Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, the 5-year survival rate being less than 5%. At the time of diagnosis, 90% of PDACs extend beyond the pancreas and distant metastases are often present. Due to aggressive growth, local expansion and early appearance of metastasis, primary PDAC tumours are local enough for curative surgical resection in only 10–20% of the cases. Adjuvant chemotherapy is indicated in these curative-treated cases, with slight improvement in survival. PDAC is considered to represent a heterogeneous group of biologically and prognostically different malignancies. Characterization of these subgroups is essential and there is an urgent need for more accurate biomarkers and targeted treatments in PDAC. In the current work, we immunohistochemically investigated the expression levels and prognostic values of oxidative stress markers (8-OHdG, Keap1, Prx I, II, III, V and VI), epigenetic histone modifiers (KDM4A, KDM4B, KDM4D and SIRT1–4), and cell-cycle regulators (p16, Rb, CDK4) and DNA-repair enzymes (FEN1 and MGMT) in the cohort of surgically treated PDAC patients. We found that Keap1 expression was associated with better pancreatic cancer-specific survival. Expression of antioxidative peroxiredoxins I, III, V and VI was also connected with a more favourable tumour characteristics and Prx I and VI showed prognostic value. When considering the biology of PDAC, we noticed that pivotal epigenetic regulation also occurred in exocrine pancreatic tissue adjacent to resection margins. Overexpression of the cell-cycle regulator CDK4 and the DNA-repair enzyme FEN1 in the whole population, and elevated expression level of MGMT in the most high-risk patients were connected with worse prognosis. The results of the study can be utilized in the future when individualized therapies are being designed for PDAC patients. Due to occurrence of the epigenetic regulation also in exocrine pancreatic tissue adjacent to resection margins, it could be evaluated in future for routine diagnostics and treatment optimization. The potential role of MGMT in the development of PDAC chemoresistance should be studied in the future. / Tiivistelmä Haiman duktaalinen adenokarsinooma (PDAC) on yksi aggressiivisimmista syöpäsairauksista. Viiden vuoden elossaoloennuste on vain lähellä 5 prosenttia. Diagnoosihetkellä 90% haiman adenokarsinoomista yltää haiman ulkopuolelle ja usein kasvain on jo lähettänyt etäpesäkkeitä. Kasvutaipumuksen sekä metastasoinnin takia kuratiivinen kirurginen hoito on mahdollista vain 10–20% tapauksista. Liitännäissolunsalpaajahoito on aiheellista näissä kuratiivistavoitteisesti hoidetuissa tapauksissa. Kuitenkin vaikutus kokonaiselossaoloaikaan on melko vähäinen. Uusimman tutkimustiedon valossa PDAC:aa pidetäänkin heterogeenisenä ryhmänä biologisesti ja ennusteellisesti erilaisia tautiryhmiä. Näiden tautiryhmien tunteminen ja tunnistaminen riittävän tarkkojen merkkiaineiden avulla olisi ensiarvoisen tärkeää, jotta hoitoja voitaisiin kohdentaa niistä hyötyville potilaille. Väitöskirjatutkimuksessa selvitimme immunohistokemiallisin menetelmin oksidatiivisen stressin merkkiaineiden (8-OHdG, Keap1, Prx I, II, III, V ja VI), epigeneettisten histonimodifikaattorien (KDM4A, KDM4B, KDM4D ja SIRT1–4) sekä solusyklin säätelijöiden (p16, Rb, CDK4) ja DNA-korjausentsyymien (FEN1 ja MGMT) ilmentymistä ja ennusteellista arvoa kirurgisesti hoidetuilla PDAC-potilailla. Tutkimuksessamme totesimme, että kasvainkudoksen Keap1-ilmentymä yhdistyi parempiennusteiseen taudinkuvaan. Antioksidatiivisten peroksiredoksiinien I, III, V ja VI ilmentyminen yhdistyi niin ikään suotuisampaan kasvaimen fenotyyppiin ja Prx I ja VI osoittivat ennusteellista arvoa. Havaitsimme lisäksi, että PDAC:n biologiaan keskeistesti vaikuttavaa epigeneettistä säätelyä tapahtuu myös malignin haimakudoksen viereisessä eksokriinisessä haimakudoksessa. Solusyklin säätelijä CDK4:n ja DNA-korjausentsyymi FEN1:n voimakas ilmentyminen koko tutkimuspopulaatiossa sekä kohonnut MGMT:n ilmentyminen korkeimman riskin potilailla yhdistyivät huonompaan taudin ennusteeseen. Väitöskirjatyön tutkimustuloksia voidaan tulevaisuudessa hyödyntää, kun tutkitaan yksilöllisiä hoitomuotoja PDAC-potilailla. Koska epigeneettistä säätelyä tapahtuu myös syövän viereisessä eksokriinisessa haimakudoksessa, voidaan tulevaisuudessa tämän kudoksen arviointia mahdollisesti käyttää rutiinisti diagnostiikassa sekä hoidon optimoinnissa. MGMT:n mahdollinen rooli PDAC:n kemoresistenssin kehittymisessä tulisi tulevaisuudessa selvittää.

