The authors would like to thank S. Burchielli (FTGM, Pisa) for her assistance with animal procedures. This work was supported by ISPRO-Istituto per lo Studio la Prevenzione e la Rete Oncologica institutional funding to LP; MFAG #17095 awarded by AIRC-Associazione Italiana Ricerca sul Cancro to&#160;LP.

All methods described here have been approved by the Italian Ministry of Health (animal protocols #754/2015-PR and #684/2018-PR).
1. Melanoma induction
NOTE: Six-week-old Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;mice [B6.Cg-Braftm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ (Braf/Pten)] were used in this study (see the Table of Materials).
<ol>
	<li>Treat the mice with 4-hydroxytamoxifen (4-HT) by applying 3 &#181;L of 5 mM 4-HT on ~1 cm2 of shaved skin of the upper back, as described previously11,16,17, for 3 days in a row. 
	NOTE: This will activate the Cre enzyme and cause a switch from wt Braf to BrafV600E and the loss of Pten. These two hits are enough to induce melanoma formation.</li>
	<li>Observe that&#160;primary tumors develop at the site of the skin painting in 2-3 weeks and reach a volume of 50-100 mm3 in 4 weeks. Also, observe metastases to other skin sites, lymph nodes, and lungs at this time point (t0).</li>
	<li>Use calipers to measure the volume of the primary tumor and of subcutaneous metastases, and ultrasound imaging to measure the volume of the inguinal lymph nodes. Repeat these measurements after one week (t1, 5 weeks after skin painting) and after two weeks (t2, 6 weeks after skin painting).</li>
	<li>At the last time point, euthanize the mice by overdosing with gaseous sevorane.</li>
	<li>Analyze the primary tumor and lymph nodes by visual inspection, then excise them for histological studies, as reported in16.</li>
</ol>
2. Imaging procedure
<ol>
	<li>Place the mouse in an induction chamber for gas anesthesia and supply 3% isoflurane in pure oxygen until the animal is fully anesthetized. Verify the depth of anesthesia by lack of response to paw pinch.</li>
	<li>Transfer the animal to a heated board - a constitutive part of the UHFUS imaging station - holding the animal in a supine position. Use a rectal probe lubricated with petroleum jelly to measure the body temperature. Adjust the board temperature to maintain the mouse&#39;s body temperature in the physiological range (36 &#177; 1&#176;C).</li>
	<li>Moisten the mouse&#39;s eyes with vet ointment to prevent dryness during anesthesia. Supply narcotic gas (1.5% isoflurane in pure oxygen) through a mouse&#39;s nose mask. Adjust the percentage of isoflurane to maintain the correct depth of anesthesia.</li>
	<li>Coat the fore and hind paws with conductive paste and tape them to the ECG plate electrodes embedded in the board. Check that the physiological parameters (heart rate, respiration signal, and core body temperature) are correctly acquired and displayed.</li>
	<li>Remove hair from both inguinal areas by applying a depilatory agent and coat them with an acoustic coupling medium.</li>
	<li>Clamp the UHFUS linear probe (40 MHz center frequency) into a specialized 3D motor embedded in the UHFUS imaging station, allowing automated and stepwise movement of the probe.</li>
	<li>Properly orient and adjust the position of the ultrasound probe to obtain short-axis images of the inguinal lymph node (left/right), and place the region of interest in the focal zone.</li>
	<li>Scan the entire volume of the inguinal lymph node as a sequence of 2D B-mode images, as described previously18. Acquire images at multiple levels of the lymph node by linear movement of the transducer with step sizes on a micrometer scale, to generate 3D data in terms of automatically respiration- and cardiac-gated cine loops.</li>
	<li>Set the image recording with the following parameters: scan distance ranging between 2 and 5 mm (depending on lymph node size); step size 44 &#181;m, with an outcome of 46-114 scan steps/lymph node slices and an acquisition time of 1-3 min per animal. Digitally store the acquired images in raw format (DICOM) for further offline analyses.</li>
	<li>At the end of the imaging session, discontinue the gas anesthesia and allow the animal to recover on the heating board in sternal recumbency. Take care of the animal until it has regained sufficient consciousness to maintain the prone position.</li>
</ol>
3. Post-processing of ultrasound images
<ol>
	<li>Open the dataset of DICOM 3D images of the left/right inguinal lymph node in the software platform for ultrasound image post-processing.</li>
	<li>Segmentation:
	<ol>
		<li>Select Multi-slice Method to visualize both the current frames and thumbnails of all frames corresponding to each image captured during the 3D acquisition.</li>
		<li>Select the thumbnail of the first frame to load it into the contouring view. In the contouring view, left-click on the mouse to drop points along the border of the lymph node. Once the desired number of points has been set (range 10-15), right-click to complete the contour.</li>
		<li>After the first contour is completed, use the thumbnail view to select the next image for contouring. If required, skip over several images (average of 3 frames) between contours to reduce the number of manual traces needed for each 3D volume. 
		NOTE: The software platform for ultrasound image post-processing will automatically generate contours between manual traces, thereby reducing the time of analysis.</li>
		<li>Repeat this process until the entire volume has been outlined. Once complete, click on Finish.</li>
	</ol>
	</li>
	<li>Generation of the 3D wireframe and volume measurement:
	<ol>
		<li>While in the 3D mode window workspace, click on the Volume measurement icon beneath the image display area to activate the surface view. 
		NOTE: The surface view creates a compilation view that maps the user-generated volume to the acquired image. The surface view can be rotated into any desired position.</li>
		<li>Take note of the volume measurement listed in the lower left-hand corner of the cube view. 
		NOTE: Segmentation and 3D volume generation can also be obtained using custom-developed software and/or freely available/commercial software for general image processing. Starting from manual segmentation, the software should provide a mathematical and/or pixel-level description of the lymph node contours. These contours would be combined in a 3D space to render the external surface of lymph nodes. All steps described in the imaging procedure and the post-processing of ultrasound images are summarized in Figure 2.</li>
	</ol>
	</li>
</ol>

