles (Figure 3 and Figure 4), and ensure secure attachment to the skin.</li>
		<li>Acquiring RCM images (Figure 2, Supplemental Figure S1, and Supplemental Figure S2)
		<ol>
			<li>Use the dermoscopy image (step 5.2.) for guiding the RCM image acquisition (Supplemental Figure S1). Select the center of the lesion and identify the topmost (brightest) layer of the skin &#8212; the anucleate layer of the stratum corneum (Supplemental Figure S1).</li>
			<li>Set the imaging depth to zero at this level (Supplemental Figure S1). 
			NOTE: This depth serves as a reference point for determining the actual z-depth of subsequent layers within the lesion.</li>
			<li>Acquire a stack in the lesion&#39;s center (Figure 2 and Supplemental Figure S1) by pressing the stack icon. Select an anatomical site from the drop-down menu: face or body. Set 4.5 &#181;m step-size and 250 &#181;m depth. 
			NOTE: Begin the stacks from the stratum corneum and end at the deepest visible layers in the dermis. Supplemental Figure S1 shows an example of how to acquire a stack, while Figure 2 gives an example of a stack.</li>
			<li>Acquire a mosaic: take the first mosaic at the dermal-epidermal junction (DEJ) (Supplemental Figure S2). Identify the DEJ layer in the stack acquired and then use the mouse to select an 8 mm x 8 mm square to cover the entire lesion. Press the mosaic icon to complete the operation (Supplemental Figure S2). Acquire at least 5 mosaics at various depths: stratum corneum, stratum spinosum, suprabasal layer, DEJ, and superficial papillary dermis.</li>
			<li>Open the DEJ mosaic to guide the acquisition of the subsequent mosaics. Click on any structure on the DEJ mosaic to bring up that area on the live view imaging. Scroll down to acquire mosaics at the dermis and then up (from the DEJ) to take mosaics in the epidermis.</li>
			<li>Get the acquired mosaics evaluated by the expert RCM reader present at the bedside to identify the region of interest and take stacks. In the absence of an expert at the bedside, capture 5 stacks: one in each quadrant and one in the center of the lesion with a homogeneous pattern on dermoscopy (steps 1.5.2.). For heterogeneous lesions, acquire additional stacks to cover all the dermoscopy features. 
			NOTE: A &#34;stack&#34; (Figure 2) is a sequential collection of high-resolution, single-frame, small field of view (FOV) images (0.5mm x 0.5 mm) acquired in depth starting from the topmost layer of the epidermis to the superficial dermis (~200 &#181;m). A &#34;mosaic&#34; (Supplemental Figure S2) is a large FOV of images obtained by stitching individual 500 &#181;m x 500 &#181;m images together in &#34;X-Y&#34; (horizontal en face plane).</li>
		</ol>
		</li>
		<li>Completing an imaging session
		<ol>
			<li>Click on Done Imaging.</li>
			<li>Detach the microscope from the plastic window. Remove the plastic window by gently holding the patient&#39;s skin taut and dispose of it. Wipe off oil on the skin with an alcohol swab.</li>
			<li>Detach the protective cone surrounding the microscope lens. Clean the tip of the objective lens with an alcohol swab to remove the ultrasound gel. Dry the objective lens with a paper towel. Reattach the plastic cone to the microscope probe. 
			NOTE: Images can be read, and a report can be generated and signed at the bedside by a trained physician. In the absence of an expert reader, a remote expert can be consulted either by transferring the images via the cloud or via a live teleconfocal session26.</li>
		</ol>
		</li>
		<li>Generating a Confocal Diagnostic Evaluation report (Supplemental Figure S3)
		<ol>
			<li>Click on New Evaluation. Enter the diagnosis from the preselected options in the dropdown menu.</li>
			<li>If another imaging session is required, select images inadequate and need to be recaptured. If a descriptive diagnosis is needed, select other and describe in the free text box at the end of the form. Enter the CPT code for billing7&#160;(Supplemental Figure S3A). Select the applicable features seen during imaging from the report checklist (Supplemental Figure S3B). Select the applicable management from the checklist. 
			NOTE: No billing code is applicable for HH-RCM imaging.</li>
			<li>Click on Finish and Sign. Generate the report as a PDF and print. Get the report signed by a physician and add it to the patient&#39;s chart for billing.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Image acquisition using the HH-RCM device (Figure 5) 
	NOTE: HH-RCM devices are capable of capturing stacks, live single-framed videos, and single-framed images.
	<ol>
		<li>Encircle the lesion identified by the physician with a paper ring. Use steps detailed in section 3. for positioning the patient and cleaning the lesion site. 
