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Assessment of cytokine mRNA expression profiles in tumor microenvironment and peripheral blood mononuclear cells of patients with high-grade serous carcinoma of the ovary
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynecology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
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2017 (English)In: Journal of Cancer Science & Therapy, ISSN 1948-5956, Vol. 9, no 5, p. 422-429Article in journal (Refereed) Published
Abstract [en]

Objective: Tumor establishment, metastatic spreading and poor survival in ovarian cancer is strongly associated with progressive derangement of the patient’s immune system. Accumulating evidence suggests that immune impairment is influenced by the production and presence of cytokines in the tumor microenvironment. Methods: Cytokine mRNA profiles in tumor tissue and peripheral blood mononuclear cells (PBMC) were analyzed in patients with high grade serous carcinoma (HGSC) of the ovary and compared it to patients with benign ovarian conditions and controls with normal ovaries. Cytokine assessment was done by real-time quantitative RT-PCR and specific primers and probes for 12 cytokines-IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β/LTA, TGF-β1, and GM-CSF chosen to distinguish between cytotoxic Th1, humoral Th2, regulatory Th3/Tr1 and inflammatory responses. Results: The cytokine mRNA response in the HGSC patients was significantly up regulated compared to patients with benign ovarian conditions and normal ovary controls confirming the immunogenicity of HGSC and implying immune recognition and reaction locally in the tumor microenvironment and systemically in the peripheral blood.There was an up-regulation of inflammatory and inhibitory cytokine mRNA promoting tumor progression, T-regulatory cell priming and T-regulatory cell-mediated immune suppression. In contrast, there was an inability to mount the crucially important IFN gamma response needed for upregulation of the cytotoxic anti-tumor response in the local microenvironment. In addition, systemic IL-4- mediated Th2 response prevailed in the peripheral blood deviating the systemic defense towards humoral immunity. Conclusions: Taken together, these results suggest local and systemic cytokine cooperation promoting tumor survival, progression and immune escape. Our study confirms and extends previous investigations and contributes to the evaluation of potential cytokine candidates for diagnostic cytokine mRNA profiles and for future therapeutic interventions based on cytokine inhibition.

Place, publisher, year, edition, pages
2017. Vol. 9, no 5, p. 422-429
Keywords [en]
Cytokines, High-grade serous ovarian carcinoma (HGSC), EOC, Tumor microenvironment, Tumor inflammation, Immune suppression
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-138256DOI: 10.4172/1948-5956.1000453OAI: oai:DiVA.org:umu-138256DiVA, id: diva2:1133365
Available from: 2017-08-15 Created: 2017-08-15 Last updated: 2021-03-02Bibliographically approved
In thesis
1. Immune modulation in serous epithelial ovarian cancer: focus on the role of tumor-derived exosomes
Open this publication in new window or tab >>Immune modulation in serous epithelial ovarian cancer: focus on the role of tumor-derived exosomes
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Serous epithelial ovarian cancer (EOC) is a potent suppressor of the immune defense. Here, we studied interactions between EOC and the immune system that lead to escape from tumor immune surveillance. We explored: 1) tumor escape from cytotoxicity by exosome-mediated modulation of the NK-cell receptors NKG2D and DNAM-1; 2) cytokine mRNA profiles in the EOC microenvironment and peripheral blood and their role in the suppression of the anti-tumor immune responses; 3) expression of long non-coding (lnc) RNAs in EOC tumors and exosomes.

We found that EOC-secreted exosomes carried MICA/B and ULBP1-3, ligands of NKG2D, and could downregulate the NKG2D receptor and impair NKG2D-mediated cytotoxicity. In contrast, the DNAM-1 receptor ligands PVR and nectin-2 were seldom found in exosomes and were not associated with the exosomal membrane leaving the DNAM-1 receptor-mediated cytotoxicity intact. We compared cytokine mRNA expression in the tumor microenvironment and in immune cells of peripheral blood in EOC patients and patients with benign ovarian conditions. EOC patients were unable to mount an IFN-gamma mRNA response needed for tumor cell elimination. Instead, there was a significant up-regulation of inflammation and immune suppression i.e. responses promoting tumorigenesis and T-regulatory cell priming that suppress anti-tumor immunity. In addition, we studied lncRNAs in tissues and sera exosomes from EOC and benign ovarian conditions aiming to assess the lncRNA(s) expression profile and look for lncRNA(s) as possible marker(s) for early diagnosis. We found a deregulated lncRNAs expression in EOC tissues that correlated well with the lncRNAs expression in exosomes. Candidate lncRNAs with the highest expression and abundance were suggested for evaluation as EOC diagnostic markers in a future large cohort study.

