This work was supported by the National Key Research Development Program of China (2021YFA1301203); the National Natural Science Foundation of China (82103031, 82103918, 81973408, 82272933); the Clinical Research Incubation Project, West China Hospital, Sichuan University (22HXFH019); the International Cooperation Project of Chengdu Municipal Science and Technology Bureau (2020-GH02-00017-HZ); Natural Science Foundation of Sichuan, 2022NSFSC1314; the &#34;1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University&#34; (ZYJC18035, ZYJC18025, ZYYC20003, ZYJC18003); and Sichuan Science and Technology Program (2023YFS0098).

The animal procedures described here were performed with the approval of the Animal Ethics Committee of West China Hospital, Sichuan University, Chengdu, Sichuan, China.
1. Induction of TBP mice
<ol>
	<li>Identify mice genotype
	<ol>
		<li>At around 3 weeks, separate the female mice from the male mice. At the same time, use ear tag clamp to fix an ear tag. Place the ear tags in the lower half and on the middle third of the ear, making sure to avoid the area with the highest concentration of capillaries.</li>
		<li>Gently but securely restrain the mice. Grasp firmly at the base of the mouse tail. Place the mice on a surface that will allow them to grip.</li>
		<li>Gently but firmly place the free hand over the shoulders, then quickly grasp the scruff of the neck between the thumb and forefinger. Hold the tail with the little finger.</li>
		<li>Swab the tail with alcohol. Use sterile scissors to snip the skin sample &#60;5 mm and put it into a clean sample container with a label. It is not necessary to remove the fur of the mice. Compress the tail with sterile sponges to attain hemostasis.</li>
		<li>Next, lyse the murine tail and perform polymerase chain reaction (PCR) to identify the genotype. 
		NOTE: After cutting one murine tail, the surface of the scissors must be wiped with alcohol cotton to avoid mutual contamination of genes between murine tails. After placing them in their cage, the mice must be watched for 5 min to look for any signs of bleeding at the wound site. Refer to Table 1 for the list of primers and the PCR settings used in this study.</li>
	</ol>
	</li>
	<li>Tamoxifen-induced TBP mice
	<ol>
		<li>Weigh 0.3 g of tamoxifen and dissolve it in 15 mL of corn oil by ultrasound (power = 20%, duration = 20 min, temperature = 4 &#176;C), at a concentration of 20 mg/mL. Store at 4 &#176;C. 
		NOTE: Tamoxifen is sensitive to light and needs to be placed in a brown container.</li>
		<li>At around 8 weeks, weigh the mice with electronic scales and give them an i.p. injection of tamoxifen at a dose of 150 mg/kg, administered twice with a 1 week interval.</li>
	</ol>
	</li>
</ol>
2. Dissection and imaging of mouse thyroid tumors and metastatic tumors
<ol>
	<li>Preparation
	<ol>
		<li>Prepare the dissection tools: sterile scissors and forceps, sterile blade, 75% alcohol spray bottle, 20 cm straightedge, vernier calipers, paper towels, and alcohol cotton.</li>
		<li>Mouse tissue fixing solution: prepare 4% paraformaldehyde tissue fixing solution.</li>
		<li>Clean a 10 cm dish filled with about 10 mL of sterile phosphate buffered saline (PBS) for temporary tissue storage, and wash the blood on the surface of the tissue.</li>
	</ol>
	</li>
	<li>Thyroid extraction
	<ol>
		<li>Euthanize the mice with carbon dioxide at a&#160;flow rate of 2.0 L/min for 5 min. Remove the mice from the cage and perform cervical dislocation.</li>
		<li>Place the mouse on the dissecting board with the ventral side facing up and the head away from the experimenter, and fix the limbs on the dissecting board with sterile syringe needles. For a more convenient removal of thyroid tissue, use another sterile syringe needle to fix the head.</li>
		<li>Disinfect the neck with three alternating rounds of a chlorhexidine or povidone-iodine scrub followed by 75% alcohol. Then, make a small incision above the center of the clavicle with sterile scissors and forceps.</li>
		<li>Continue the incision midline up to the mouth. Find the submandibular gland and remove it to expose the anatomical location of the thyroid, which is placed near the thyroid cartilage and trachea.</li>
		<li>Find the thyroid, carefully dissect the thyroid from the rest of the neck region with sterile scissors, then put the removed thyroid in a 10 cm dish filled with 10 mL of sterile PBS. 
		NOTE: During removal of the thyroid gland, be gentle and slow and avoid cutting the neck blood vessels. If the neck vessels are cut, the neck will be immediately filled with blood, and the blood must be promptly removed with alcohol sponges to expose the anatomical location of the thyroid. Before removing the thyroid tissue, carefully observe the characteristics of the left and right lobes of the thyroid (size, shape, etc.) to avoid being unable to distinguish between the left and right lobes of the thyroid after removal.</li>