<ol>
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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=Prospective+cohort+study+of+ultrasound+surveillance+of+regional+lymph+nodes+in+patients+with+intermediate-risk+cutaneous+melanoma.">Prospective cohort study of ultrasound surveillance of regional lymph nodes in patients with intermediate-risk cutaneous melanoma.</a> British Journal of Surgery. 106 (6), 729-734 (2019).</li><li>Ipenburg, N. A., Thompson, J. F., Uren, R. F., Chung, D., Nieweg, O. 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M., Bosenberg, M. W., Phillips, W., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+AKT+dependency+displayed+by+mouse+models+of+BRAFV600E-initiated+melanoma.">Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma.</a> Journal of Clinical Investigation. 123 (12), 5104-5118 (2013).</li><li>Hooijkaas, A. I., Gadiot, J., vander Valk, M., Mooi, W. J., Blank, C. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+BRAFV600E+in+an+inducible+murine+model+of+melanoma.">Targeting BRAFV600E in an inducible murine model of melanoma.</a> American Journal of Pathology. 181 (3), 785-794 (2012).</li><li>Steinberg, S. 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T., Rutkowski, J. L., Feuerstein, G. Z., Wang, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+2-and+3-dimensional+assessment+of+tumor+volume+and+vascular+density+by+ultrasonography+in+a+transgenic+mouse+model+of+mammary+carcinoma.">Correlation between 2-and 3-dimensional assessment of tumor volume and vascular density by ultrasonography in a transgenic mouse model of mammary carcinoma.</a> Journal of Ultrasound in Medicine. 29 (4), 587-595 (2010).</li></ol>

The authors have nothing to disclose.

The data obtained in this study attest the ability of ultrasound imaging to monitor the metastatic involvement of inguinal lymph nodes of the Braf/Pten mouse model of metastatic melanoma. As shown previously16, this technique is especially useful to assess the efficacy of drug treatment. This is because it allows the monitoring of the change in lymph node volume in the same animal over time, by comparing the measurements collected at t1 and&#160;t2 with those colle...

