We thank Ms. J. Shinohara and I. Nieda for technical and clerical work.&#160;Also, we thank Drs. Shiro Matsumoto, Hidenori Haruta, Kentaro Kurashina, and Kazuya Takahashi for their cooperation for sample acquisition in operating room.&#160;This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science (17K10606).

LDN were obtained from patients enrolled in this study and were approved by the Institutional Review Board of Jichi Medical University.
1. Isolation of LDN from Abdominal Cavity Lavages and NET Detection
<ol>
	<li>Sample acquisition
	<ol>
		<li>Infuse 1,000 mL of normal sterile saline directly into the abdominal cavity before wound closure in patients who have undergone abdominal surgery due to gastrointestinal malignancy. 
		NOTE: Samples were obtained from patients who underwent a gastrectomy, colectomy, or esophagectomy without bias based on age or sex. Saline was transferred into a container and poured into the whole abdomen within one minute. This is routinely performed as a postoperative peritoneal washing without significant effects on patients.</li>
		<li>Lavage the abdominal cavity extensively for at least 1 min. 
		NOTE: It is recommended that the infused fluid is slowly stirred with the surgeon&#39;s hands so that the samples are uniform.</li>
		<li>Recover 200 mL of lavage fluid with four 50 mL syringes. 
		NOTE: Sometimes a rubber connector is used to take up the fluids.</li>
	</ol>
	</li>
	<li>Perform purification of peritoneal LDN using granulocyte specific mAb, CD66b16. 
	NOTE: Because the intermediate layer after density gradient centrifugation contains many mononuclear cells, positive selection of polymorph neutrophils using CD66b mAb was performed.
	<ol>
		<li>Transfer the peritoneal lavage fluid to a 50 mL tube. 
		NOTE: In this step, pass the fluids through a 100 &#181;m nylon filter to remove impurities.</li>
		<li>Centrifuge the peritoneal fluid at 270 x g for 7 min at RT.</li>
		<li>Resuspend the pellets in 5 mL of PBS with 0.02% EDTA.</li>
		<li>Carefully overlay the 5 mL of cell suspension on a 3 mL density gradient solution.</li>
		<li>Centrifuge at 1,700 x g for 15 min at RT without any breaks.</li>
		<li>Harvest the ~2 - 3 mL of solution containing the intermediate layers (Figure 1A) using a pipette and mix it with 10 mL of PBS with 0.02% EDTA.</li>
		<li>Centrifuge the peritoneal fluid at 400 x g for 7 min at RT and discard the supernatant.</li>
		<li>Add another 10 mL of PBS with 0.02% EDTA, and centrifuge at 270 x g for 7 min at RT. Discard the supernatant.</li>
		<li>Dissolve the cell pellet (1 x 107) in 60 &#181;L of buffer for the magnetic cell separation kit.</li>
		<li>Add 20 &#181;L of Fc block to the pellets and incubate for 10 min at 4 &#176;C.</li>
		<li>Add 20 &#181;L of the anti-CD66b conjugated with microbeads and incubate for an additional 10 min at 4 &#176;C.</li>
		<li>Wash and re-suspend the pellets in 500 &#181;L of MACS buffer.</li>
		<li>Apply the cell suspension to a magnetic column in the magnetic field of a suitable magnetic separator and collect the flow-through containing CD66b(-) cells by washing the column with 15 mL of buffer.</li>
		<li>Remove the column from the magnetic separator and immediately flush out the magnetically labeled CD66b(+) cells by firmly pushing the plunger into the column. 
		NOTE: The CD66b(+) cell population consists mostly of neutrophils as determined by FACS analysis indicating that &#62;95% of the CD66(+) fraction are positive for CD11b, CD15, and CD16, but negative for CD14.</li>
	</ol>
	</li>
	<li>Generation of NETs
	<ol>
		<li>After centrifugation at 270 x g for 7 min at RT, re-suspend the isolated LDN (5 x 106) in 1 mL of RPMI1640 with 10% FCS.</li>
