Kanser kök hücre lizatı ile yüklenmiş dendritik hücrelerin antitümör immün yanıtlarının incelenmesi
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Dosyalar
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kanserin kökü olarak ifade edilen "kanser kök hücre"lerini ortadan kaldırmaya
yönelik vücudun kendi savunma hücrelerinin kullanılması, kanser immünterapisinin
en önemli devrimlerin birisidir. Kanser kök hücreleri (KKH), kendini yenileme ve
tümörü başlatma kapasitelerine sahip eşsiz bir küçük alt popülasyonu temsil
etmektedir. Kanser immünoterapisinin temel amacı, KKH'lere karşı güçlü bir
bağışıklık tepkisinin uyarılmasıdır. Güçlü ve aynı zamanda karmaşık olan bağışıklık
sistemi hücre, doku ve organların birlikteliğini yansıtan bir ordudan oluşmaktadır.
Ordunun en önemli üyelerinden biri olan dendritik hücreler (DH), doğal ve adaptif
bağışıklık arasında önemli bir köprü gören profesyonel hücrelerdir. Matürasyon
sürecini takiben, DH’ler anti-tümör yanıtlarının indüksiyonu ve sürdürülmesinde
merkezi rol oynamaktadır. Bununla birlikte, kanser hastalarında DH-bazlı tedavinin
terapötik etkinliği yeterince tatmin edici değildir ve yeni stratejilerin geliştirilmesine
ihtiyaç vardır. DH’lerin yüzeyinde bulunan sialik asitler antijenlerin alınması,
işlenmesi, T-hücrelerine sunulması ve immünolojik sinapsların oluşturulmasında
oldukça kritik öneme sahiptir.
Bu çalışmada, i)sialik asitleri uzaklaştırıldıktan sonra KKH antijenleri ile muamele
edilerek matüre edilen DH’lerdeki değişikliklerin çeşitli yönlerle incelenmesi, ii)
KKH’lerine karşı immün yanıtın değerlendirilmesi amaçlanmıştır. Meme kanser kök
hücreleri (CD44+/ CD24-/low) floresanla aktive hücre ayırma (FACS) cihazı ile izole
edildikten sonra KKH antijenleri içeren lizat elde edilmiştir. Kemik-iliğinden izole
edilen DH’ler CD11c kullanılarak karakterize edilmiştir. DH’lerin yüzeyindeki sialik
asitler nöraminidaz yardımıyla uzaklaştırıldıktan sonra KKH lizatı ile muamele
edilmiştir. KKH lizatının dendritik hücreler üzerine olan etkileri canlılık, apoptoz ve
hücre döngüsü analizleri yapılarak değerlendirilmiştir. İmmatür (imDH) ve matür
(mDH) hücrelerdeki CD40, CD80 ve CD86 gibi kostimülatör moleküllerdeki
değişimler akım sitometrisi kullanılarak analiz edilmiştir. Çeşitli sinyal yolakları ile
ilişkili genler PCR ile incelenmiştir. Dendritik hücrelerde meydana gelen biyokimyasal değişimler (glikojen, lipid, protein, nükleik asit) ATR-FTIR
yardımıyla analiz edilmiştir. Hücrelerde meydana gelen morfolojik değişimler SPM
ve histokimyasal boyamalar ile incelenmiştir. Hücrelerdeki IL-12 üretimindeki
değişimler ELISA yöntemi ile analiz edilmiştir. In vivo çalışmalarında, meme kanser
modeli oluşturularak imDH ve mDH’lerin immün yanıt etkileri çeşitli parametreler
ile değerlendirilmiştir.
Çalışmadan elde edilen veriler, imDH’lere ile kıyaslandığında mDH’lerin daha etkili
immün yanıt oluşturduğunu göstermiştir. KKH antijenleri ile muamele edilen
DH’lerin podozom yapılar oluşturduğu ve aktin, gelsolin, talin, Arp/2, WASp gibi
hücre iskelet elemanlarının antijen alınımında etkin rollerinin olduğu gösterilmiştir.