		NOTE: Select the size of the paper ring (5-15 mm) based on the lesion size to define the boundary of the lesion and ensure imaging is done within the lesion. If a paper ring is not available, use paper tape to define the lesion.</li>
		<li>Remove the plastic cap covering the microscope lens. Apply a pea-sized amount of ultrasound gel to the objective lens of the HH-RCM and cover it with the plastic cap (Supplemental Figure S3A). Add a generous drop of mineral oil to the side of the plastic cap that will be touching the skin. 
		NOTE: Increase the amount of oil for very dry skin, if needed.</li>
		<li>Press the probe to the lesion site on the skin with firm pressure. Use the z-depth controls on the HH-RCM device to move up and down at various depths within the lesion (Supplemental Figure S3B). Acquire multiple single-frame images and stacks in the regions of interest. Take stacks as described in step 1.5.4.3.</li>
		<li>For large lesions where the WP-RCM device cannot be attached, take continuous videos at various layers by moving the HH-RCM probe over the entire lesion surface. Click on the video capture symbol to do so. Record the movement of blood cells within vessels, if needed. 
		NOTE: These videos can be later stitched using software to provide large FOV images similar to mosaics.</li>
		<li>Press Done Imaging after the imaging session is complete. Clean the lesion with an alcohol swab to remove the oil. Remove the ultrasound gel from the objective lens of the probe by cleaning it with an alcohol wipe and reattaching the plastic cap. 
		&#8203;NOTE: Unlike the WP-RCM device, which can be operated by a technician, the HH-RCM should be operated by an RCM reader who can interpret images in real time to navigate within the lesion and arrive at a correct diagnosis.</li>
	</ol>
	</li>
</ol>
2. Combined RCM-OCT device and imaging protocol
NOTE: There is only one prototype of the RCM-OCT device. This device has a handheld probe and can be used on all body surfaces, similar to the HH-RCM device. It acquires RCM stacks (similar to the RCM device) and OCT rasters (a video of sequential, cross-sectional images22). Both RCM and OCT images are in gray scale. RCM images have an FOV of ~200 &#181;m x 200 &#181;m, while the OCT image has an FOV of 2 mm (in width) x 1 mm (in depth). Below is the image acquisition protocol using the RCM-OCT device, along with their clinical indications. Figure 6 shows an image of the RCM-OCT device, while Figure 7 shows the software system of the RCM-OCT device.
<ol>
	<li>Lesion selection
	<ol>
		<li>Look for dermoscopically equivocal pink or pigmented lesion to rule out BCC.</li>
		<li>Assess the depth of BCC for management, and assess the residual BCC post treatment.</li>
	</ol>
	</li>
	<li>Positioning the patient for imaging: Imaging a single lesion may require up to 20 minutes with the RCM-OCT device. The device is also a handheld probe similar to the HH-RCM device and, thus, can be moved freely over the lesion. For details of patient positioning, refer to section 1.4. above.</li>
	<li>Preparation of the site for imaging: When using this probe, ensure that the boundary of the lesion is free of excessive hair and topical impurities and is clearly defined. Refer to step 1.4.1. above for more detail.</li>
	<li>Image acquisition using the RCM-OCT device (Figure 6 and Figure 7)
	<ol>
		<li>Prepare the probe similarly to the one used for the HH-RCM (steps 1.6.1-1.6.2.)</li>
		<li>Acquire images in line imaging mode and raster mode.
		<ol>
			<li>Click on imaging settings (Figure 7A). Select the line imaging mode to acquire an RCM image (cellular resolution) (Figure 7B). Set the step size to 5 &#181;m and the number of steps to 40 (Figure 7A).</li>
			<li>Click on Grab. Acquire stacks following step 1.5.4.3. Once complete, click on the Freeze button.</li>
			<li>Click on imaging settings. Select raster mode to acquire a correlative OCT video for the lesion architecture (Figure 7B). Switch to the technician tab (Figure 7C). Once complete, click on the Grab button (Figure 7A) and immediately press the save button.</li>
			<li>Acquire multiple stacks and videos based on the physician&#39;s interest.</li>
			<li>Clean the lesion and machine as described in step 1.6.5.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Ucalene Harris has no competing financial interest. Dr. Jain is a consultant on Enspectra Health Inc. Dr. Milind Rajadhyaksha is a former employee of and owns equity in Caliber ID (formerly, Lucid Inc.), the company that manufactures and sells the VivaScope confocal microscope. The VivaScope is the commercial version of an original laboratory prototype that was developed by Dr. Rajadhyaksha when he was at Massachusetts General Hospital, Harvard Medical School.