		<li>In sterile PBS, remove the blood from the surface of the thyroid tissue with sterile scissors and carefully cut off the trachea. Then, put the thyroid tissue on a sterile cloth, and measure the size of the left and right lobes of the thyroid gland with vernier calipers.</li>
		<li>Divide the left and right lobes of the thyroid gland into two parts with a sterile blade. Put one part into 2 mL of 4% paraformaldehyde solution for fixation, and put the other part into liquid nitrogen for preservation.</li>
	</ol>
	</li>
	<li>Extraction of lung and liver
	<ol>
		<li>Disinfect the abdomen with three alternating rounds of a chlorhexidine or povidone-iodine scrub and 75% alcohol. Then,&#160;pinch just above the mouse&#39;s penis and&#160;make a small incision, cut along the midline of the abdomen to the subclavian bone, and expose the abdominal cavity.</li>
		<li>Find the liver in the upper part of the abdomen and carefully remove it. Place the liver into sterile PBS.</li>
		<li>Carefully cut the diaphragm along the ribs to expose the thoracic cavity. Next, grab hold of the sternum and pull up to widen the space even more. Find the lung and remove it. Put the removed lung into sterile PBS.</li>
		<li>Grossly observe whether there are metastases in the lung and liver. Count the number of metastases and take a digital record. After that, use a sterile blade to divide the lung and liver tissues into two parts. Put one part into 3 mL of 4% paraformaldehyde solution for fixation and another part into liquid nitrogen for preservation.</li>
		<li>Tissue dehydration
		<ol>
			<li>Put the tissues into the dehydration box. Put the dehydration box into the basket at gradient alcohol dehydration as follow: 70% alcohol for 1 h; 80% alcohol for 1 h; 95% alcohol for 30 min; 95% alcohol for 30 min; anhydrous ethanol I for 30 min; anhydrous ethanol II for 30 min; xylene I for 20 min; xylene II for 20 min; paraffin wax I for 30 min; paraffin wax II for 1 h; paraffin wax III for 30 min.</li>
		</ol>
		</li>
		<li>Embed the wax-impregnated tissues in the embedding machine.
		<ol>
			<li>Put the melted wax into the embedding frame first. Before the wax solidifies, remove the tissue from the dehydration box and put it into the embedding frame, then attach the label.</li>
			<li>Allow the wax to cool at -20 &#176;C on the freezing table. After the wax solidifies, remove the wax block from the embedding frame and trim it.</li>
		</ol>
		</li>
		<li>Place the trimmed wax block on a paraffin slicer, set at a thickness of 5 &#181;m and a size of 2 cm x 2 cm. After sectioning, allow the sections to float on a spreader with warm water at 45 &#176;C to flatten the tissue. Pick up the tissue with a slide, and bake the slices in an oven at 65 &#176;C for 2 h.</li>
		<li>After the water dries and the wax melts, remove the slide with the tissue and use it for hematoxylin and eosin (HE) and immunohistochemical (IHC) staining16. Make sure that the sections are adhered to the slides for staining.</li>
	</ol>
	</li>
</ol>
3. HE staining of the primary tumor and lung
<ol>
	<li>Dewaxing
	<ol>
		<li>Put the slides with the sections in xylene for 10 min.</li>
		<li>Move into anhydrous alcohol (100%) (two bottles) for about 3 min each.</li>
		<li>Move into 95% alcohol (two bottles) for about 3 min each.</li>
		<li>Move into 80% alcohol for about 3 min.</li>
		<li>Move into 50% alcohol for about 3 min.</li>
		<li>Move into the tap water, and wash away the alcohol for about 1 min.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Move the slides into hematoxylin for 8 min.</li>
		<li>Move the slides into the water for 1 min to wash away the hematoxylin; the tissue changes from blue to red.</li>
		<li>Move the slides into 1% hydrochloric acid alcohol for about 30 s.</li>
		<li>Move the slides into the water, washing for 5 min.</li>
		<li>Move the slides into eosin for 90 s, then wash them with water for 5 min.</li>
		<li>Move the slides into 50% alcohol, 80% alcohol, 95% alcohol (two bottles), and anhydrous alcohol (100%) (two bottles), for 1 min each.</li>
		<li>Move the slides into xylene for 5 min.</li>
		<li>After the slides are dry, seal them with neutral resin.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

Critical steps within the protocol for thyroid tumor dissection 
During dissection, the anatomical location of the thyroid gland needs to be correctly understood. The thyroid gland is a butterfly-shaped gland located on the dorsal side of the submandibular gland near the thyroid cartilage and the trachea. During the procedure, severing the blood arteries on both sides of the neck was carefully avoided.
Modification and troubleshooting of the mATC breed<br...