Our studies of EOC tissue and EOC exosomes highlight the immunosuppressive tumor microenvironment and the complex tumor exosome-mediated network of immunosuppressive mechanisms, and provide a mechanistic explanation of the observation that NKG2D-mediated cytotoxicity does not function in EOC patients and is partially replaced by the accessory DNAM-1 dependent cytotoxic pathway. The deregulated lncRNAs expression in EOC tissues and exosomes might serve for diagnostic purposes but could also be a potential risk of spreading tumor-derived lncRNAs in EOC exosomes to recipient cells throughout the body.

Place, publisher, year, edition, pages
Umeå: Umea University, 2017. p. 68
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1901
Keywords
ovarian cancer, EOC, HGSC, exosomes, NKG2D, MICA/B, ULBP, DNAM-1, PVR, nectin-2, lncRNAs, immune suppression, anti-tumor immunity, NK-cell cytotoxicity, cytokines, Th1, Th2, T regulatory response, inflammation, tumor microenvironment
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-138264 (URN)978-91-7601-731-9 (ISBN)
Public defence
2017-09-15, Horsal D, Unod T9, Norrland's Universitetssjukhus, Umea, 09:00 (English)
Opponent
Supervisors
Available from: 2017-08-25 Created: 2017-08-16 Last updated: 2018-06-09Bibliographically approved
2. Mechanisms for immune escape in epithelial ovarian cancer
Open this publication in new window or tab >>Mechanisms for immune escape in epithelial ovarian cancer
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tumors develop mechanisms to subvert the immune system, constituting immune escape. Epithelial ovarian cancer (EOC), the deadliest of all gynecological malignancies, uses a variety of mechanisms to undermine immune surveillance, aiding its establishment and metastatic spreading. Despite progress in oncoimmunology, a lot remains unknown about the cancer-immune system interplay. The aim of this thesis was to study tumor-mediated mechanisms for immune escape in EOC patients, focusing on the role of cytokines and EOC- derived exosomes. 

Cytokines are key molecules regulating immune effector functions in health and disease. We used real-time RT-qPCR and a set of primers and probes for 12 cytokines, discriminating between different immune responses and compared the cytokine mRNA expression profiles locally in the TME and systemically in peripheral blood immune cells of EOC patients, to women with benign ovarian conditions and women with normal ovaries. The cytokine mRNA expression was in general most prominent in EOC patients, confirming the immunogenicity of EOC. We found significant dominance of inflammatory and immunosuppressive/ regulatory cytokines, known to promote tumor progression by priming and activating T regulatory cell-mediated immune suppression. In contrast, IFN-γ, crucially important for evoking a cytotoxic anti-tumor response, was not upregulated. Instead, a systemic increase of IL-4 prevailed, deviating the immune defense towards humoral immunity. With regard to our cytokine study, we performed comparative analyses of cytokine mRNA versus protein expression in the EOC cell lines OVCAR-3 and SKOV-3. We found that cytokine mRNA signals were universally detected, and in some instances translated into proteins, but the protein expression levels depended on the material analyzed and the method used. Due to the high sensitivity of real-time RT-qPCR, we suggest that cytokine mRNA expression profiles can be used for some instances, such as in studies of mechanistic pathways and in comparisons between patient groups, but cannot replace expression at the protein level. 