In this article, we have described protocols for image acquisition using in vivo RCM and RCM-OCT devices. Currently, there are two commercially available RCM devices: A wide-probe or arm-mounted RCM (WP-RCM) device and a handheld RCM (HH-RCM) device. It is crucial to understand when to use these devices in clinical settings. Cancer type and location are the main factors determining the selection of the device.
The WP-RCM device is well-suited for lesions on flat and gently undulating ...

Traditionally, the diagnosis of skin cancer relies on visual inspection of the lesion followed by a closer look at suspicious lesions using a magnifying lens called a dermatoscope. A dermatoscope provides subsurface information that increases sensitivity and specificity over that of visual inspection for diagnosing skin cancers1,2. However, dermoscopy lacks cellular detail, often leading to a biopsy for histopathological confirmation. The low and variable (67% to 97%) specificity of dermoscopy3 results in false positives and biopsies that turn out to show benign lesions on pathology. A biopsy is not only an invasive procedure that causes bleeding and pain4 but is also highly undesirable on cosmetically sensitive regions such as the face due to scarring.
To improve patient care by overcoming existing limitations, many noninvasive, in vivo imaging devices are being explored5,6,7,8,9,10,11,12,13,14,15,16,17,18. RCM and OCT devices are the two main optical noninvasive devices that are used for diagnosing skin lesions, especially skin cancers. RCM has acquired Current Procedural Terminology (CPT) billing codes in the USA and is being increasingly used in academic tertiary care centers and some private clinics7,8,19. RCM images lesions at near-histological (cellular) resolution. However, images are in the en-face plane (visualization of one layer of skin at a time), and the depth of imaging is limited to ~200 &#181;m, sufficient to reach the superficial (papillary) dermis only. RCM imaging relies on the reflectance contrast from various structures in the skin. Melanin imparts the highest contrast, making pigmented lesions bright and easier to diagnose. Thus, RCM combined with dermoscopy has significantly improved diagnosis (sensitivity of 90% and specificity of 82%) over dermoscopy of pigmented lesions, including melanoma20. However, due to a lack of melanin contrast in pink lesions, especially for BCCs, RCM has lower specificity (37.5%-75.5%)21. A conventional OCT device, another commonly used noninvasive device, images lesion up to 1 mm in depth within the skin and visualizes them in a vertical plane (similar to histopathology)9. However, OCT lacks cellular resolution. OCT is primarily used for diagnosing keratinocytic lesions, especially BCCs, but still has lower specificity9.
Thus, to overcome the existing limitations of these devices, a multi-modal RCM-OCT device has been built22. This device incorporates RCM and OCT within a single, handheld imaging probe, enabling the simultaneous acquisition of co-registered en-face RCM images and vertical OCT images of the lesion. OCT provides architectural detail of the lesions and can image deeper (up to a depth of ~1 mm) within the skin. It also has a larger field of view (FOV) of ~2 mm22 compared to the handheld RCM device (~0.75 mm x 0.75 mm). RCM images are used to provide cellular details of the lesion identified on OCT. This prototype is not yet commercialized and is being used as an investigational device in clinics23,24,25.
Despite their success in improving the diagnosis and management of skin cancers (as supported by the literature), these devices are not yet widely used in clinics. This is mainly due to the paucity of experts who can read these images but is also due to the lack of trained technicians who can acquire diagnostic-quality images efficiently (within a clinical time frame) at the bedside8. In this manuscript, the goal is to facilitate the awareness and eventual adoption of these devices in clinics. To achieve this goal, we familiarize dermatologists, dermatopathologists, and Mohs surgeons with images of normal skin and skin cancers acquired with the RCM and RCM-OCT devices. We will also detail the utility of each device for the diagnosis of skin cancers. Most importantly, the focus of this manuscript is to provide step-by-step guidance for image acquisition using these devices, which will ensure good-quality images for clinical use.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Crystal Plus 500FG mineral oil</td>
			<td>STE Oil Company, Inc.</td>
			<td></td>
			<td>A food grade, high viscous mineral oil used with our various devices during in vivo imaging.</td>
		</tr>
		<tr>
			<td>RCM-OCT</td>
			<td>Physical Science Inc.</td>
			<td>-</td>
			<td>A &#8220;multi-modal&#8221; combined RCM-OCT device simultaneously images skin lesions in both horizonal and vertical modes.</td>
		</tr>
		<tr>
			<td>Vivascope 1500</td>
			<td>Caliber I.D.</td>
			<td>-</td>
			<td>A wide-probe RCM (WP-RCM) device that attaches to the skin to campture in vivo devices.</td>
		</tr>
		<tr>
			<td>Vivascope 3000</td>
			<td>Caliber I.D.</td>
			<td>-</td>
			<td>A hand-held RCM (HH-RCM) device that is moved across the skin to capture in vivo images.</td>
		</tr>
	</tbody>
</table>