Matürasyon aşamasından sonra, CD86, CD80 ve CD40 kostimülatör moleküllerde
artış tespit edilmiştir. DH’lerin farklılaşma ve fonksiyonu ile ilişkili Notch, MAPK
ve interlökin sinyalizasyonunda önemli değişimler meydana gelmiştir. Nükleik asit,
lipid, karbonhidrat ve protein yapılarında önemli farklılıkların olduğu ve
matürasyonda lipit dropletlerinde azalış glikojen miktarında artışın olduğu tespit
edilmiştir. Morfolojik analizler hücre büyüklüğü, yüzey alanı, pürüzlülük özelliklerin
değiştiğini göstermiştir. Matürasyondan sonra, IL-12 üretiminde artış meydana
gelmiştir. Anti-tümör immün yanıtlarının incelenmesi için yapılan kanser modelinde,
mDH ile aşılanan farelerde tümör hacmi ve ağırlığında önemli bir azalmanın olduğu
tespit edilmiştir. Bununla birlikte, proliferasyon ile ilişkili belirteçlerde azalma,
apoptotik indekste ise artış belirlenmiştir. imDH ile kıyaslandığında, mDH’lerde
CD4 ve CD8 oranlarında önemli artışların olduğu belirlenmiştir. mDH ile aşılamanın
metastazı engellediği gösterilmiştir. Epitelial mezenkimal geçişle ilişkili Snail, Slug
ve Twist gibi belirteçlerin ifadelerinde azalma tespit edilmiştir. Çalışmadan elde
edilen veriler, sialik asit uzaklaştırma ve KKH antijenleri ile etkileşimin DH’lerde
önemli moleküler, biyokimyasal, morfolojik ve fonksiyonel değişimler yarattığını
göstermiştir. Bu değişimler, DH’lerin anti-kanser immün yanıtlarının artırılmasına
katkı sağlamıştır.
The use of the body's own defensive cells to eliminate the "cancer stem cells" as the root of cancer is one of the most important revolutions of cancer immunotherapy. Cancer stem cells (CSCs) represent a unique small subpopulation that possess capacity for self-renewal and tumor initiation. The main purpose of cancer immunotherapy is to stimulate a strong immune response to CSCs. The immune system is powerful and also complex and consists of an army that reflects the relationship between cells, tissues and organs. Dendritic cells (DCs), one of the most important members of the army, are professional cells that serve as an important bridge between innate and adaptive immunity. Following the maturation process, DCs play an essential role role in the induction and maintenance of anti-tumor responses. However, the therapeutic efficacy of DC-based therapy in cancer patients is not satisfactory and new strategies need to be developed. Sialic acids on the surface of DCs are very critical in uptake, processing, and presentation to T-cell of antigen and formation of immunological synapses. The goal of this study is twofold: i) to investigate changes in DCs treated with CSCs antigens after removal of sialic acids in various aspects, ii) to evaluate of the immune response to CSCs. Breast cancer stem cells (CD44+/CD24-/low) were isolated by fluorescence activated cell sorting (FACS) and the lysates containing the antigens of CSCs were obtained. DCs isolated from bone-marrow were characterized using CD11c. After removal of the sialic acids from the surface of the DCs by neuraminidase, the DCs were treated with the CSCs lysate. The effects of CSCs lysate on dendritic cells were evaluated by cell viability, apoptosis and cell cycle assays. Changes in co-stimulatory molecules such as CD40, CD80 and CD86 in immature (imDC) and mature (mDC) cells were analyzed using flow cytometry. The genes associated with various signaling pathways were evaluated by PCR. Biochemical (glycogen, lipid, protein, nucleic acid) changes in the dendritic cells were analyzed using ATR-FTIR. Morphological changes in cells were examined visually by SPM and histochemical staining. Changes in IL-12 production in the cells were analyzed by ELISA assay. In the in vivo experiments, the immune response effects of imDC and mDCs were evaluated by using a variety of parameters. The data obtained from the study show that mDCs exhibit a more effective immune response compared to imDCs. It has been shown that DCs treated with CSCs antigens form podosome structures and cell skeleton elements such as actin, gelsoline, talline, Arp/2, WASp play an important role in antigen uptake. After the maturation, there was an increase in the expression of co-stimulatory molecules such as CD86, CD80 and CD40. Significant changes in Notch, MAPK and interleukin signaling pathways, which are known to be associated with the differentiation and function of DCs, have been identified. There were significant differences in nucleic acid, lipid, carbohydrate and protein structures, and a decrease in lipid droplets and an increase in glycogen after maturation were determined. Morphological analyzes showed significant changes in the size, surface area and roughness properties of the cells. After maturation, there was an increase in IL-12 production. In vivo tumor model for the investigation of anti-tumor immune responses, it was found that the mice vaccinated with mDC had a significant decrease in both tumor volume and weight. However, a decrease in proliferation-related markers and an increase in apoptotic index were determined. Significant increases in CD4 and CD8 ratios were found in mDCs compared to imDCs. Vaccination with mDCs has been shown to inhibit metastasis. A reduction in the expression of markers such as Snail, Slug and Twist associated with epithelial mesenchymal transition has been identified. The data obtained from this study showed that sialic acid removal and loading with CSCs antigens provide important molecular, biochemical, morphological and functional changes. These changes contributed to the increase of anti-cancer immune responses of DCs.
