The authors have no acknowledgments.

This study uses the EVIS Exera II endoscopy tower with a BF-UC180F EBUS endoscope (Figure 1) to demonstrate the scope and the Surgical Science Simulator (ENDO mentor suite) with the GI-Bronch Mentor software from Simbionix, Essential EBUS Case 6, when performing the EBUS procedure in the simulation-based setting. No patients are included in the study as the entire procedure is performed on the Surgical Science Simulator (ENDO mentor suite). Prior to the EBUS procedure, a complete bronchoscopy is performed using a regular bronchoscope to make sure the bronchial tree has been visualized systematically and to identify key anatomical positions where the underlying lymph node stations must be located (Figure 2).
1. Handling of the endoscope
NOTE: The EBUS endoscope is handled similarly to the bronchoscope. However, it is important to note that contrary to the bronchoscope, the EBUS endoscope gives an oblique angle view as the ultrasound transducer reduces the visibility (Figure 3).
<ol>
	<li>Hold the endoscope in the left hand with the left thumb on the steering lever.</li>
	<li>Hold the distal end of the endoscope in the right hand and enter the trachea through the nasal or oral cavity. When the vocal cords are visualized at the bottom of the picture (Figure 3B), administer 2 mL of 2% lidocaine twice via the simulator by pressing the appropriate button on the screen, and pass the vocal cords with care.</li>
	<li>Administer a further 2 mL of 2% lidocaine in the trachea as well as in the right and left main bronchus, respectively.</li>
</ol>
2. Anatomy
<ol>
	<li>After having inspected the bronchial tree, retract the bronchoscope and change to the EBUS scope. Turn on the ultrasound transducer and locate the six anatomical EBUS landmarks in the order mentioned below.
	<ol>
		<li>Locate Landmark 1 = station 4L
		<ol>
			<li>Locate station 4L at the left side of the trachea, just cranial to the carina. To find station 4L, turn the endoscope counterclockwise in the trachea and locate it between the arch of the aorta and the left pulmonary artery, sometimes referred to as &#34;the Mickey Mouse window&#34; (Figure 4).</li>
		</ol>
		</li>
		<li>Locate Landmark 2 = Station 7
		<ol>
			<li>Locate station 7 between the right pulmonary artery and the left atrium below the carina. Place the EBUS scope in the right or the left main bronchus and turn the endoscope facing medially (Figure 5).</li>
		</ol>
		</li>
		<li>Locate Landmark 3 = Station 10L
		<ol>
			<li>Locate station 10L adjacent to the left main bronchus cranial to the left upper lobe. Place the endoscope in the left main bronchus or the left upper lobe and look upwards. The upper border of the left pulmonary artery forms the boundary between stations 4L and 10L (Figure 6).</li>
		</ol>
		</li>
		<li>Locate Landmark 4 = Station 10R
		<ol>
			<li>Locate station 10R on the lateral wall of the right main bronchus, just caudal to the inferior border of the azygos vein. The upper border is the lower border of the azygos vein. Place the endoscope in the right main bronchus or the right upper lobe bronchus and look upwards (Figure 7).</li>
		</ol>
		</li>
		<li>Locate Landmark 5 = The azygos vein
		<ol>
			<li>To find the azygos vein, retract the endoscope slightly cranially and turn the transducer clockwise in the trachea. Turn the transducer counterclockwise to visualize the azygos vein draining into the superior vena cava (Figure 8).</li>
		</ol>
		</li>
		<li>Locate Landmark 6 = Station 4R
		<ol>
			<li>To find station 4R, retract the endoscope further cranially from the azygos vein and turn the transducer clockwise in the trachea. Locate station 4R to the right or in front of the trachea above the lower border of the azygos vein, which marks the border between station 10R and 4R (Figure 9).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After having located the six landmarks, look for other lymph node stations, i.e., stations 2R, 2L, 11R, and 11L, and other structures of clinical importance. At least stations 4L, 7, and 4R should be biopsied3.</li>
	<li>When the relevant lymph node is localized, ask the assistant for the biopsy equipment. The biopsy equipment includes a sheath protecting the needle, connected to a handle that can be locked onto the endoscope. Inside the sheath is the needle, and inside the needle is the stylet. When inserting the needle into the working channel, keep the steering lever in a neutral position as shown in the video to avoid damage to the endoscope. 
	NOTE: The needle used here comes with the simulator. However, the recommended needle size for this procedure is 21 G.</li>
	<li>Adjust the sheath so it is visible at the end of the endoscope; however, no more than 1-2 mm.</li>
	<li>Turn the transducer towards the bronchial wall so the lymph node is visualized on the left side of the ultrasound picture. Now, perform the biopsy.</li>
	<li>After puncturing the lymph node with the needle, ask the assistant to remove the stylet and then apply suction to the needle by pressing the appropriate button on the screen. The needle must be moved back and forward several times.</li>
	<li>Remove the suction and retract the needle tip while inside the sheath. Ensure that the distal end of the endoscope is not flexed and remains in a neutral position to prevent damage to the endoscope. Each lymph node must be punctured at least three times10,11.</li>
	<li>After the final biopsy, check for bleeding. Inspect the place for biopsy with the white light view and stay for a few seconds. If no bleeding is observed, retract the endoscope.</li>
</ol>

