Yazarlar yararlı tartışmalar ve teknik yardım için Kadın Hastalıkları ve Doğum Anabilim Dalı üyeleri ve Kanser Biyoloji teşekkür ederiz. Bu çalışma Bilim Teşvik (JSPS) için Japan Society tarafından desteklenen Hibe-in-Aid Bilimsel Araştırma (15K15604 H. Kajiyama için).

Tedavi protokolü Nagoya Üniversitesi tarafından kabul edilen hayvan deney için kuralları takip eder. Hücre süspansiyonları hazırlanması 1. <ol><li> İnsan yumurtalık berrak hücreli karsinoma hücre hattı. <ol><li> (100 birim / ml streptomisin: 0.1 mg / ml penisilin),% 10 fetal sığır serumu (FBS) ve penisilin / streptomisin ile RPMI-1640 ortamı içinde ES-2 hücreleri 12 koruyun. % 5 CO2 nemlendirilmiş inkübatör içinde 37 ° C &#39;de kültür hücreleri. </li><li> % 0.05 tripsin /% 0.02 EDTA çözeltisi (tripsin / EDTA) logaritmik büyüme fazında hücreler toplanır. <ol><li> (- 25 cm 2 başına 4 ml, 2 + Ca 2 + / Mg olmadan PBS 3), sonra bir kez PBS ile yıkama hücreleri, hücre kültürü geminin eski ortamı çıkarın. </li><li> Daha sonra, 25 cm tripsin / EDTA 2 &#39;ye 1 ml. tripsin / EDTA ile hücre mono tabakasının karşılamak için hücre kültür kabı döndürerek. kaldırma ve tripsin / EDTA atıldıktan sonra, hücre dönüş3 inkübatör kültür gemi - hücreleri 5 dakika kadar veya ayrılır. </li><li> Bir mikroskop altında hücreleri incelemek hücreler, hücre kültürü damar ve yüzen yüzeyinden ayrılır sağlamak. </li><li> Tripsin (25 cm2 başına 5 mi) inaktive ve hücrelerin yeniden askıya almak için büyüme ortamı içeren taze serum ekleyin. 15 ml konik tüp yeniden askıya hücreleri aktarın. </li><li> 5 dakika boyunca 300 x g&#39;de santrifüjleyin. 4 ° C&#39;de ya da oda sıcaklığında santrifüjleme ya da gerçekleştirir. Dikkatle süpernatant kaldırmak ve (25 cm 2 başına 5 ml) büyüme ortamı ile hücreleri askıya yeniden. </li></ol></li><li> (Tripan mavi eksklüzyonu ile, örneğin,) hücre sayımı, daha sonra 2 x 10 8 hücre / ml bir yoğunlukta büyüme ortamında yeniden askıya. buz üzerinde asılı hücreleri koyun. </li></ol></li><li> Çözülme dondurulmuş hücre dışı matris (ECM) bir buz banyosu içinde hidrojel alikotları tabanlı. </li><li> 1 (nihai hücre c: 1 bir oranda ECM bazlı hidrojel alikotları hücre süspansiyonu ekleyinbağlı yoğunlaşma: 1 x 10 5 hücre / orta-ECM tabanlı hidrojel ul) ve iyice karıştırın. kullanılana kadar bir buz banyosu içinde hazırlanan hücre süspansiyonları yerleştirin. </li></ol> Hücre süspansiyonları, 2. Ortotopik aşılama NOT: özel bir paketi gerektirmez Cerrahi. Cerrahi ayrı ve en az aktiviteye sahip odasının bir bölgesinde bir laboratuar sıra üzerine gerçekleştirilebilir. Bir laminar kapağı da kullanılabilir. Steril cerrahi aletler kullanılmalıdır ve makas cilt kesi yapmak için kullanılmamalıdır. <ol><li> Bu ortotopik aşılama modeli için yaş 8 haftalık dişi çıplak farelerin (BALB / c nu / nu) kullanın. </li><li> (-% 4 2) İlk olarak, izofluran ile inhalasyon kullanarak fare uyutmak. Firma ayak tutam üzerine çekilmesi eksikliği doğrulayarak anestezi derinliğini kontrol edin. Fare uyuşturulduktan sonra, gerekli kurumasını önlemek için gözlere bir mülayim steril oftalmik merhem uygulayın. </li><li> Bir operat üzerinde anestezi fare yerleştirinaşağı sahne göbek tarafı ing. Cerrahi parkı alkol ve iyot bazlı veya klorheksidin bazlı fırçalayın