This work was supported in part by grants from the National Natural Science Foundation of China (81772777), Shanghai Science and Technology Commission Medical Guidance Project (18411963700), Clinical Research Plan of SHDC (No. SHDC2020CR4079); Shanghai Pujiang Talents Project (17PJ1401400). We thank the timely help given by Fenghua Ma from radiology department for MR images and Chao Wang from pathology department for pathologic images from Obstetrics and Gynecology Hospital of Fudan University.

All surgery procedures related to the patient described here were approved by the Ethics Committees of Obstetrics &#38; Gynecology Hospital of Fudan University in Shanghai, China. Informed consent was acquired from the patient.
1. Indications for SLN mapping with ICG in EC patients
<ol>
	<li>Ensure that patients are clinically diagnosed with primary EC confined to the uterus with low-risk factors (grade 1 or 2 endometrioid carcinomas, pre-surgical endometrial lesion &#8804;2 cm, and myometrial invasion &#60;50%).</li>
	<li>Ensure that patients are not allergic to the developer (ICG).</li>
	<li>Ensure that patients have not experienced retroperitoneal lymph node dissection for any reason before the disease.</li>
	<li>Ensure that the patients understand the SLN surgical procedures and have signed the surgical consent forms.</li>
	<li>Ensure that the patients cannot perform systematic lymphadenectomy but SLN mapping for surgical staging for any reason.</li>
	<li>Ensure that the patients are enrolled in the clinical trial into the SLN mapping group if they are with intermediate-high risk EC.</li>
</ol>
2. Patient preparation
<ol>
	<li>Preoperative bowel preparation
	<ol>
		<li>Provide laxatives for bowel preparation and enema to patients who have undergone multiple pelvic and abdomen surgeries. 
		NOTE: Intestinal preparation is not required for patients without bowel surgery history.</li>
	</ol>
	</li>
	<li>Preoperative diet preparation
	<ol>
		<li>Avoid solid food for 8 h before the operation, prevent a semi-liquid diet for 6 h before the procedure, and stop drinking for 2 h before the procedure.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Use intravenous agents (e.g., propofol) to produce unconsciousness and add some neuromuscular blockers by inhalation or intravenous routes to achieve the needed depth of anesthesia. Use a combination of administrations for anesthetic maintenance to ensure the patient to be unconscious and fully relaxed with stable vital signs throughout the operation. 
		NOTE: The usage of anesthetics will be following the rules of the anesthesiology department of each center.</li>
	</ol>
	</li>
	<li>Position
	<ol>
		<li>Once the patient is anesthetized, allow the patient to be in the lithotomy position with Trendelenburg position.</li>
	</ol>
	</li>
	<li>Prepare the skin at the surgical site. Perform skin preparation with an antiseptic preparation (e.g., povidone-iodine) in an organized fashion from the intended site of the incision radiating out to the edges of the intended area of skin exposure, with the upper boundary of flat xiphoid process, side borders of mid-axillary lines, and lower boundary of the upper thigh (Figure 1).</li>
</ol>
3. Fluorescent dye preparation
<ol>
	<li>Indocyanine green (ICG) preparation
	<ol>
		<li>Dissolve 25 mg of ICG into 20 mL of sterile water for a final concentration of 1.25 mg/mL and shake gently.</li>
	</ol>
	</li>
</ol>
4. ICG injection
<ol>
	<li>Use a 2 mL syringe (with the needle size of 0.6 mm x 32 mm TW LB) to inject 0.5 mL of the prepared ICG at 3 o&#39;clock and 9 o&#39;clock of the ectocervix at the superficial (2-3 mm) and deep (1-2 cm) cervix, respectively (Figure 2). 
	&#8203;NOTE: In some cases, the ectocervix positions of 6 and 12 o&#39;clock can also be selected. ICG should be avoided when injected into Nessler&#39;s cyst.</li>
</ol>
5. Intraoperative SLN identification
<ol>
	<li>Preparation before development
	<ol>
		<li>Grasp the skin around the umbilicus with two towel forceps to elevate the abdominal wall. Make an incision of about 10 mm wide in the skin of the umbilicus and through the fascia and peritoneum.</li>
		<li>Insert a 10 mm trocar through the umbilicus, producing the pneumoperitoneum with intraperitoneal pressure of about 13-14 mmHg. Insert a laparoscope (with light source and cameras) via the trocar.</li>
		<li>Make three other small incisions of ~5 mm wide in the lower abdomen and insert 5 mm trocars through the abdominal wall into the cavity. Insert instruments for manipulation via the other three 5 mm trocars (Figure 1).</li>
		<li>Identify the fallopian tubes under laparoscopy. Grasp and elevate the tubes close to fimbriae and tie a ligature around the tube with a 2-0/T silk suture. 
		NOTE: This step is used to prevent endometrial tumor cells from entering the abdominal cavity along the fallopian tubes from the uterine cavity.</li>
		<li>Take the peritoneal washings with 100 mL of normal saline before any significant handling or manipulation of the uterus. Collect the washing fluid for cytology.</li>