Exosomes are nanometer-sized vesicles of endosomal origin, released by virtually all cells, participating in normal and pathological processes. Like many tumors, EOC is a great exosome producer. We isolated exosomes from EOC ascitic fluid and supernatant from tumor explant cultures to study their effect on the NK cell receptors NKG2D and DNAM-1, involved in tumor killing. We found that EOC exosomes constitutively expressed NKG2D ligands on their surface while DNAM-1 ligand expression was rare and not associated with the exosomal membrane. Consistently, the major cytotoxic pathway of NKG2D-mediated killing was dysregulated by EOC exosomes while the accessory DNAM-1- mediated pathway remained unchanged. Our results provide a mechanistic explanation to the previously made observation that in EOC patients, tumor killing is only dependent on the accessory DNAM-1 pathway. Following these iii iv results, we studied NKG2D-mediated cytotoxicity in vivo in EOC patients before and after surgery. We found that the serum exosomes isolated from EOC patients were able to downregulate the NKG2D receptor and suppress NKG2D-mediated cytotoxicity in NK cells from healthy donors, in a similar way as exosomes from EOC ascites. We also found that surgery of the primary EOC tumor has a beneficial effect on the patients’ anti-tumor cytotoxic immune response. One mechanistic explanation could be a decrease in circulating NKG2D ligand- expressing exosomes, thus improving the cytotoxic NK cell function. 

In conclusion, our results contribute to the understanding of the mechanisms responsible for tumor immune escape in general, and in EOC patients in particular, and might be useful in developing novel antitumor therapies. Our studies highlight the prevailing immunosuppression in the local TME and the immunosuppressive role of EOC exosomes. Furthermore, they support the notion that cancer surgery is also a way of removing exosome-producing cells and reducing the serum concentration of immunosuppressive exosomes, thus boosting the patients’ cytotoxic anti-tumor response. 

Abstract [sv]

Äggstockscancer är den dödligaste av alla gynekologiska cancersjukdomar. Sjukdomen ger sällan några symtom hos kvinnan förrän den har spridit sig och upptäcks således oftast sent. Därför är det viktigt att genom forskning försöka förstå mekanismerna bakom cancerns spridning och hitta markörer för tidigare diagnostik och nya sätt att behandla sjukdomen. En del av vårt immunförsvar är programmerat för att kunna känna igen och eliminera förändrade eller skadade celler, exempelvis cancerceller. Ibland utvecklar cancerceller en förmåga att nedreglera och undkomma immunförsvaret. När det sker märker inte kroppen att en tumör bildas och sprids. Syftet med denna avhandling var att studera biologiska mekanismer som ger äggstockscancer förmåga att inaktivera immunförsvaret. Äggstockscancer är nämligen en tumörtyp som har utvecklat flera mekanismer för att nedreglera kroppens immunförsvar i syfte att undvika upptäckt. Det är ännu inte helt klarlagt hur detta går till. För att försöka förstå hur äggstockscancer kan undkomma immunförsvaret har vi i detta forsknings- projekt undersökt cytokiner i tumörvävnad och exosomer som tumören utsöndrar. Cytokiner är proteiner som agerar som lösliga signalmolekyler. Exosomer är mycket små ”signalbubblor” som innehåller olika molekyler som också finns i deras modercell. Cytokiner och exosomer utsöndras av såväl kroppens friska som sjuka celler för att cellerna ska kunna kommunicera med varandra utan att vara i direkt kontakt. 

I det första delarbetet kartlades nivåerna av cytokin-mRNA, dvs. det genetiska förstadiet till cytokinproteiner, i tumörvävnad och i cirkulerande vita blodkroppar från kvinnor med äggstockscancer, och jämfördes med motsvarande uttryck hos kvinnor med godartade tillstånd i äggstockarna och kvinnor med normala äggstockar. Vi fann förhållandevis högre uttryck av cytokin-mRNA hos cancerpatienterna vilket tyder på att immunförsvaret i större utsträckning är aktiverat hos dem. Vidare dominerade nedreglerande och inflammatoriska cytokin-mRNA hos kvinnor med äggstockscancer. Cytokin-mRNA som är viktiga för vårt eget cancerförsvar och kroppens egna mördarceller visades vara nedreglerade. Mönstret sågs också i cirkulerande vita blodkroppar, vilket visar att tumören har en förmåga att påverka immunförsvaret, inte bara lokalt i tumören, utan i hela kroppen. 

