Bu çalışma hibe hiçbir şekilde desteklenmiştir. SNUBH Araştırma Fonu 14-2014-017. Yazarlar bu yazının onun pro bono düzenleme J. Patrick Barron, Profesör Emeritus, Tokyo Üniversitesi Tıp Fakültesi ve Yardımcı Doçent, Seul Ulusal Üniversitesi Bundang Hastanesi&#39;ne borçluyuz.

NOT: Tüm hayvan çalışması protokolleri Seul Ulusal Üniversitesi Bundang Hastanesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (BA1308-134 / 072-01) tarafından onaylanmıştır. Hyaluronik asit-seramid 1. N- (HACE) <ol><li> Iki kez damıtılmış su (DDW) 60 ml tetra-n-butilamonyum hidroksit (TBA) 12.21 hiyalüronik asit (HA) oligomer mmol ve 9.77 mmol çözünürleştirilir. 30 dakika boyunca karıştırın. </li><li> DS-Y30 bağlayıcı sentez, 25 ml tetrahidrofuran (THF) içinde trietilamin 8.59 DS-Y30 seramid mmol 9.45 mmol eritin. THF içerisinde 4-klorometilbenzoil klorür 8.59 mmol ile karıştırın. 60 ° C&#39;de 6 saat karıştırılır. </li><li> Bir THF ve asetonitril DS-Y30 bağlayıcının sentez HA-TBA 8.10 mmol ve 0.41 mmol çözülür (4: 1, hac / hac). 40 ° C&#39;de 5 saat boyunca karıştırılır. </li><li> bir filtre maddesi ile filtre ile yabancı maddelerin çıkarın ve vakum buharlaştırma ile organik çözücü ortadan kaldırır. (Bir diyaliz membranı kullanılarak ürünün arıtılmasımolekül ağırlığı kesme: 3.5 kDa) ve liyofilize edilir. </li></ol> Nanopartiküllerin 2. Hazırlık <ol><li> HB 1 mg ve dimetil sülfoksit (DMSO) 0.5 ml paklitaksel 1 mg çözülür ve vorteks karıştırma 5 dakika ile DDW 0.5 ml karıştırılır. Daha sonra, 5 dakika daha vorteksleyin-karıştırılarak bu karışım içinde tavşan çözünür. </li><li> bir azot gazı akımı altında yumuşak bir 4 saat boyunca 70 ° C de çözücü ısı ortadan kaldırmak için. </li><li> DDW 1 ml HACE, HB oluşan bir film ve paklitaksel yeniden süspanse edin. kapsülsüz ilaçlar kaldırmak için bir şırınga filtresi (0.45 mikron gözenek boyutu) ile Filtresi. </li></ol> 3. İn Vitro Fototoksisite <ol><li> Akciğer kanseri hücre hatlarında nanopartiküllerinin Alım <ol><li> RPMI-1640 maddesi içeren% 10 (h / h) cenin sığır serumu ve% 1 (ağ / hac), penisilin-streptomisin hazırlayın. </li><li> Her / gözenek 1 x 10 5 hücre yoğunluğunda 24-kuyulu hücre kültürü kaplarına Tohum A549 hücreleri (kopyalarıngrubu). Nemlendirilmiş bir% 5 CO2 ve% 95 hava atmosferinde 37 ° C &#39;de 24 saat süreyle inkübe edilir. </li><li> hücre yapışmasının ardından orta kaldırmak ve fosfat tamponlu tuz (PBS) 1 ml ekleyerek hücreleri yıkayın. </li><li> 2 uM / ml HB bir son konsantrasyona PBS içinde nano-tanecikleri içinde çözülür. Daha sonra, karanlıkta 4 saat her bir PBS boş NP, HB-NPlerin veya HB-p-NP 1 ml kuluçkaya bırakılır. </li><li> çözeltinin Tümünü çıkarın ve soğuk PBS 1 ml ekleyerek hücreleri yıkayın. bir kez daha yıkama adımı yineleyin. Taze kültür ortamı. </li></ol></li><li> Hücre canlılığı deneyi <ol><li> PDT lif altında hücre kültürü plakası yerleştirin (kuyuya PDT fiber mesafenin 1 cm). Lazer güvenlik gözlükleri takın ve değişik süreler için karanlıkta bir PDT lazer (630 nm, 400 mW / cm2) ile hücrelerin aydınlatmak: 0, 5, 10, 20, 30, ve 40 saniye (0, 2, 4 , 8, 12 ve 16 J / cm2). Daha sonra, karanlıkta, 24 saat süre ile hücrelerin inkübe edin. </li><li>orta aspire ve soğuk PBS 1 