The use of the body's own defensive cells to eliminate the "cancer stem cells" as the root of cancer is one of the most important revolutions of cancer immunotherapy. Cancer stem cells (CSCs) represent a unique small subpopulation that possess capacity for self-renewal and tumor initiation. The main purpose of cancer immunotherapy is to stimulate a strong immune response to CSCs. The immune system is powerful and also complex and consists of an army that reflects the relationship between cells, tissues and organs. Dendritic cells (DCs), one of the most important members of the army, are professional cells that serve as an important bridge between innate and adaptive immunity. Following the maturation process, DCs play an essential role role in the induction and maintenance of anti-tumor responses. However, the therapeutic efficacy of DC-based therapy in cancer patients is not satisfactory and new strategies need to be developed. Sialic acids on the surface of DCs are very critical in uptake, processing, and presentation to T-cell of antigen and formation of immunological synapses. The goal of this study is twofold: i) to investigate changes in DCs treated with CSCs antigens after removal of sialic acids in various aspects, ii) to evaluate of the immune response to CSCs. Breast cancer stem cells (CD44+/CD24-/low) were isolated by fluorescence activated cell sorting (FACS) and the lysates containing the antigens of CSCs were obtained. DCs isolated from bone-marrow were characterized using CD11c. After removal of the sialic acids from the surface of the DCs by neuraminidase, the DCs were treated with the CSCs lysate. The effects of CSCs lysate on dendritic cells were evaluated by cell viability, apoptosis and cell cycle assays. Changes in co-stimulatory molecules such as CD40, CD80 and CD86 in immature (imDC) and mature (mDC) cells were analyzed using flow cytometry. The genes associated with various signaling pathways were evaluated by PCR. Biochemical (glycogen, lipid, protein, nucleic acid) changes in the dendritic cells were analyzed using ATR-FTIR. Morphological changes in cells were examined visually by SPM and histochemical staining. Changes in IL-12 production in the cells were analyzed by ELISA assay. In the in vivo experiments, the immune response effects of imDC and mDCs were evaluated by using a variety of parameters. The data obtained from the study show that mDCs exhibit a more effective immune response compared to imDCs. It has been shown that DCs treated with CSCs antigens form podosome structures and cell skeleton elements such as actin, gelsoline, talline, Arp/2, WASp play an important role in antigen uptake. After the maturation, there was an increase in the expression of co-stimulatory molecules such as CD86, CD80 and CD40. Significant changes in Notch, MAPK and interleukin signaling pathways, which are known to be associated with the differentiation and function of DCs, have been identified. There were significant differences in nucleic acid, lipid, carbohydrate and protein structures, and a decrease in lipid droplets and an increase in glycogen after maturation were determined. Morphological analyzes showed significant changes in the size, surface area and roughness properties of the cells. After maturation, there was an increase in IL-12 production. In vivo tumor model for the investigation of anti-tumor immune responses, it was found that the mice vaccinated with mDC had a significant decrease in both tumor volume and weight. However, a decrease in proliferation-related markers and an increase in apoptotic index were determined. Significant increases in CD4 and CD8 ratios were found in mDCs compared to imDCs. Vaccination with mDCs has been shown to inhibit metastasis. A reduction in the expression of markers such as Snail, Slug and Twist associated with epithelial mesenchymal transition has been identified. The data obtained from this study showed that sialic acid removal and loading with CSCs antigens provide important molecular, biochemical, morphological and functional changes. These changes contributed to the increase of anti-cancer immune responses of DCs.
Açıklama
Anahtar Kelimeler
Kanser Kök Hücre, Dendritik Hücre Aşısı, İmmünterapi, Sialik Asit, Matürasyon, İmmün Yanıt, Cancer Stem Cell, Dendritic Cell Vaccine, Immunotherapy, Sialic Acid, Maturation, Immune Response