Melanoma is an aggressive form of skin cancer that often spreads to other skin sites (subcutaneous metastases), as well as to lymph nodes, lungs, liver, brain, and bones1. In the last decade, new drugs have been introduced into clinical practice and have contributed to improving the life expectancy of metastatic melanoma patients. However, limitations remain, including variable time to and degree of response, severe side effects, and the insurgence of acquired resistance1. Therefore, it is crucial to detect metastatic spreading at its early stages, i.e., when it gets to the local lymph nodes.
A biopsy of the local lymph nodes (sentinel lymph nodes) is usually performed to check for the presence of melanoma cells. However, ultrasound imaging is taking hold as a non-invasive method of detecting metastatic involvement, as it outperforms clinical evaluation and can help avoid an unnecessary biopsy2,3,4. Furthermore, ultrasound imaging seems appropriate for lymph node surveillance, especially in the case of advanced age and/or comorbidities5,6. The features that are detected by ultrasound analysis and allow the differentiation between normal and metastatic lymph nodes comprise increased size (volume), change of shape from oval to round, irregular margin, altered echogenic pattern, and altered (increased) vascularization7.
Tyr::CreER+,BrafCA/+,Ptenlox/lox genetically engineered mice (Braf/Pten mice) have recently been made available to the scientific community as a tissue-specific and inducible model for metastatic melanoma8. In this animal model, primary tumors develop very quickly: they become visible within 2-3 weeks after the induction of the switch from wild-type (wt) Braf to BrafV600E and of the loss of Pten, while they reach a volume of 50-100 mm3 within 4 weeks. In the following 2 weeks, the growth of the primary tumor is accompanied by a progressive increase in metastatic burden in other skin sites, lymph nodes, and lungs.
Braf/Pten mice have been extensively used for multiple purposes including the dissection of signaling pathways involved in melanomagenesis9,10, the identification of melanoma cells of origin11,12,13, and the testing of new therapeutic options in terms of both targeted therapy and immunotherapy8,14,15,16. Specifically, we used Braf/Pten mice to demonstrate that attenuated Listeria monocytogenes (Lmat) works as an anti-melanoma vaccine. When systemically administered in the therapeutic setting, Lmat is not associated with overall toxicity as it selectively accumulates at tumor sites. Furthermore, it causes a remarkable decrease in primary melanoma mass and a reduction in metastatic burden in the lymph nodes and lungs. At the molecular level, Lmat causes apoptotic killing of melanoma cells, which is due, at least in part, to non-cell-autonomous activities (recruitment on-site of CD4+ and CD8+ T lymphocytes)16.
When Braf/Pten mice are used for melanoma modeling, the growth of primary tumors and subcutaneous metastases can be monitored by caliper measurements. However, the involvement of lymph nodes and lungs needs to be investigated using an alternative technique, possibly a non-invasive one that allows researchers to follow the same animal over time. This paper describes the use of ultrasound imaging (Figure 1), coupled with a subsequent 3D volumetric analysis of the obtained data, for the longitudinal monitoring of the increase in size (volume) of inguinal lymph nodes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-hydroxytamoxifen</td>
			<td>Merck</td>
			<td>H6278</td>
			<td>drug used for tumor induction</td>
		</tr>
		<tr>
			<td>B6.Cg-Braftm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ (Braf/Pten) mice</td>
			<td>The Jackson Laboratory</td>
			<td>013590</td>
			<td></td>
		</tr>
		<tr>
			<td>Blu gel</td>
			<td>Sooft Ialia</td>
			<td></td>
			<td>ophthalmic solution gel</td>
		</tr>
		<tr>
			<td>BRAFV600E antibody</td>
			<td>Spring Bioscience Corporation</td>
			<td>E19290</td>
			<td></td>
		</tr>
		<tr>
			<td>IsoFlo (isoflorane)</td>
			<td>Zoetis</td>
			<td></td>
			<td>liquid for gaseous anaesthesia</td>
		</tr>
		<tr>
			<td>MLANA antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>M2-7C10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma gel</td>
			<td>Parker</td>
			<td></td>
			<td>electrode gel</td>
		</tr>
		<tr>
			<td>Transonic gel clear</td>
			<td>Telic SAU</td>
			<td></td>
			<td>ultrasound gel</td>
		</tr>
		<tr>
			<td>Veet</td>
			<td>Reckitt Benckiser IT</td>
			<td></td>
			<td>depilatory cream</td>
		</tr>
		<tr>
			<td>Compact Dual Anesthesia System</td>
			<td>Fujifilm, Visualsonics Inc.</td>
			<td></td>
			<td>Isoflurane-based anesthesia system equipped with nose cone and induction chamber</td>
		</tr>
		<tr>
			<td>MX550S</td>
			<td>Fujifilm, Visualsonics Inc.</td>
			<td></td>
			<td>UHFUS linear probe</td>
		</tr>
		<tr>
			<td>Vevo 3100</td>
			<td>Fujifilm, Visualsonics Inc.</td>
			<td></td>
			<td>UHFUS system</td>
		</tr>
		<tr>
			<td>Vevo Imaging Station</td>
			<td>Fujifilm, Visualsonics Inc.</td>
			<td></td>
			<td>UHFUS imaging station and Advancing Physiological Monitoring Unit endowed with heated board</td>
		</tr>
		<tr>
			<td>Vevo Lab</td>
			<td>Fujifilm, Visualsonics Inc.</td>
			<td></td>
			<td>software platform for ultrasound image post-processing</td>
		</tr>
	</tbody>
</table>

analysis of lymph node volume by ultra-high-frequency ultrasound imaging in the braf/pten genetically engineered mouse model of melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

After skin painting of Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;mice with 4-HT, Cre activity is induced, due to which there is a switch at the genomic level from wt Braf to BrafV600E, while Pten is lost (Figure 3A). In 2-3 weeks, mice develop on-site primary tumors with 100% penetrance. After four weeks from 4-HT treatment (t0), primary tumors reach a volume of 50-100 mm3, and their growth can be measured by calipers for an additiona...

Watch this Scientific Journal Video about Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma at JoVE.com

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

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2.8K Views.  National Research Council (IFC-CNR). Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Video: Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been recommended for low-risk endometrial cancer (EC) patients by authoritative international guidelines, but it has not been performed broadly in China and worldwide. This work aims to describe detailed SLN mapping and biopsy procedures to promote the clinical application. SLN mapping and postoperative pathologic ultrastaging were conducted in a patient with low-risk EC using indocyanine green (ICG) dye to track the SLNs under laparoscopy and resecting them completely for ultrastaging. In conclusion, this protocol describes details of ICG injection, and SLN mapping and biopsy in EC patients based on the experiences gained during clinical practice.

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been ...

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...