I delarbete II undersökte vi sambandet mellan cytokinernas mRNA respektive proteinuttryck. Proteinet är aktivt i kroppen medan mRNA är dess genetiska förstadium. Eftersom cytokinproteiner endast uttrycks kortvarigt och lokalt samt påverkas mycket av hur ett prov tas och senare hanteras i laboratoriet, är det svårt att dra slutsatser av en proteinanalys. I en modell med odlade cancerceller jämförde vi skillnaden mellan cytokinernas mRNA- och proteinuttryck. Vi fann att metoden för proteinanalys har ett snävt spann för att kunna upptäcka protein. Om inget protein kan detekteras behöver det inte betyda att det inte finns utan provet kan behöva spädas eller koncentreras. Analys av mRNA är mindre v vi ömtåligt och har hög träffsäkerhet. Vi fann att äggstockscancerceller producerade cytokin-mRNA som kan leda till mätbara proteinnivåer. Det finns vissa fördelar med mRNA-analysen som gör att den kan användas t ex för att kartlägga en individuell cytokin mRNA-profil som kan användas vid diagnostik eller behandling. 

I delarbete III analyserades exosomer utsöndrade av äggstockscancer i form av odlade celler, vävnad, blod och ascites. Ascites är vätska i bukhålan som kvinnor med äggstockscancer kan utveckla. Vi ville undersöka om exosomer utsöndrade av cancerceller påverkar kroppens mördarceller. Vid äggstockscancer förefaller en aktiverande signalväg i mördarcellerna delvis vara utslagen. Vår hypotes var att exosomerna orsakar detta. Signalen går via något som kallas ligand- receptorsystem. En ligand är en molekyl som kan binda till en mottagarmolekyl (receptor) fäst på mördarcellernas yta. När liganden binder till receptorn får mördarcellen en signal om att den ska aktiveras och döda en målcell. Vi fann att äggstockscancer-exosomer uttrycker ligander för receptorn NKG2D på sin yta. Det är den viktigaste signalvägen i avdödandet av cancerceller. På så vis agerar exosomerna ”lockbete” och lurar mördarcellerna att nedreglera sina NKG2D- receptorer. Därmed aktiveras inte mördarcellerna för att döda cancerceller. En mindre effektiv receptor, DNAM-1, verkar istället dominera avdödande av äggstockscancerceller. Vi fann att dess ligander inte uttrycks på exosomernas yta och således lämnas denna bana opåverkad. 

Om en tumör kan opereras bort i sin helhet förbättras kvinnans prognos avseende överlevnad. I delarbete IV ville vi studera effekten av kirurgi genom att undersöka blodprover tagna före och efter operationen. Vi undersökte hur en operation påverkar mängden exosomer i blodet, de molekyler som exosomerna uttrycker och om effektiviteten hos kvinnans mördarceller påverkas. Oavsett om hela tumören opererats bort eller inte kunde vi hos samtliga patienter se en förbättrad funktion hos mördarcellerna efter operationen. En möjlig förklaring till detta är den minskade mängden exosomer i blodet som kunde observeras hos en grupp patienter efter kirurgi. Dessa kvinnor hade dessutom ett högre uttryck av NKG2D receptorn hos mördarcellerna efter operationen. 

Sammanfattningsvis har vi funnit nya mekanismer för hur äggstockscancer nedreglerar och undkommer immunförsvaret. Förhoppningsvis kan denna nya kunskap utgöra ytterligare en pusselbit i sökandet efter nya diagnostiska verktyg, nya effektiva behandlingar och i förlängningen bidra till en förbättrad överlevnad för kvinnor som drabbas av äggstockscancer. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2021. p. 68
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2119
Keywords
human, ovarian cancer, high-grade serous cancer, EOC, HGSC, HGSOC, tumor microenvironment, immune escape, immune suppression, cytokines, exosomes, NKG2D, MICA/B, ULBP1-3, DNAM-1, surgery
National Category
Obstetrics, Gynecology and Reproductive Medicine Cancer and Oncology Immunology in the medical area
Research subject
Obstetrics and Gynaecology
Identifiers
urn:nbn:se:umu:diva-180917 (URN)978-91-7855-484-3 (ISBN)978-91-7855-483-6 (ISBN)
Public defence
2021-03-26, Betula, målpunkt L0, plan 0, NUS, UMEÅ, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2021-03-05 Created: 2021-03-02 Last updated: 2021-03-04Bibliographically approved

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