combining reflectance confocal microscopy with optical coherence tomography for noninvasive diagnosis of skin cancers via image acquisition

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological confirmation. While the sensitivity of dermoscopy is high, the lower specificity results in 70%-80% of the biopsies being diagnosed as benign lesions on histopathology (false positives on dermoscopy).
Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) imaging can noninvasively guide the diagnosis of skin cancers. RCM visualizes cellular morphology in en-face layers. It has doubled the diagnostic specificity for melanoma and pigmented keratinocytic skin cancers over dermoscopy, halving the number of biopsies of benign lesions. RCM acquired billing codes in the USA and is now being integrated into clinics.
However, limitations such as the shallow depth (~200 &#181;m) of imaging, poor contrast for nonpigmented skin lesions, and imaging in en-face layers result in relatively lower specificity for the detection of nonpigmented basal cell carcinoma (BCCs) &#8212; superficial BCCs contiguous with the basal cell layer and deeper infiltrative BCCs. In contrast, OCT lacks cellular resolution but images tissue in vertical planes down to a depth of ~1 mm, which allows the detection of both superficial and deeper subtypes of BCCs. Thus, both techniques are essentially complementary.
A &#34;multi-modal,&#34; combined RCM-OCT device simultaneously images skin lesions in both en-face and vertical modes. It is useful for the diagnosis and management of BCCs (nonsurgical treatment for superficial BCCs vs. surgical treatment for deeper lesions). A marked improvement in specificity is obtained for detecting small, nonpigmented BCCs over RCM alone. RCM and RCM-OCT devices are bringing a major paradigm shift in the diagnosis and management of skin cancers; however, their use is currently limited to academic tertiary care centers and some private clinics. This paper familiarizes clinicians with these devices and their applications, addressing translational barriers into routine clinical workflow.

Reflectance confocal microscopy (RCM) 
Image interpretation on RCM: 
The RCM images are interpreted in a manner that mimics the evaluation of histopathology slides. Mosaics are evaluated first to get the overall architectural detail and identify areas of concern, akin to the evaluation of histology sections on scanning magnification (2x). This is followed by zooming in on the mosaic for evaluation of the cellular details, similar to evaluating slides at high magnification (20x)...

Watch this Scientific Journal Video about Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition at JoVE.com

Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition

Here, we describe protocols for acquiring good-quality images using novel, noninvasive imaging devices of reflectance confocal microscopy (RCM) and combined RCM and optical coherence tomography (OCT). We also familiarize clinicians with their clinical applications so that they can integrate the techniques into regular clinical workflows to improve patient care.

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological ...

combining-reflectance-confocal-microscopy-with-optical-coherence

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JoVE Journal

Cancer Research

2.1K Views.  Memorial Sloan Kettering Cancer Center. Here, we describe protocols for acquiring good-quality images using novel, noninvasive imaging devices of reflectance confocal microscopy (RCM) and combined RCM and optical coherence tomography (OCT). We also familiarize clinicians with their clinical applications so that they can integrate the techniques into regular clinical workflows to improve patient care.

Video: Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...