ml ekleyerek hücreleri yıkayın. bir kez daha yıkama adımı yineleyin. </li><li> Her bir oyuğa sitotoksisite ölçüm solüsyonu 10 ul ekle. 2 saat boyunca karanlıkta inkübe edin. </li><li> Bir mikrolevha okuyucu kullanılarak 450 nm&#39;de absorbans ölçülür. </li></ol></li><li> mikroskopik analiz <ol><li> PDT lif altında hücre kültürü plakası yerleştirin (kuyuya PDT fiber mesafenin 1 cm). Lazer güvenlik gözlükleri takın ve 0, 20 veya 40 saniye (0, 8, ya da 16 J / cm2) için karanlıkta bir PDT lazer (630 nm, 400 mW / cm2) hücreleri aydınlatır. Daha sonra, karanlıkta, 24 saat süre ile hücrelerin inkübe edin. </li><li> orta aspire ve soğuk PBS 1 ml ekleyerek hücreleri yıkayın. bir kez daha yıkama adımı yineleyin. </li><li> anneksin V-FITC 50 ul ve 4 &#39;, 6-diamidino-2-fenilindol (DAPI / ml 1.5 ug) 50 ul ekle. Yavaşça plakayı çalkalayın ve karanlıkta, oda sıcaklığında 15 dakika boyunca inkübe edin. NOT: anneksin V-FI kullanınfloresan mikroskop kiti TC. </li><li> soğuk PBS 1 ml ekleyerek hücreleri yıkanır. bir kez daha yıkama adımı yineleyin. Taze PBS hücrelerin tutun. 100X büyütmede ışık mikroskobu kullanılarak apoptotik hücreleri tanımlamak. </li></ol></li><li> Flöresanla aktive edilen hücre tasnif (FACS) analizi <ol><li> PDT lif altında hücre kültürü plakası yerleştirin (kuyuya PDT fiber mesafenin 1 cm). Lazer güvenlik gözlükleri takın ve 0, 20 veya 40 saniye (0, 8, ya da 16 J / cm2) için PDT lazer (630 nm, 400 mW / cm2) hücreleri aydınlatır. Daha sonra, karanlıkta, 24 saat süre ile hücrelerin inkübe edin. </li><li> orta aspire ve soğuk PBS 1 ml ekleyerek hücreleri yıkayın. bir kez daha yıkama adımı yineleyin. </li><li> Ve transfer 100 ul; 1 × bağlama tamponu, 1 ml hücrelerin tekrar (0.1 M HEPES / NaOH (pH 7.4), 1.4 M NaCl, ve 25 mM CaCl2 9 kısım saf su 10x bağlanma tamponu 1 bölüm seyreltilmiş) 5 ml numune çözeltisininkültür tüpü. </li><li> anneksin V-FITC 5 ul ve propidyum iyodür (PI) 5 ul ekle. Tüp hafifçe karıştırın ve karanlıkta, oda sıcaklığında (RT) 15 dakika boyunca inkübe edin. 1 × bağlama tamponu 400 ul ekleyin. NOT: floresan mikroskop kiti anneksin V-FITC ve PI kullanın. </li><li> FACS 14 ile apoptotik hücreleri tanımlamak. PI ve anneksin V-FITC uyarma lazer çizgileri sırasıyla 488 nm ve 635 nm vardır. sırasıyla 610 ± 20 nm ve 660 ± 20 nm PI ve anneksin V-FITC floresan emisyonunu ölçün. 10.000 olaylar başına Akış sitometresindeki edinilen hücreleri toplayın. </li></ol></li></ol> Tümör taşıyan farelerde 4. in vivo anti-kanser etkisi <ol><li> Akciğer kanseri kaynaklı fare modeli <ol><li> 0.1 ml RPMI-1640 ortamı içinde 1 x 10 6 A549 hücreleri hazırlamak; buz tutmak. </li><li> Bir ksilazin bir IP enjeksiyonu ve tiletamin ve zolazepam (1 karışımı ile fareler anestezi: 2, 1 mL /kilogram). nazikçe fare cildin küçük bir kat kısma doğru anesthetization onaylayın. anestezi altında iken kuruluğunu önlemek için gözleri veteriner merhem kullanın. NOT: Hiçbir hareket görülürse, hayvan deneyleri başlatmak için yeterli anestezi olduğunu. </li><li> (- 7 haftalık, 20-22 g 6) ve BALB / c erkek çıplak farelerin sol yanları içine hücreleri enjekte edilir. </li><li> onlar kafes etrafında hareket başlayana dek fareler gözlemleyerek tutun. NOT: sternum yatma korumak için yeterli bilinci yerine kadar gözetimsiz bir hayvan bırakmayın. Tamamen iyileşene kadar diğer hayvanların şirkete ameliyat geçirmiş bir hayvan iade etmeyin. spesifik patojen free (SPF) koşullar altında fareler tutun. </li><li> Her gün kaliperler ile tümör boyutunu ölçmek. Tümör boyutu hacminde yaklaşık 200 mm 3 ulaştığında denemeyi başlatmak, (genişlik 2 × uzunluk) / 2 olarak tümör hacmi (mm3) hesaplayın. </li&gt; </ol></li><li> Antikanser etkinlik çalışması <ol><li> Rasgele 4 gruba (her grupta n = 10) olarak fareler bölün. </li><li> (: 2, 1 mL / kg 1) ksilazin bir IP enjeksiyonu ve tiletamin ve zolazepam karışımıyla fareler anestezi. nazikçe fare cildin küçük bir kat kısma doğru anesthetization onaylayın. anestezi altında iken kuruluğunu önlemek için gözleri veteriner merhem kullanın. NOT: Hiçbir hareket görülürse, hayvan deneyleri başlatmak için yeterli anestezi olduğunu. </li><li> 2 mg / ml HB bir son konsantrasyona PBS içinde nano-tanecikleri içinde çözülür. İki kez günlerde 0 ve 7 kuyruk damarından (2 mg / kg HB gibi) aracılığı ile PBS, ücretsiz HB, HB-NP, ya da HB-P-NP enjekte edilir. </li><li> onlar kafes etrafında hareket başlayana dek fareler gözlemleyerek tutun. NOT: sternum yatma korumak için yeterli bilinci yerine kadar gözetimsiz bir hayvan bırakmayın. Tamamen kadar diğer hayvanların şirkete ameliyat geçirmiş bir hayvan dönüş yokiyileşti. Karanlık ve özel SPF koşullarında kafesleri tutun. </li><li> (: 2, 1 mL / kg 1) 24 saat, her enjeksiyondan sonra, ksilazin bir IP enjeksiyonu ve tiletamin ve zolazepam karışımı ile fareler anestezi. nazikçe fare cildin küçük bir kat kısma doğru anesthetization onaylayın. anestezi altında iken kuruluğunu önlemek için gözleri veteriner merhem kullanın. NOT: Hiçbir hareket görülürse, hayvan deneyleri başlatmak için yeterli anestezi olduğunu. </li><li> (Tümöre PDT fiber mesafenin 1 cm) PDT fiber altında tümör sitesini yerleştirin. , Lazer güvenlik gözlükleri takın düğmesini kapatın ve iki kez 1 gün ve 8 500 sn (200 J / cm2) için PDT lazer (630 nm, 400 mW / cm2) ile tümör aydınlatmak. </li><li> onlar kafes etrafında hareket başlayana dek fareler gözlemleyerek tutun. sternal yatma korumak için yeterli bilinci yerine kadar gözetimsiz bir hayvan bırakmayın. undergon sahip bir hayvan dönüş yokdiğer hayvanların şirkete e cerrahisi tamamen iyileşene kadar. </li><li> SPF koşullarında kafesleri tutun. lazer tedavisi sonrası 24 saat boyunca karanlıkta kafesleri koruyun. </li><li> Görme tümör hacmi ve tümör sitesinde her gün değişiklikleri izlemek. Kaliperler ile tümör boyutunu ölçmek ve (genişlik 2 × uzunluk) / 2 (mm 3) olarak hacmini hesaplayın. PDT sonra tümör yüzey değişikliklerine kontrol etmek için her gün tümör sitelerin fotoğraf çekmek. </li><li> günde 16, izofluran kullanarak, terminal anestezi ile grup başına beş fareler kurban. </li><li> Forseps ve makas ile, dış deriyi kesip tümörler 15 maruz kalmaktadır. Dikkatle hasat. ,% 10 formalin içinde bunları düzeltmek parafin onları gömmek ve histolojik analiz 16 hematoksilen ve eozin (H &amp; E) ile leke. </li><li> izleme 45 gün sonra, izofluran kullanarak, terminal anestezi kalan fareler kurban. </li></ol></li></ol>