Thyroid cancer is one of the most common endocrine malignancies1, originating from the thyroid epithelium. In recent years, the incidence of thyroid cancer has increased rapidly worldwide2. Thyroid cancer can be divided into distinct types according to the degree of tumor cell differentiation. On the basis of clinical behavior and histology, thyroid carcinomas are divided into well-differentiated carcinomas, including papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC), poorly differentiated carcinoma (PDTC), and undifferentiated or anaplastic carcinoma of the thyroid (ATC)3. In contrast to PTC, which is a common type with mild behavior and better prognosis4, ATC is a rare and highly aggressive malignancy that accounts for 2% to 3% of all thyroid tumors5. Although ATC is rare, it is responsible for approximately 50% of thyroid cancer-related deaths, with dismal survival (6-8 months)6,7. Over 50% of ATC cases are diagnosed as lung metastasis8. In addition to the aggressive nature of ATC, limited effective treatment has been developed in the clinic. Therefore, ATC patients have a bleak prognosis9,10,11. This suggests that further in-depth studies are urgently needed on the molecular mechanisms underlying the development of ATC and treatment.
The tumorigenesis of ATC is a dynamic dedifferentiated process. The difficulty in collecting human tumor samples at each stage in clinical studies has hindered the understanding of the mechanism of development from well-differentiated to undifferentiated carcinomas. In contrast, the use of murine ATC models (mATC) favors the collection of mATC samples in the whole tumorigenesis course. Therefore, we can better understand the mechanisms of tumor formation by analyzing the dynamic dedifferentiated process. In addition, the heterogeneity of clinical ATC samples has also contributed to the difficulty in understanding the molecular mechanism. Nevertheless, mice shared the same genetic backgrounds and were maintained in similar living environments, ensuring each tumor&#39;s consistency. This facilitates exploring the generalized role of ATC development12,13,14. Additionally, mATC is an in situ tumor model that can restore the influence of the anatomic location and tissue-specific microenvironment. As such, compared with commonly used immunodeficient mice, mATC is a spontaneous murine model with an intact immune system and immune microenvironment.
Therefore, we constructed conditionally induced mATC with the C57BL/6 strain, which is a murine model capable of reproducing the pathological features of dedifferentiated thyroid carcinoma. Based on this model, we gave a brief overview of the molecular basis, construction ideas, pathological features, and applications of mATC. In addition, we observed and reported tumor growth, survival time, metastasis, and pathological features of mATC. We believe this will be an informatic overview to assist other researchers in using this model easier.
We constructed a conditional inducible mATC model, as first reported by McFadden15; initially, we constructed mice: TPO-cre/ERT2, Brafflox/wt, and Trp53flox/wt. Specifically, TPO-cre/ERT2 mice included the human thyroid peroxidase (TPO) promoter (a thyroid-specific promoter), driving the expression of a cre-ERT2 fusion gene (a cre recombinase fused to a human estrogen receptor ligand binding domain). Cre-ERT2 is usually confined to the cytoplasm and enters the nucleus only when exposed to tamoxifen, which induces cre to exert recombinant enzyme activity. When the mice are crossed with mice carrying loxP-flanked sequences, after tamoxifen-induction, cre-mediated recombination deletes the floxed sequences in the thyroid cells to achieve the purpose of knocking out or knocking in specific genes.
In addition, Brafflox/wt mice are a knock-in allele of human Braf based on the cre-loxP system. Brafflox/wt&#160;murine transcript is encoded by endogenous exons 1-14 and loxP-flanked human exons 15-18. After cre-mediated excision of the floxed regions, the mutant exon 15 (modified with a V600E amino acid substitution linked with constitutively active BrafV600E in human cancers) and the endogenous exons 16-18 are used to generate the transcripts. Furthermore, Trp53flox/wt&#160;mice are knockout alleles of human Trp53 and&#160;have loxP sites flanking exons 2-10 of Trp53.&#160;When crossed with mice with a cre recombinase, cre-mediated recombination deletes the floxed sequence to knock out Trp53. Then, TPO-cre/ERT2, Brafflox/w, and Trp53flox/wt mice were crossed to obtain TB (TPO-cre/ERT2; Brafflox/wt) mice and TBP (TPO-cre/ERT2; Brafflox/wt; Trp53flox/wt) mice, which could be used to generate PTC and ATC. After approximately 8 weeks, the mice were induced by an intraperitoneal (i.p.) administration of 150 mg/kg tamoxifen dissolved in corn oil for two administrations. Tumor growth could be monitored by high-frequency ultrasonography (the first time point of ultrasonography was recorded as Day 0). Initial ultrasonography was performed 40 days after tamoxifen introduction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x&#160;Citrate antigen retrieval solution (PH 6.0)</td>
			<td>MXB</td>
			<td>Cat# MVS-0101</td>
			<td></td>
		</tr>
		<tr>
			<td>50x EDTA antigen retrieval solution(pH 9.5)</td>
			<td>ZSGB-GIO</td>
			<td>Cat# ZLI-9071</td>
			<td></td>
		</tr>
		<tr>
			<td>Brafflox/wt mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase-3</td>
			<td>Beyotime</td>
			<td>Cat# AC033</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>Cell Signaling Technology</td>
			<td>Cat# 98941; RRID:AB_2756376</td>
			<td></td>
		</tr>
		<tr>
			<td>CD206</td>
			<td>Cell Signaling Technology</td>
			<td>Cat# 24595; RRID:AB_2892682</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber for anesthesia induction</td>
			<td>RWDlifescience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced DAB chromogenic kit</td>
			<td>MXB</td>
			<td>Cat# DAB-2031</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin staining solution</td>
			<td>ZSGB-GIO</td>
			<td>Cat# ZLI-9613</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Abcam</td>
			<td>Cat# 100790; RRID:AB_10675322</td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3</td>
			<td>Cell Signaling Technology</td>
			<td>Cat# 12653; RRID:AB_2797979</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully enclosed tissue dehydrator</td>
			<td>Leica Biosystems</td>
			<td>ASP300S</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin staining solution</td>
			<td>ZSGB-GIO</td>
			<td>Cat# ZLI-9610</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCore Arcadia fully automatic tissue embedding machine</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Beyotime</td>
			<td>Cat# AF1738</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating Slicer</td>
			<td>RWDlifescience</td>
			<td>&#160;Minux S700</td>
			<td></td>
		</tr>
		<tr>
			<td>SPlink detection kits (Biotin-Streptavidin HRP Detection Systems)</td>
			<td>ZSGB-GIO</td>
			<td>Cat# SP-9001</td>
			<td></td>
		</tr>
		<tr>
			<td>TPO-cre/ERT2 mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trp53flox/wt mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cell crusher</td>
			<td>Ningbo Xinyi Ultrasound Equipment Co., Ltd</td>
			<td>JY92-IIN</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Keppler</td>
			<td>KL-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 3100</td>
			<td></td>
		</tr>
	</tbody>
</table>