hem birkaç kez dezenfekte edin. </li><li> dikey sağ kaburga kemer ve femur arasında omurga yakınında ~ 2 cm kesi olun. insizyon bölgesinde yaklaşık steril bir cerrahi örtü kullanın. açılış korumak için kelepçeler ile kazıma cilt yüzeyleri düzeltmek. Not: Retraktörler yerine kelepçeler de kullanılabilir. </li><li> Sonraki, karın boşluğu açmak için ilk kesi altında parietal periton ikinci bir kesi (yaklaşık 1 cm) yapmak. çizi karın yüzeyleri kelepçe. Karın boşluğunda yumurtalık kaldırmak için bir kelepçe ile ipsilateral yumurtalık ve fallop tüpü çevreleyen yağ dokusu klibi. Dikkatle gazlı bez yumurtalık ve fallop tüpü yerleştirin. Sonra, mikroskop altında fare göbek tarafı aşağıya bakacak şekilde yerleştirin. </li><li> hemen enjeksiyon öncesi (1/2 cc 29 G) bir insülin şırınga hücre süspansiyonu yerleştirin. Özenle hazırlanmış hücre süspansiyonu (enjekte 1- Yumurtalık içine 2 ul). Birkaç saniye için ECM bazlı hidrojel jelleşmesini sağlamak için yumurtalık içinde iğnesinin tespit edilmesi. jelleşme sonra, enjekte edilen tümör hücreleri, yumurtalığın içinde kalır. </li><li> Dikkatle vücuda yumurtalık döner. steril tıbbi ipek ile ikinci kesi dikin ve sonra yara klipleri ile cilt mühür. </li><li> Cerrahiden sonra, farenin, ilk 24 saatlik bir süre için bir analjezik (örn, meloksikam, kas içinden (İM) ya da deri altından (sc), 0.2 mg / kg) uygulanır. </li></ol> 3. Post-cerrahi tedavisi <ol><li> sternal yatma korumak için yeterli bilinci yerine kadar fare sıcak tutmak için sıcak bir paketi ile temiz kağıt üzerinde ayrı bir kafes içinde her fare yerleştirin. Sonra, yetiştirilmesi ve izlenmesi için kafeslerine hayvanları dönün. Cerrahi tedavi sonrası 7 gün - 3 günlük fareler izleyin. </li><li> Ameliyat sonrası farelerin izlenmesi <ol><li> ağrı belirtileri durumunda, analge idareSICS günlük olarak (örneğin, meloksikam, İM, veya SC, 0.2 mg / kg). </li><li> Fareler enfeksiyon (örneğin, kızarıklık, şişlik, akıntı) belirtileri varsa, bir anti-enfektif reaktif (örn, penisilin, im, 100.000 IU / kg) ile günlük enjeksiyon yönetmek. </li></ol></li><li> ameliyat sonrası tedavi sonrası 14 gün - cilt kapatma malzemelerdir 10 çıkarın. </li></ol> Tümörlerin ve Metastazlarının 4. Analizi NOT: Tümör enjekte over 2 oluşacaktır - aşılamadan sonra 3 hafta. <ol><li> Karbon dioksit fareler kurban, ve daha sonra analiz için numune 14,16-20 (örneğin, tümörler, metastazlar, organları) 13-15 toplar. Not: bir in vivo görüntüleme sistemi, mevcut ise ışıldaması (örneğin, lusiferaz) ya da floresan proteini (örneğin GFP) üretimi enzimler taşıyan hücrelerin Bu protokol kullanılabilir. Bu modifiye protokol için yararlı bilgiler sağlayacaktırprimer tümörden kanser hücrelerinin yayılmasını dinamiği. </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Hayvan modelleri, kanser hücrelerine karşı tümör gelişimi, ilerlemesi, metastaz ve ilaç etkinliğinin mekanizmalarını analiz için esastır. insan yumurtalık kanseri hücreleri olan farelere, aynı zamanda bir hayvan modeli olarak kullanılmıştır. Burada, ortotopik yerinde yumurtalık tümörü hücre uygulama için daha az invaziv bir prosedür açıklanmaktadır. dorsal zayıf bir retroperitoneal yaklaşımla yumurtalık içine bu prosedür, aşılama kullanan diğer sık ​​kullanılan tekniklere gör...

Epitelyal over kanseri (EOC) jinekolojik maligniteler 1 arasında kansere bağlı ölüm oranı en yüksek oluşturmaktadır. EOC öncelikle nedeniyle geç semptomatoloji, kötü prognoz ile ilişkilidir ve genellikle birden çok intraperitoneal saçınımlı ve uzak metastaz 2-4 ile ilişkilidir. Periton yaygınlaştırma çok adımlı bir işlemdir. İlk olarak, tümör hücrelerinin birincil lezyonlarla ayırmak ve karın boşluğuna göç ederler. Tümör hücrelerinin periton mesothelium eklemek, onlar Mesothelium 5,6 ile dokuları işgal etmeye başlar. Daha iyi tümör biyolojisi (örneğin, kanser ilerleme ve terapötik yanıt) anlamak için, fare modelleri bilgi hazinesi sağlar. İnsan kanser hücreleri bir ksenograft yaygın insan kanser hücreleri, intraperitonal, intravenöz ve ortotopikal, deri altından aşılanır olan fare modellerinde kullanılmaktadır. Bir orthotopically g bir hayvan modelirafted tümör daha verimli ve doğru bir şekilde heterotopically aşılanmış tümörlü bir hayvan modeli ile karşılaştırıldığında insanlarda tümör ortamı yansıtan sonuçlar üretebilir. Bu nedenle, birçok insan tümörleri için, ortotopik transplantasyon modelleri 7-11 kurulmuştur. Burada, ventral aşılamadan oranla invazivliğini sınırlı dorsal kanadını, bir retroperitoneal yaklaşımla insan EOC hücrelerinin ortotopik aşılama açıklar. Bu teknik EOC yararlı bilgiler, periton yayılması özellikle mekanizmasının çeşitli sağlayabilir.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Matrigel matrix&#160;</td>
			<td style="width:151px;">BD</td>
			<td style="width:163px;">354234</td>
			<td style="width:163px;">ECM-based hydrogel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin syringe</td>
			<td>TERUMO</td>
			<td>SS-05M2913</td>
			<td>1/2cc, 29Gx1/2&#34;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suturing needle with suture</td>
			<td></td>
			<td></td>
			<td>11mm, 3/8, Nylon6-0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reflex 7mm Wound Clip Applier</td>
			<td>CellPoint Scientific</td>
			<td>204-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Reflex 7mm wound clips</td>
			<td>CellPoint Scientific</td>
			<td>203-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with ovarian carcinoma cells

o periton yayılmasını gösterir çünkü Epitelyal over kanseri (EOC) kötü prognoz ile ilişkilidir. prognozu iyileştirmek için, periton yayılmasını kontrol etmek önemlidir. Tümör hücreleri primer lezyonlardan ayırmak ve mesothelium eklemek nasıl Ancak, hala belirsizdir. Uygun bir hayvan modelinin kurulması in vivo periton yayılım mekanizmasının bir anlayış kazanmak için gereklidir. Bu çalışmada, biz periton ve uzak organlara da dahil olmak üzere metastaz gelişimine fare yumurtalık yüzeyine EOC hücrelerinin lokal enjeksiyon işlemi tanıtmak. yaş 8 haftalık dişi çıplak fareler (BALB / c nu / nu) kullanılmıştır. Bir görüş mikroskobik alan altında, EOC hücreleri dorsal zayıf bir retroperitoneal yaklaşımla fare yumurtalıkların enjekte edildi (orta-ekstrasellüler matriks (ECM) 1 x 10 5 hücre / ul hidrojel / tek taraflı yumurtalık / fare tabanlı). Önerilen bu yöntem le olanfare için ss invaziv prosedür ve yumurtalık zarar en aza indirir. Burada, EOC özgün ve metastatik tümör oluşumu gelişiminde metodolojik adımları açıklar.

<html> Altı çıplak fareler bu protokol açıklandığı gibi onların yumurtalıklar, insan berrak hücreli karsinom hücre hattı ES-2 ile aşılanmış vardı. Şekil 1A&#39;da gösterildiği gibi, 2 hafta sonra, 6 farelerinin 5 ES-2 hücreleri ile aşılandı yumurtalık tümör vardı, ama (bir kontrol olarak kullanılmıştır) aşılaması olmayan karşı yumurtalık tümör yoktu. Bunun yanında, 5 farelerin 2 asit ile birden periton disseminations göstermişti...

Watch this Scientific Journal Video about Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells at JoVE.com

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

over kanseri periton yayılması çok adımlı süreci açıklamak için, mevcut çalışma bu tümör hücreleri ile ortotopik aşılamadan yoluyla orijinal tümör gelişimi ve periton metastazı bir fare deneysel modeli sunar.

Orijinal Tümör Geliştirme Murin Deneysel Modeli ve Periton Metastaz<em&gt; yoluyla</emYumurtalık Kanseri Hücreleri ile&gt; Ortotopik aşılama

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

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Cancer Research

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

10.4K Görüntüleme.  Bell Research Center for Reproductive Health and Cancer.  over kanseri periton yayılması çok adımlı süreci açıklamak için, mevcut çalışma bu tümör hücreleri ile ortotopik aşılamadan yoluyla orijinal tümör gelişimi ve periton metastazı bir fare deneysel modeli sunar. 

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Tümör metastaz üzerinde Hipoksi Test Etkisinin Vivo Modeli

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Kanser Araştırmaları

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.

Bir Fare Ortotopik Mesane Tümör Modeli ve Tümör Tespit Sistemi

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 ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Kolorektal kanserli bir Ortotopik Fare Modelinde Dolaşan Tümör Hücrelerinin İzolasyonu

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Tümör metastazı çalışmaya acellular ve hücresel akciğer modeli

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Skuamöz Hücre Karsinomu Hücre Tümörü bağışıklık profil oluşturma için fareler ve tedaviye yanıt değerlendirme intramucosal aşı

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

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.

Insan Üromotorial Hücre Karsinomu ve kolorektal kanser tümörü büyüme ve spontan Metasrik için hasta türevi Ortootopik xenograft modelleri

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Akım moleküler Analizi ve metastatik potansiyel değerlendirme için sirkülasyon tümör hücrelerinin mikromanipülasyon

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Akciğer Skuamöz Karsinom Progresyonunu Modellemek Için Çok Boyutlu Coculture Sistemi

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Tümör Hücrelerinin Lateral Kuyruk-Damar Enjeksiyonu Sonrasında Akciğer Metastazının Patolojik Analizi

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primer Kemik Tümörleri ve Kemik Metastazlarının Solid Tümör Grefti İmplantasyonu ile Modellenmesi

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...