		<li>Open the fluorescence mode of the endoscopic fluorescence imaging system (see step 5.2). Lift unilateral pelvic infundibulum ligament and uterus to reveal the lateral peritoneum and look for the fluorescence lymphatic vessels. 
		&#8203;NOTE: If there are no fluorescence lymphatic vessels identified, it is allowed to inject ICG repeatedly.</li>
	</ol>
	</li>
	<li>SLN mapping
	<ol>
		<li>Endoscopic fluorescence imaging system preparation (PINPOINT).</li>
		<li>Adjust the fluorescence mode to near-infrared laser (excitation light, 805 nm) for the best field of view. 
		NOTE: The endoscopic fluorescence imaging system has several modes, such as HD (High definition) white light, SPY fluorescence, PINPOINT fluorescence, and SPY CSF mode, and the surgeon can switch to another mode if necessary.</li>
	</ol>
	</li>
	<li>Identify SLNs under a fluorescence laparoscopy.
	<ol>
		<li>Hold an ultrasonic scalpel and toothless forceps with each hand (performed by the surgeon). 
		NOTE: The assistant grasps toothless forceps to cooperate with the surgeon.</li>
		<li>Lift and open the peritoneum, separate the sub-peritoneal fat and connective tissues to reveal the necessary structures, including the ureter, internal iliac vessels, and obturator nerves according to the indication of green fluorescence lymphatic lines, as well as to protect them from injury. 
		NOTE: An ultrasonic scalpel with activation is used to cut the tissues, while an inactivated scalpel can be used for blunt dissection.</li>
		<li>Observe the SLNs covered by the peritoneum about 5-10 min after ICG injection. 
		&#8203;NOTE: Typically, SLNs present along with the fluorescence lymphatic vessels from both sides of the cervix to at least the height of the common iliac blood vessel. Occasionally, the SLNs emerge beside the abdominal aorta. PINPOINT equipment has multiple modes, including white light, fluorescence, and black and white modes. Switching between multiple modes helps to determine the location of SLNs.</li>
	</ol>
	</li>
</ol>
6. Intraoperative SLN biopsy
<ol>
	<li>Locate the SLN, the first mapped lymph node along the drainage from parametrium in each hemi-pelvis, and use multiple modes to confirm it. Fully expose the SLNs for complete resection (Figure 3).</li>
	<li>Grasp and elevate the SLN by toothless forceps and perform a complete resection of the lymph node along the periphery of the lymph node in white light. 
	NOTE: Avoid lymph node damage, which might cause contamination with tumor cells.</li>
	<li>Place the removed SLNs in the obturator fossa or put them into a small, simple bag. After the uterus is completely removed and taken out through the vagina, bilateral SLNs are taken out entirely through the vagina rather than through the 5 mm trocar opening to avoid fragmentation.</li>
	<li>Send the removed lymph nodes to the pathology department for pathological ultrastaging.</li>
	<li>During the operation, resect the enlarged or suspicious lymph nodes simultaneously and send them for pathological examination.</li>
	<li>Record the locations of SLNs on standardized intraoperative data collection forms.</li>
	<li>If SLN mapping fails on one or both sides, perform side-specific lymphadenectomy according to the NCCN guidelines. 
	NOTE: If there is no fluorescent node in the hemipelvis, continue to seek the mapped node along the lymphatic vessels until the para-aortic region due to the possibility of mapping in unexpected locations. A failed SLN mapping on either side means that any fluorescent green node is not visible with or without fluorescent green lymphatic vessels under the fluorescence laparoscope system on either side of the pelvis and para-aortic region.</li>
	<li>Perform a total hysterectomy plus bilateral salpingectomy. 
	&#8203;NOTE: Since this was a 28-year-old patient with early-stage endometrioid cancer who had a strong desire for fertility preservation and normal-appearing ovarian cancer and had no apparent family history of breast/ovarian cancer or Lynch Syndrome, ovarian preservation could be considered.</li>
	<li>Indwell a drainage tube for potential residual fluid in the pelvis.</li>
	<li>Remove the laparoscope and release the gas from the abdomen. Close the incisions in the umbilicus and lower abdomen with a single stitch by 2-0/T silk suture.</li>
</ol>
7. Postoperative SLN ultrastaging20
<ol>
	<li>Cut the SLN along the maximum diameter and micro-sectioned 50 &#181;m apart to get three hematoxylin-eosin (H&#38;E) slides and one immunohistochemistry (IHC) slide with anti-cytokeratin antibody (AE1/AE3). 
	NOTE: Ultrastaging entails thin serial sectioning of the gross SLN. Cytokeratin IHC is not essential. No standard protocol is for ultrastaging.</li>
	<li>Lymph node metastases were described as macrometastases (&#62;2 mm), micrometastases (0.2-2.0 mm), or isolated tumor cells (ITCs) according to the classification of the American Joint Committee on Cancer (AJCC)21.</li>
	<li>Report the postoperative pathology with details about the tumor size, histologic type and grade, myometrial invasion, lymphovascular space involvement, cervical stromal involvement, number and status of per lymph nodes, and the cytology of peritoneal washings.</li>
</ol>