Simulation-Based Training of EBUS-TBNA

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	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer.">Cancer.</a> , (2022).</li><li>Kutob, L., Schneider, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+staging.">Lung cancer staging.</a> Surgical Pathology Clinics. 13 (1), 57-71 (2020).</li><li>Vilmann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+endobronchial+and+esophageal+endosonography+for+the+diagnosis+and+staging+of+lung+cancer:+European+Society+of+Gastrointestinal+Endoscopy+(ESGE)+Guideline,+in+cooperation+with+the+European+Respiratory+Society+(ERS)+and+the+European+Society+of+Thoracic+Surgeons+(ESTS).">Combined endobronchial and esophageal endosonography for the diagnosis and staging of lung cancer: European Society of Gastrointestinal Endoscopy (ESGE) Guideline, in cooperation with the European Respiratory Society (ERS) and the European Society of Thoracic Surgeons (ESTS).</a> Endoscopy. 47 (6), 545-559 (2015).</li><li>Konge, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulator+training+for+endobronchial+ultrasound:+a+randomised+controlled+trial.">Simulator training for endobronchial ultrasound: a randomised controlled trial.</a> European Respiratory Journal. 46 (4), 1140-1149 (2015).</li><li>Liam, C. K., Lee, P., Yu, C. 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M., 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=Systematic+and+combined+endosonographic+staging+of+lung+cancer+(SCORE+study).">Systematic and combined endosonographic staging of lung cancer (SCORE study).</a> The European Respiratory Journal. 53 (2), 1800800 (2019).</li><li>Andersen, A. G., 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=Preparing+for+reality:+A+randomized+trial+on+immersive+virtual+reality+for+bronchoscopy+training.">Preparing for reality: A randomized trial on immersive virtual reality for bronchoscopy training.</a> Respiration. 102 (4), 316-323 (2023).</li><li>Naur, T. M. H., Nilsson, P. M., Pietersen, P. I., Clementsen, P. 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A., 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=British+Thoracic+Society+guideline+for+diagnostic+flexible+bronchoscopy+in+adults:+accredited+by+NICE.">British Thoracic Society guideline for diagnostic flexible bronchoscopy in adults: accredited by NICE.</a> Thorax. 68 (Suppl 1), 1-44 (2013).</li><li>Nilsson, P. M., Naur, T. M. H., Clementsen, P. F., Konge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulation+in+bronchoscopy:+current+and+future+perspectives.">Simulation in bronchoscopy: current and future perspectives.</a> Advances in Medical Education and Practice. 8, 755-760 (2017).</li><li>Cold, K. M., Clementsen, P. 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The authors have nothing to disclose.

We hereby propose a systematic approach to the EBUS-TBNA procedure by splitting the anatomy into six landmarks to help guide the endoscopist through the bronchial maze. Furthermore, we demonstrate how to perform needle aspiration in a systematic way possible to repeat every time to standardize the procedure.
Even though the simulation-based setting is a safe environment, the endoscopist should be aware of some critical steps in the procedure. Initially, it is important to know the oblique angl...