The tumor microenvironment (TME) comprising immune cells and stromal components, such as fibroblasts and vessels, emerged as one of the most significant predictors of patient survival in a variety of solid tumors. With T cells representing the major cellular effector cells of the adaptive immune system and B cells orchestrating the humoral immune response, both cell types acquire crucial roles in the antitumor immune response. Thus, a high abundance of tumor-infiltrating CD8+ T cells and B cells has been generally associated with longer survival, while immunosuppressive subsets such as regulatory T cells (Treg) and M2-polarized macrophages are frequently linked to poor prognosis. Besides the frequency, also the spatial organization emerged as a clinically relevant parameter. Hence, the formation of T and B cells in tertiary lymphoid structures (TLS) was found to favor improved clinical outcome of patients. It was further reported that besides the prognostic value, the baseline immune architecture harbors the ability to predict the response to immunotherapies such as immune checkpoint inhibitor treatment and even chemotherapy. In turn, standard cytotoxic treatment regimens like radio- and chemotherapy, as well as novel immunotherapeutic or targeted approaches, exhibit distinct effects on various immune cells. Depending on the tumor entity, therapy, and immune cell subsets, differing modulation of infiltrating immune cells after therapy was observed. While previous studies mainly investigated an altered abundance of T and B cells, changes in functional orientation and composition of lymphocyte populations are gaining increasing relevance. In this thesis, the aim was to uncover the phenotype, frequency, composition, spatial distribution, clinical significance, and therapeutic modulation of the T cell compartment in soft tissue sarcoma (STS), and B cell populations in pancreatic ductal adenocarcinoma (PDAC). Due to the low incidence and heterogeneous nature of STS, detailed analyses of distinct CD8+ and CD4+ T cell subsets are lacking. To assess the effect of multimodal treatment, comprising radiotherapy and locoregional hyperthermia with or without chemotherapy, on the immune architecture, the patient cohort included matched pre- and post-therapy tissue samples. By assessing both the peritumoral and intratumoral region, additional information about the spatial distribution of STS-infiltrating T cells was gained. In PDAC, the T cell compartment and its therapeutic modulation has been explored in detail recently, but equivalent insight into the B cell landscape is missing. Going beyond the abundance of pan B cells, the aim was to identify proliferating B and T cells, germinal center (GC) B cells, plasmablasts, and plasma cells to investigate their modulation by neoadjuvant chemo(radio)therapy (NeoTx). Further insight into the spatial composition was gained by analyzing different regions (intratumoral and peritumoral) and tissue compartments (epithelial, stromal, TLS). To achieve this, three novel multiplex immunohistochemistry panels were established enabling simultaneous staining of six markers plus DAPI. For CD4+ T helper (Th) cells, the master transcription factors for Th1 (T-box expressed in T cells), Th2 (GATA-binding protein 3), Th17 (retinoic acid receptor-related orphan receptor T), and Treg (Forkhead box protein 3) were included in addition to CD3 and the proliferation marker Ki67. The CD8+ T cell panel comprised the phenotypic marker CD8, the immune checkpoint molecules programmed cell death protein 1 and lymphocyte-activation gene 3 as well as the activation-associated molecules granzyme B and 4-1BB, in addition to Ki67. It was thus found that post-treatment STS samples displayed moderately reduced frequencies of both CD8+ and CD3+ T cells in comparison to the pretreatment biopsy. The Th cell landscape was dominated by Th2 cells, whose density was significantly reduced upon multimodal therapy and a moderate redistribution favoring Th1 and Th17 cells was observed. While high frequencies of CD3+ and CD8+ T cells in the posttreatment tissues were associated with significantly longer disease-free survival, these populations held no prognostic value in the biopsy obtained prior to treatment, suggesting a reshaping of the TME upon therapy. Furthermore, the spatial distribution, reflected by the ratio of intra- to peritumoral CD8+ T cells, emerged as an independent prognostic factor for the risk of recurrence. In PDAC, B cell subsets were identified by staining for CD3, CD20, Ki67, the transcription factor B cell lymphoma 6, and the plasma cell markers CD38 and CD138. While CD3+ T cells were unaffected, significantly lower frequencies of proliferating B cells, GC B cells, plasmablasts, and plasma cells were observed in the NeoTx group compared to patients undergoing primary resection (PR). Furthermore, neoadjuvant-treated patients exhibited a significantly lower abundance of TLS, which was validated in an independent cohort. These results indicate that NeoTx differentially affects distinct immune cell subsets, and that B cellmediated antitumor immunity may be inhibited by chemo(radio)therapy. Spatial analysis further revealed that plasma cell accumulations frequently localized close to TLS, being accompanied by C-X-C motif chemokine ligand 12-expressing fibroblasts. Furthermore, patients with TLS exhibited significantly higher plasma cell frequencies, suggesting that TLS can foster the generation of plasma cells whose migration is then guided by fibroblastic tracks. Lastly, a prognostic value of pan T and B cells was observed only in the PR group, while these populations provided no clinical significance in neoadjuvant-treated patients. However, proliferating Ki67+CD20+ B cells emerged as an independent prognostic factor for a lower risk of death in the NeoTx group, suggesting a restorative post-treatment TME in these patients. Altogether, this thesis provided novel insights into the TME of STS and PDAC and its therapeutic alteration. Spatial analyses further enabled an improved understanding of the immune architecture and potential cell-cell interactions within the TME. In addition, strong associations with patient survival highlight the enormous significance of the TME and may guide future therapy development. Although the results do not encourage a concomitant application of cytotoxic therapy regimens and immunotherapy, patients may benefit from sequential combination treatments. An enhanced understanding of the immunomodulatory effects of NeoTx is pivotal for overcoming the immunosuppressive TME of STS and PDAC by refining existing treatment regimens and developing novel therapy approaches in order to improve the long-term outcome of patients.