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The authors have nothing to disclose.

Bu çalışmada en önemli adım doğru lazer koşullarının seçilmesi olduğunu: dalga boyu, güç ve ışınlama süresi. Belirli photosensitizer uygun ışık uygun dalga boyu PDT için gereklidir. Biz çıkış gücü, birçok pilot çalışmalara dayanarak 400 mW / cm 2 olarak belirlenmiştir başka önemli faktör oldu hypocrellin B için uygun olan bir 630-nm lazer kullanıldı. 400 mW / cm 2 altında çıkış güçleri herhangi bir terapötik etkisini göstermek için çok zayıf iken ı...

Photodynamic therapy (PDT) is composed of three major factors: photosensitizers, light, and oxygen. PDT is reported as a promising treatment for various cancers3. When the photosensitizers are administered into the cancer patient, they selectively accumulate in the tumor tissues. When the proper wavelength of light is applied, the highly reactive singlet oxygen and other free radicals lead to tumor cell damage4. 
Lung cancer was introduced as one of the first applications for PDT in the early 1980s5. PDT provides several advantages in treating lung cancer. Since PDT is a non-invasive and non-surgical treatment, it is an attractive alternative choice for the patients in whom surgical resection is inappropriate.
There have been many challenges to enhance the cancer-targeting efficacy of the photosensitizers. Increasing photosensitizer accumulation in cancer sites and decreasing accumulation in normal tissues are the identical goals for the cancer-targeting studies. A variety of targeted drug delivery systems, such as polymers, liposomes, and nanoparticles are adopted as photosensitizer carriers6-8. In our previous studies, nanoparticles effectively increased the cancer-targeting abilities of the photosensitizers9,10. Nanoparticles are ideal cancer-targeting carriers since they possess both passive and active targeting abilities. The leaky tumor vessels provide opportunity for nano-sized carriers to accumulate easily in tumors, which is well-known as the enhanced permeability and retention (EPR) effect11,12. The interaction between the nanoparticles and the specific receptors on cancer cells enables active cancer targeting. In this study, we prepared hyaluronic acid-based nanoparticles to interact with CD44, the major hyaluronic acid receptor that is overexpressed on lung cancer cells13. 
To maximize the anticancer efficacy, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may permit an increased therapeutic effect. Furthermore, decreased doses of both the photosensitizer and the anticancer drug can diminish adverse effects. We selected hypocrellin B (HB) as a novel photosensitizer in the present study. HB is isolated from Chinese medicinal fungus Hypocrella bambuase. Shang et al. reported that HB-based PDT possesses a higher anticancer efficacy when compared to hematoporphyrin derivative-based PDT1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for various cancers, including lung cancer2. 
Herein, we compared the anticancer efficacies of photosensitizer (hypocrellin B, HB) solution, photosensitizer-encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>oligo hyaluronic acid</td>
			<td>Bioland Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Y30 (ceramide 3B; mainly N-oleoyl-phytosphingosine)</td>
			<td>Doosan Biotech Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>adipic acid dihydrazide</td>