Lung cancer is one of the most common cancers worldwide with 2.21 million cases in 2020, and the most frequent cause of cancer death with 1.80 million deaths in 20201. As with most cancers, fast and accurate diagnosis of lung cancer is crucial to be able to offer the best treatment, which in cases with a localized disease with no or little spreading to mediastinal lymph nodes can be surgical removal of the tumor. In order to be able to confirm or invalidate the suspicion of malignancy and to determine the Tumor-Node-Metastasis (TNM)-classification if lung cancer is confirmed2, it is extremely important to have good and representative biopsies from the suspected tumor or lymph nodes.
Among the invasive techniques, flexible bronchoscopy combined with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) plays a key role3. However, it is a complex technical procedure, and the success is dependent on the competence of the operator4. Anatomic orientation can easily be lost if the endoscopist does not know the anatomy of the mediastinum. Knowledge of endosonographic anatomy and its relation to the TNM lung cancer classification system is therefore crucial. In the case of lung cancer, if no tumor cells are found in any lymph node stations, the disease is classified as N0 disease and is often operable and thus, potentially curable. In the case of a right-sided lung tumor, the disease is classified as N1 disease if tumor cells are solely found in station 10R and could be operable and thus potentially curable. However, if tumor cells are found in station 4R, the disease is classified as N2 disease, and the patient can only be offered life-prolonging chemotherapy5. Three borders should therefore be remembered as they are important for treatment and prognosis.
(i)The left border of the trachea is the border between stations 4R and 4L. 
(ii)The upper border of the left pulmonary artery is the border between stations 4L and 10L. 
(iii)The lower border of the azygos vein is the border between stations 4R and 10R6.
To be qualified to perform EBUS-TBNA in the diagnostic process of possible lung cancer, it is therefore essential that EBUS-TBNA is thoroughly trained in a simulator-based setting based on a structured training curriculum before being performed on patients. Therefore, a stepwise approach relying on the six anatomical landmarks is used in the EBUS-certified training program offered by the European Respiratory Society (ERS)7.
We demonstrate the stepwise structured guide in a simulation-based setting at Copenhagen Academy for Medical Education and Simulation (CAMES), Denmark8, on how to perform EBUS-TBNA with the EBUS endoscope relying on the six anatomical landmarks9 as a guide.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EVIS Exera II endoscopy tower with a BF-UC180F EBUS endoscope</td>
			<td>Olympus</td>
			<td>https://medical.olympusamerica.com/products/bf-uc180f-ebus-bronchoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>ENDO mentor suite</td>
			<td>Surgical Science</td>
			<td>https://simbionix.com/endo-mentor-suite/</td>
			<td>Surgical Science Simulator</td>
		</tr>
		<tr>
			<td>GI-Bronch Mentor software</td>
			<td>Simbionix</td>
			<td>https://simbionix.com/simulators/gi-mentor/</td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic endobronchial ultrasound - the six landmarks approach

Lung cancer is the leading cause of cancer mortality globally. To ensure the correct diagnosis and staging in relation to treatment options, it is crucial to obtain valid biopsies from suspected tumors and mediastinal lymph nodes and accurate identification of the mediastinal lymph nodes regarding the Tumor-Node-Metastasis (TNM)-classification. Flexible bronchoscopy combined with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is essential in the workup and diagnosis of patients suspected of lung cancer. EBUS-TBNA from mediastinal lymph nodes is a technically difficult procedure and has been identified as one of the most important procedures that should be integrated into a simulation-based training program for invasive pulmonologists. More specific guidelines that govern training in EBUS-TBNA are needed to meet this demand. We hereby propose a systematic, stepwise approach with specific attention to six landmarks that support the endoscopist when navigating through the bronchial maze. The stepwise approach relying on the six landmarks is used in the EBUS-certified training program offered by the European Respiratory Society (ERS).

The above-mentioned structured approach to an EBUS-TBNA procedure has been taught at CAMES since 2016 as part of the EBUS-certified training program offered by the European Respiratory Society (ERS)7. The 6 landmarks approach is based on a validated assessment tool for measuring competency in EBUS-guided transbronchial needle aspiration4. By performing EBUS-TBNA in a structured way, as shown above, no important lymph nodes will be missed, and the diagnostic accuracy will be...

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Author Spotlight: Demonstrating Systematic Endobronchial Ultrasound to New Endoscopists 

Endobronchial ultrasound-guided sampling using transbronchial needle aspiration plays a key role in staging and diagnosing lung cancer. We propose a systematic stepwise approach dividing the procedure into six landmarks that should be taught to new operators.

Systematic Endobronchial Ultrasound - The Six Landmarks Approach

Lung cancer is the leading cause of cancer mortality globally. To ensure the correct diagnosis and staging in relation to treatment options, it is crucial ...

systematic-endobronchial-ultrasound-the-six-landmarks-approach

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Cancer Research

1.7K Views.  Rigshospitalet, University of Copenhagen. Endobronchial ultrasound-guided sampling using transbronchial needle aspiration plays a key role in staging and diagnosing lung cancer. We propose a systematic stepwise approach dividing the procedure into six landmarks that should be taught to new operators. 

Video: Author Spotlight: Demonstrating Systematic Endobronchial Ultrasound to New Endoscopists 

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

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 ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

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 ...

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 ...

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.

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 ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

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 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 ...