Lokale Invasion und Ausbreitung von Tumorzellen entlang von Nerven und Gefäßen limitieren den Erfolg kurativer Therapien von Patienten mit Pankreaskarzinom (PDAC). Der axon guidance Faktor Slit2 und seine Robo-Rezeptoren steuern die Navigation von Nerven und Gefäßen sowie die Motilität von Epithelzellen. Sie stellen somit attraktive Regulatoren der klinisch bedeutsamen Ausbreitungswege des PDAC dar. Zielsetzung der vorgelegten Arbeit war die Charakterisierung der Expression von Slit2 im PDAC und seiner Funktion für Tumorwachstum und -ausbreitung. Quantitative Analysen belegten eine deutliche Reduktion der Slit2 mRNA Expression in humanen PDAC Proben im Vergleich zu gesundem Gewebe. Zudem korrelierten Slit2 mRNA-Werte unterhalb des Medians mit einer höheren Inzidenz lymphatischer Metastasierung und einem gesteigerten Prozentsatz befallener Lymphknoten. Die Slit2-Rezeptoren Robo1 und 4 wiesen hingegen vergleichbare Immunreaktivität im Tumor und gesundem Gewebe auf, wobei eine differentielle Lokalisation in Epithelien, Nerven und Gefäßen zu beobachten war. Die Re-Expression von Slit2 in Slit2-defizienten Zelllinien führte zu einer Hemmung der gerichteten Migration und Invasion. Der Robo1-Rezeptor knockdown hingegen stimulierte die Motilität von Tumorzellen mit endogener Slit2 Expression. Slit2-konditioniertes Medium aus Tumorzellen hemmte die Lamellipodienbildung und die Migration von Endothelzellen. In orthotopen humanen Xenograft-Modellen und einem murinen, syngenen Tumormodell reduzierte die Re-Expression von Slit2 in PDAC Zellen Tumorwachstum, Invasion, Metastasierung und Angiogenese. Zudem verminderte die Induktion von Slit2 in PDAC Zellen deren gerichtete Migration entlang aussprießender Neuriten in einem ex vivo Model. Die vorliegenden Daten weisen Slit2 die Funktion eines Tumorsuppressors im duktalen Pankreaskarzinom zu. Ein Verlust der Slit2-Robo Aktivität könnte somit Metastasierung und neuronale Invasion fördern und einen aggressiveren Phänotyp begünstigen. / Early dissemination of pancreatic ductal adenocarcinoma (PDAC) via vascular routes and neural invasion limits curative therapy, suggesting a central role for the interaction of tumor cells with blood vessels and nerves in the tumor stroma. Slit2 and its Robo receptors constitute a system of guidance cues that function in axon guidance, angiogenesis and epithelial morphogenesis, respectively. Here, we studied the expression of Slit2 in PDAC and its function for tumor growth and dissemination. Slit2 mRNA expression was reduced in specimens of human PDAC as compared to non-transformed pancreas and low Slit2 mRNA expression correlated with a higher incidence and a higher extent of lymphatic metastasis. In contrast, the Slit2 receptors Robo1 and Robo4 were uniformly present in clinical samples of PDAC and healthy pancreas and displayed differential localization on epithelial tumor cells, nerves and tumor vasculature. Stable or inducible re-expression of Slit2 in Slit2-deficient PDAC cell lines inhibited directed migration and invasion. Conversely, Robo1-knockdown stimulated the motility of PDAC cells with endogenous Slit2 expression. Tumor cell derived Slit2, furthermore, suppressed lamellipodia formation and migration of primary endothelial cells. In vivo studies in orthotopic human xenograft and mouse syngeneic pancreatic cancer models revealed that re-expression of Slit2 in PDAC cells inhibited tumor growth, invasion, metastasis and angiogenesis. In addition, induction of Slit2 in PDAC cells impaired the unidirectional migration along outgrowing neurites in ex vivo co-cultures of tumor cells and dorsal root ganglia. These data provide evidence for a functional role of Slit2 as a tumor suppressor in human PDAC. A loss of Slit2-Robo activity as observed in human PDAC samples, might consequently promote metastasis and neural invasion and favors a more aggressive phenotype.

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