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B., 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=Survival+of+patients+with+uterine+carcinosarcoma+undergoing+sentinel+lymph+node+mapping.">Survival of patients with uterine carcinosarcoma undergoing sentinel lymph node mapping.</a> Annals of Surgical Oncology. 23 (1), 196-202 (2016).</li><li>Soliman, P. T., 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=A+prospective+validation+study+of+sentinel+lymph+node+mapping+for+high-risk+endometrial+cancer.">A prospective validation study of sentinel lymph node mapping for high-risk endometrial cancer.</a> Gynecologic Oncology. 146 (2), 234-239 (2017).</li></ol>

The authors have nothing to disclose.

SLN mapping and biopsy is a more selective and tailored lymph node dissection approach that has been applied in the clinic for nearly 20 years. In the field of EC, SLN mapping and biopsy have been increasingly recommended by several guidelines due to their high diagnostic efficiency in early-stage EC, achieving overall and bilateral detection rates of 89%-95% and 52%-82%, respectively, with a sensitivity of 84%-100% and negative predictive value of 97%-100%22. The present study reports a typical E...

Endometrial cancer (EC) is one of the most common diseases in gynecological oncology, and its incidence is rising1,2. Surgery is the first-line treatment for early-stage EC3,4. The evaluation of lymph node metastasis is an essential part of surgical staging in EC. The Gynecologic Oncology Group (GOG) study 33 demonstrated that lymph node metastases are associated with poor prognosis5.
As a new and essential technique for evaluating lymph node metastasis, sentinel lymph node (SLN) mapping and biopsy have emerged in recent years and have been recommended to be employed for patients with apparent uterine-confined EC according to the National Comprehensive Cancer Network (NCCN) guidelines for uterine cancer6,7,8,9,10. SLN mapping has also been extensively applied in tumors such as breast cancer11, lung cancer12, thyroid cancer13, and melanoma14. Pathologic ultrastaging has achieved good performance in colorectal and gynecological cancer15,16,17 and is recommended by the authoritative European guidelines18. Although principles of SLN mapping for EC staging have been provided in international guidelines10,19, there are no detailed manipulations of surgery in other present works.
This work presents the protocol for detailed SLN mapping and biopsy with ICG in a 28-year-old female who had been clinically diagnosed with early-stage EC, thus, improving and promoting the diagnosis of patients.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mL syringe</td>
			<td>Becton, Dickinson and Company, USA</td>
			<td>301940</td>
			<td></td>
		</tr>
		<tr>
			<td>Coagulation forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instrument Factory</td>
			<td>D0A010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Harmonic scalpel</td>
			<td>Soering GmbH</td>
			<td>SONOCA190</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin-eosin staining</td>
			<td>Beyotime Biotechnologies Corporation,CN</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunohistochemical cytokeratin staining</td>
			<td>MXB Biotechnologies Corporation, CN</td>
			<td>Kit-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine green (ICG)</td>
			<td>Dandong Medical Innovation Pharmaceutical Corporation.</td>
			<td>3599-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pararaffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>Pinpoint Endoscopic Fluorescence Imaging system</td>
			<td>Novadaq Technoloies Bonita, Springs, FL</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol Injection</td>
			<td>Fresenius Kabi Deutschland GmbH D-61346 Bad Homburg v.d.H., Germany</td>
			<td>H20030124</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Braided non-absorbable suture</td>
			<td>Shanghai Ethicon</td>
			<td>SA854G</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk thread</td>
			<td>Beijing AKIN AIEN International Medical Technology Co.,Ltd.</td>
			<td>VCPP71D</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction-Irrigation Tube</td>
			<td>Shanghai SMAF</td>
			<td>YX980D</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothless forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instrument Factory</td>
			<td>J41010</td>
			<td></td>
		</tr>
		<tr>
			<td>Trocar</td>
			<td>ETHICON Co,. Ltd, USA</td>
			<td>B10LT, B5LT</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The patient in the present case was a 28-year-old female with irregular vaginal bleeding for 2 years, and she was diagnosed with an abnormality of the endometrium 4 months ago. Transvaginal ultrasound examination revealed a heterogeneous endometrial thickness with an adnexal mass. Abdominopelvic magnetic resonance imaging (MRI) demonstrated a 51 mm x 56 mm x 88 mm mass with a clear boundary within the uterine cavity under a high T2WI&#160;signal in a local hospital. Then, she was evaluated by hysteroscopy, and the endome...

Watch this Scientific Journal Video about Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy at JoVE.com

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

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 ...

sentinel-lymph-node-mapping-biopsy-for-endometrial-cancer-at-early

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

11.0K Views.  Obstetrics and Gynecology Hospital of Fudan University. This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

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

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening

Here, we present the detailed protocol adopted in the BioMILD screening trial to perform the circulating microRNA signature classifier test for early lung cancer detection.

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the ...

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

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 ...