		<li>Culture the LDN on a 6-well poly-L-lysine coated plate (6 cm in diameter) for 2 h at 37 &#730;C in 5% CO2.</li>
		<li>Add the green fluorescent counterstain (a membrane-impermeable dye to stain the nucleus and chromosomes) at the final concentration of 5 &#181;M.</li>
		<li>Immediately observe the morphology of NETs (the extracellular DNA components expelled from the LDN under fluorescence microscopy).</li>
	</ol>
	</li>
</ol>
2. Staining of Tumor Cells with Red Fluorescent Cell Linker Dye
<ol>
	<li>Prepare human cancer cell lines MKN45, NUGC, and OCUM-1.</li>
	<li>Wash 1 x 107 cells with PBS + 0.02% EDTA in a 15 mL tube and centrifuge at 270 x g for 7 min at RT.</li>
	<li>Add 1 mL of solution for dye staining to the pellets, and pipette gently.</li>
	<li>Dissolve 4 &#181;L of Red Fluorescent Cell Linker dye in 1 mL of solution for staining and mix with the cell suspension from step 2.2.</li>
	<li>Incubate the pellets for 4 min at RT.</li>
	<li>Add 4 mL of DMEM (10% FBS) to stop the staining reaction.</li>
	<li>Centrifuge 270 x g for 7 min and discard the supernatant.</li>
	<li>Repeat steps 2.6 and 2.7 twice.</li>
	<li>Check with a fluorescence microscope (excitation = 551 nm, emitting = 567 nm) that the tumor cells are stained red.</li>
</ol>
3. Analysis of Tumor Cell Adhesion to NETs
<ol>
	<li>Re-suspend the red fluorescent stained tumor cells (1 x 106) in 1 mL of RPMI 1640 supplemented with 0.1% BSA.</li>
	<li>Add the tumor cells to the LDN culture that produced the NETs as described in step 1.3.</li>
	<li>Incubate for 5 min at 37 &#730;C, which enables the tumor cells to contact the NETs. 
	NOTE: Long incubation induces tumor cell adhesion to LDN or to the plates.</li>
	<li>Remove the medium and gently wash the wells by adding 2 mL of prewarmed media (0.1% BSA + RPMI 1640) and swirling the dish.</li>
	<li>Repeat the washing procedure in step 3.4 twice. 
	NOTE: Since NETs weakly attach to the plate, washing should be done as gently as possible to avoid removal of the NETs themselves.</li>
	<li>Add the green fluorescent dye for staining the nucleus and chromosomes at a final concentration of 5 &#181;g/mL for visualization of the NETs.</li>
	<li>Observe the NETs and attached tumor cells using the appropriate filters (green, excitation = 504 nm, emitting = 523 nm; red, excitation = 551 nm, emitting = 567 nm).</li>
	<li>Merge the figures to show the tumor cells that are trapped by the NETs. 
	NOTE: In some experiments, DNA degradation enzyme was added to the LDN culture (final concentration of 100 U/mL) 5 min before co-incubation for 5 min.</li>
</ol>
4. Time Lapse Video Analysis of the Trapped Tumor Cells
<ol>
	<li>Culture the peritoneal LDN in a 35 mm round dish coated with poly-L-lysine as described in step 1.3.</li>
	<li>Add the green fluorescent dye for staining the nucleus and chromosomes to visualize NETs as described in step 1.3.</li>
	<li>After 1 min of incubation, remove the media and add 2 mL of 0.1% BSA + RPMI 1640.</li>
	<li>Remove the media and add unstained 1 x 106 MKN45 cells suspended in 0.1% BSA + RPMI 1640. Incubate for 5 min at RT.</li>
	<li>Remove any unattached tumor cells by gently washing as described in step 3.4 and add DMEM with 10% FCS supplemented with 100 u/mL Penicillin and 100 &#181;g/mL streptomycin.</li>
	<li>Mount the culture dish in a whole-view cell observation system and select the appropriate field in which tumor cells are trapped by the NETs.</li>
	<li>Continue to co-culture for an additional 2 days and take continuous photos of the field every 6 min under normal light and fluorescence, which detects MKN45 cells and NETs, respectively.</li>
	<li>Superimpose the images at each timepoint and construct time-lapse videos using Image Viewer software.</li>
</ol>