spontaneous murine model of anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

We induced mATC to investigate tumor growth, mouse survival time, and pathological characteristics. After induction, the mice were immediately sacrificed, and samples (thyroid, lung, and liver) were collected once one of the following conditions were found: 1) respiratory distress caused by tumor compression; 2) decreased appetite and abnormal vocalization; 3) unusually lethargy; and 4) body weight loss of over 20%. During the sampling process, we found that all mice (12/12) successfully formed tumors after induction. We...

Watch this Scientific Journal Video about Spontaneous Murine Model of Anaplastic Thyroid Cancer at JoVE.com

Spontaneous Murine Model of Anaplastic Thyroid Cancer

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the ...

spontaneous-murine-model-of-anaplastic-thyroid-cancer

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Cancer Research

1.5K Views.  Sichuan University and Collaborative Innovation Center. Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

Video: Spontaneous Murine Model of Anaplastic Thyroid Cancer

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating investigators to analyze and interpret genes, samples and clinical data across different types of cancer. Herein, we describe a practical operation procedure, taking ID1 (Inhibitor of DNA binding proteins 1) as an example, to characterize the expression patterns of biomarker and survival predictors of breast cancer based on pooled clinical datasets derived from online accessible databases, including ONCOMINE, bcGenExMiner v4.0 (Breast cancer gene-expression miner v4.0), GOBO (Gene expression-based Outcome for Breast cancer Online), HPA (The human protein atlas), and Kaplan-Meier plotter. The analysis began with querying the expression pattern of the gene of interest (e.g., ID1) in cancerous samples vs. normal samples. Then, the correlation analysis between ID1 and clinicopathological characteristics in breast cancer was performed. Next, the expression profiles of ID1 was stratified according to different subgroups. Finally, the association between ID1 expression and survival outcome was analyzed. The operation procedure simplifies the concept to integrate multidimensional data types at the gene level from different databases and test hypotheses regarding recurrence and genomic context of gene alteration events in breast cancer. This method can improve the credibility and representativeness of the conclusions, thereby, present informative perspective on a gene of interest.

Here, we present a protocol to explore the biomarker and survival predictor of breast cancer based on the comprehensive analysis of pooled clinical datasets derived from a variety of publicly accessible databases, using the strategy of expression, correlation and survival analysis step by step.

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...