			<td>Sigma Aldrich</td>
			<td>A0638</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide</td>
			<td>Sigma Aldrich</td>
			<td>39391</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(chloromethyl)benzoyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>270784</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>T0546</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filter</td>
			<td>Sartorius Stedim Biotech GmbH</td>
			<td>17762</td>
			<td>15 mm, RC, PP, 0.45 &#181;m</td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-GeBAflex tubes</td>
			<td>Gene Bio-Application Ltd.</td>
			<td>D070-12-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>Taihua Corporations</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite (Filter agent)</td>
			<td>Sigma Aldrich</td>
			<td>6858</td>
			<td>See step 1.4</td>
		</tr>
	</tbody>
</table>

anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Biz yukarıda belirtilen tekniklerle HB-NP ve HB-p-NP hem hazırlandı. HB-NP ve HB-P-NPS ortalama çapları sırasıyla 220,9 ± 3.2 nm ve 211,9 ± 1.6 nm idi. Işık ışınlama ve ardından PBS boş NP, HB-NPlerin ve HB-p-NPlerin ile inkübasyon 4 saat (0 J 16 / cm2) sonra A549 hücrelerinin hücre canlılığı ışığı yok. Şekil 1 &#39;de gösterilmektedir, HB-P-NP-tedavi hücreler 81.28 ±...

Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Akciğer Kanseri Hedefli Nanopartiküller ile Fotodinamik Tedavi antikanser Etkinliği

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted

Research

JoVE Journal

Cancer Research

8.9K Görüntüleme.  Seoul National University Bundang Hospital. Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Video: Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

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.

Akciğer tümör hücre işe alım tahlil

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

Kanser Araştırmaları

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pankreas Kanseri Floresan Ortotopik Fare Modeli

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Bir Vakum Stabilize Görüntüleme Penceresi ile Akciğer uzun vadeli Yüksek Çözünürlüklü Intravital Mikroskopi

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

İnsan hipofiz adenom doku Proteom analizi için kütle spektrometresi yöntemleri ile birleştiğinde iki boyutlu Jel Elektroforez

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Tümör hücre görselleştirme akciğer dokusu içinde dolaşan bir akciğer kolonizasyon tahlil kurulması

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

Preklinik antikanser Coumarin OT48 pulcuklarının, koloni oluşumu deneyleri ve zebra balığı Xenografts Bioactivity değerlendirilmesi

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Antikanser antikor etkinliğinin Antineoplastic uyuşturucu tarafından kullanılmasının muhtemelen: antikor-uyuşturucu Synergism kombinasyon Dizin denklem kullanılarak tespiti

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Mezenkimal kök hücre izolasyon hamuru doku ve kanser hücreleri ile ortak kültür ilişkileri incelemek için

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma içinde alanları terapi tedavi tümör klinik uygulama

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Üç Afatinib dirençli akciğer adenokarsinoma PC-9 hücre hatları Afatinib artan dozlarda ile geliştirilen kurulması ve karakterizasyonu

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...