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The authors declare that they have no competing financial interests.

Previous studies have reported that circulating tumor cells can be trapped by NET substrates in vivo10,11. Metastatic breast cancer cells have been shown to stimulate neutrophils and induce formation of NETs, which assists in tumor cell growth in the target organ17. In addition, we found that short-term cultures of LDN from postoperative lavage fluid produced massive NETs that could efficiently entrap tumor cells without further s...

Polymorph nuclear neutrophils in circulating blood are typically separated from mononuclear cells through the density gradient preparation method. However, some neutrophils known as low-density neutrophils (LDN), with CD11b(+), CD15(+), CD16(+), and CD14(-) phenotypes, are co-purified with mononuclear cells. The relative number of LDN significantly increases in various pathological conditions including autoimmune diseases1,2, sepsis3, and cancer4,5. Previous studies have shown that LDN are a phenotypically and functionally distinct class of neutrophils6. It should be noted that LDN in circulating blood are more likely to produce neutrophil extracellular traps (NETs) than normal density neutrophils2,7. NETs are web-like structures composed of nucleic acids, histones, proteases, and granular and cytosolic proteins, and they can efficiently entrap and destroy pathogens8.
Recently, NETs have been shown to capture not only microbes, but also platelets and circulating tumor cells which can assist in thrombus formation9 and tumor metastases10,11. However, the molecular mechanisms behind the adhesive interactions between NETs and platelets or tumor cells are still unclear. More recently, an in vitro adhesion assay revealed that myeloid leukemia cells (K56212) and lung carcinoma cells (A54913) attach to NETs via &#946;1 and &#946;3 integrins. The authors used NET stock isolated from neutrophils and activated by phorbol 12-myristate 13-acetate (PMA) as the adhesion substrate14. Although this assay allows detection of real interactions with NET components in the absence of neutrophils, it is arguable whether the &#34;cell-free NET stock&#34; isolated by high-speed centrifugation retains the molecular structure identical to NETs produced in vivo. Recently, we found that peritoneal lavage fluid after abdominal surgery contained many mature LDN, which generated massive NETs and attached to tumor cells causing peritoneal metastases15. In this study, we successfully examined the adhesion of tumor cells to intact NETs without any physical manipulation. Here, we show details of a technique to detect adhesive interactions between NETs and free tumor cells.

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare, SWEDEN</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>StraightFrom&#8482; Whole Blood CD66b MicroBeads</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-104-913</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Rinsing Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Magnetic Separator</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-042-501</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX green nucleic acid stain 5mM solution in DMSO</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9691</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium-high glucose (DMEM)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5mol/l-EDTA Solution (pH 8.0)</td>
			<td>nacalai tesque, Japan</td>
			<td>06894-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td>gibco by life technologies, Mexico</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin lyophilized powder, &#8805;96% (agarose gel electrophoresis)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies Japan</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmocin Prophylactic</td>
			<td>InvivoGen, San Diego, CA-USA</td>
			<td>ant-mpp</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington, Lakewood NJ)</td>
			<td>LS002138</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine-Coated MICROPLATE 6Well</td>
			<td>IWAKI, Japan</td>
			<td>4810-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine-Coated MICROPLATE 24Well</td>
			<td>IWAKI, Japan</td>
			<td>4820-040</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein stereomicroscope</td>
			<td>BX8000, Keyence, Osaka Japan</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole view cell observation system</td>
			<td>Nikon, Kanagawa, Japan</td>
			<td>BioStudio (BS-M10)</td>
			<td></td>
		</tr>
		<tr>
			<td>MKN45 human gastric cancer cell line</td>
			<td>Riken, Tukuba Japan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NUGC-4 human gastric cancer cell line</td>
			<td>Riken, Tukuba Japan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OCUM-1 human gastric cancer cell line</td>
			<td>Osaka City University, Japan</td>
			<td>N/A</td>
			<td>Gift from Dr. M.Yashiro</td>
		</tr>
	</tbody>
</table>

neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

In the 2-hour culture, CD66b(+) LDN derived from peritoneal lavage fluid showed string structures stained with green-fluorescent dye for nuclear and chromosome (Figure 1B), while CD66b(-) mononuclear cells did not (Figure 1C). However, when the LDN cultures were pretreated with 100 U/mL DNase I, the characteristic structure was destroyed (Figure 1D), indicating that they were composed of extracellula...

Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

neutrophil-extracellular-traps-generated-low-density-neutrophils

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Cancer Research

8.7K Views.  Jichi Medical University. Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Video: Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

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Chapters in this video

Lung Tumor Cell Recruitment Assay

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells