INTRODUCTION: Melanoma is the most deadly skin cancer with mortality dependent on the extent and location of metastases. Lymphatic metastasis occurs early in melanoma, and tumor-associated lymphatic vessel area correlates with melanoma progression. Recently, the discovery of checkpoint inhibitors has drastically changed the treatment strategy and survival rates in melanoma. Neuropilin-2 (NRP2) is a potential common target in melanoma cells, tumor-associated lymphatic endothelial cells (LECs) and tumor-infiltrating lymphocytes (TILs). NRP2 is a cell surface receptor with competing stimulatory ligands (VEGF-A/-C) and inhibitory ligands (SEMA3F/G). AIM: The goal of this study was to investigate the role of NRP2 in both melanoma cells and the melanoma microenvironment (LECs, TILs) and to examine the effect of semaphorin 3F (SEMA3F) on the tumor cells as well as an immune modulator. RESULTS: Mouse and human melanocytes expressed NRP2 but not other vascular endothelial growth factor (VEGF) receptor tyrosine kinases in vitro and in vivo. NRP2 protein expression, as analyzed by immunohistochemistry, was upregulated in human metastatic melanoma sections. Treatment of melanoma cells in vitro with SEMA3F inhibited migration and phosphorylation of protein kinase B (AKT) but did not inhibit cell viability. SEMA3F also increased programmed cell death-ligand 1 (PD-L1) expression in melanoma. Syngeneic B16F10 melanoma did not grow in global NRP2 knockout (KO) mice but did grow in wild-type mice. In addition, mice inoculated with B16F10 were treated with SEMA3F or phosphate-buffered saline (PBS) by mini-osmotic pumps. Resulting tumors were analyzed histologically for microvessel density and presence of TILs (number and subtype). CONCLUSIONS: Expression of NRP2 protein positively correlated with melanoma progression in human patient samples. NRP2 functions differently in melanoma tumor cells than in host stromal cells (endothelial cells [ECs], LECs). In melanoma, NRP2 is not a VEGF receptor but responds to the ligand, SEMA3F. Alternately, NRP2 appears to be an important VEGF-A/-C co-receptor in tumor-associated angiogenesis and lymphangiogenesis, as demonstrated in the NRP2 transgenic mice studies. SEMA3F inhibited tumor cell migration but increased PD-L1 expression. Systemic treatment with purified SEMA3F protein in melanoma preclinical trials inhibited melanoma growth and microvessel density. Taken together, these results suggest that exploiting the NRP2/SEMA3F signaling axis may be a novel treatment strategy to be used in combination with existing immunotherapy in melanoma. / 2020-07-03T00:00:00Z

Cancer immunotherapy has become a leading treatment modality in metastatic diseases. Although this novel therapy has changed the therapeutic algorithms and patients' outcomes in multiple malignancies, certain proportions of patients still fail to respond to these approaches. In our studies, we aimed to address the main mechanisms of tumor resistance to cancer immunotherapy. We have systematically defined the main challenges in adoptive cell transfer. We have focused on two key mechanisms of the tumor resistance to immunotherapy: poor trafficking of adoptively transferred immune cells into tumors, and the death receptor-induced apoptosis of the tumor-infiltrating immune cells. In our work, we have gone beyond the tumor tissue and searched for the immune cell populations and novel targets that would help to challenge the two mechanisms of resistance. Our data uncovered the therapeutic potential of the paratumoral tissue compartments and, thus, provided new avenues on how to challenge solid tumors by immunotherapy.

Breast cancer (BC) was not initially considered an immunogenic tumor; however, recent data show that immune-related factors are associated with patient prognosis and the response to treatment. Several large adjuvant clinical trials have shown that tumor infiltrating lymphocytes (TIL) are significantly associated with a better prognosis and can also predict responsiveness to pre-operative chemotherapy, particularly in the triple negative (TN) & HER2+ BC subtypes (Carsten Denkert et al. 2010; Loi et al. 2013a). Recently, the presence of ectopic lymph node-like structures characterized by distinct T and B cell zones, called tertiary lymphoid structures (TLS), were identified adjacent to the tumor (Gu-Trantien et al. 2013) in 60% of BC (Buisseret et al. 2017b) and linked with a good prognosis (Gu-Trantien et al. 2013). The mechanisms involved in TLS formation and activities and their impact on tumor immunity is relatively unknown. TIL infiltration and TLS formation are likely regulated, in part, by transcription factors (TF) that control cytokine/chemokine production within the tumor microenvironment (TME) (Pimenta and Barnes, 2014). One such TF, the forkhead box protein 1 (FOXP1) is abnormally expressed in various human tumors and has a known role in regulating immune cell functions. Contradictory data on FOXP1 expression together with a lack of information on its immune regulation led us to explore its role in this tumor type. The first part of this thesis research focused on FOXP1-mediated regulation in BC. Gene/protein analysis was examined in the four BC molecular subtypes, revealing its enriched expression in estrogen receptor positive (ER+) tumors (Luminal A/B). Luminal BC is generally less infiltrated compared with frequently high TIL infiltration in ER negative (ER-) tumors (i.e. HER2+ and TN) [reviewed in (Solinas et al. 2017a) and (Loi et al. 2014)]. We found that high FOXP1 expression in a cohort of untreated primary BC was significantly associated with a lower TIL and fewer TLS compared to FOXP1 low (FOXP1lo) tumors. This observation led us to investigate the effect of FOXP1 on cytokines and chemokines potentially involved in TIL recruitment and/or TLS formation. BC cancer cell lines were used to silence [MCF7; FOXP1hi] or overexpress [MDA-MB-231; FOXP1lo] FOXP1 expression. FOXP1 repression upregulated a number of cytokines and chemokines involved in T and B cell migration and function, while FOXP1 overexpression repressed a majority of the same factors. Expression analysis of the major T and B cell cytokine and chemokine genes was performed for FOXP1lo and FOXP1hi primary BC. These data reveal that FOXP1hi BCs have significant decreases in CXCL9, CXCL10, CXCL11, CXCL13, CX3CL1, CCL20, IL2, IL21, granzyme B and IFNγ and high levels of the immunosuppressive cytokines, IL10 and TGFβ. We next performed a lymphocyte migration assay using primary tumor supernatants prepared from FOXP1lo and FOXP1hi BC finding significantly decreased migration of total CD45+ lymphocytes, B cells, helper (CD4+) and cytotoxic (CD8+) T cells using FOXP1hi compared to FOXP1lo SN. Overall, our data suggest that FOXP1 plays an important role in repressing anti-tumor immune responses by negatively regulating TIL migration directed by specific cytokines and chemokines.The second part of this thesis research focused on the role FOXP1 plays in BC TLS. FOXP1 expression in T and B cell TIL and TLS was evaluated using RT-qPCR, multicolor flow cytometry, immunofluorescence (IF) and immunohistochemistry (IHC) and fresh, fixed and frozen breast tissues. Based on the FOXP1 expression two types of TLS were identified in BC: 1) TLS containing a germinal center (GC-TLS) and 2) TLS lacking a GC (non-GC-TLS). Examination of proteins specifically associated with active humoral immune responses allowed us to identify GC-TLS but not non-GC-TLS as functional. Gene expression analysis of micro-dissected tissues revealed distinct immune profiles that characterize B cell follicles in tonsils and spleen as well as aggregates, non-GC-TLS and GC-TLS in BC. This analysis further demonstrates that ongoing cell-mediated immune responses are associated with GC-TLS. The findings from this thesis research add important information to our understanding of how immune responses are initiated and maintained in BC and provide further insight into the identification and organization of functional immune responses at the tumor site. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Melanoma is a highly malignant neoplasia with high rates of metastasis in humans and dogs. Regardless of being considered a highly immunogenic neoplasm, the function of the immune system is hampered by the expression of immune checkpoint molecules by the cancer cells. In contrast, soft tissue sarcomas are poorly immunogenic, as Tumor infiltrating Lymphocytes are lacking, or when present they are usually at the periphery of the tumor. Still, soft tissue sarcomas are considered immunosuppressed. Checkpoint molecules from the PD-axis are overexpressed in numerous human malignant neoplasia and have recently gained attention with a few reports in canine tumors. Immunotherapies against these checkpoint molecules have shown great efficacy in humans, but in order to determine translational approaches into canine patients, more research is needed. Here we determined the gene expression of Programed Death receptor-1, and its ligands PD-L1 and PD-L2 in canine tumors with two distinct immune profiles. Our results show that regardless of their immune profiles, melanoma versus soft tissue sarcoma, checkpoint molecules expression was higher in malignant tumors with a higher grade. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal ultrasound focused therapy that induces mechanical stress to the cells, leading to liquefactive necrosis. Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of CD3+ T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasia, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia. / Doctor of Philosophy / Melanoma is a highly malignant tumor and very resistant to therapy for humans and dogs. At the same time, this neoplasia is usually highly infiltrated by cells from the immune system. However, this immune infiltration is often inhibited by molecules expressed by the melanoma cells. In contrast, soft tissue sarcoma is considered poorly immunogenic, as they often contain low levels of immune cell infiltrates but are still considered immune suppressed. In this study, we determined the expression of molecules that inhibit the effect of T lymphocytes, specifically Programed cell death receptor-1, PD-Ligand 1, and PD-Ligand 2 for these neoplasms with distinct immune profiles. We encounter that despite their immune profiles, the expression of these molecules is higher in malignant tumors. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal focused ultrasound therapy that induces mechanical stress to the cancerigenous cells, leading to its death (necrosis). Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasms, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia.

Abstract Cancer treatments have remarkably improved over the past years since targeted therapies and immunotherapy have been introduced to the field of oncology. The benefit of these new therapies is often limited, however, by de novo or acquired therapy resistances, which should be noticed when making clinical decisions. In this current work, we studied the prognostic and predictive values of several immunological markers in metastatic HER2-positive breast cancer treated with trastuzumab, because trastuzumab is still given to patients according to the HER2 status only, without certainty of tumor response. We also determined the role of HER2 and HER3 for cancer stem cells (CSC) in ALK translocated non-small cell lung cancer (NSCLC) cell lines since the CSCs are causing therapy resistance and cancer recurrence. The results demonstrated that a high number of cytotoxic T cells, together with a high number of M1-like macrophages in the center of the tumor (CT), are promising and independent prognostic factors in HER2-positive breast cancer. These markers together also can predict the progression of the disease and the length of trastuzumab discontinuation in tumor response. Expression of HER2 and HER3 increased the stem-like properties of ALK translocated NSCLC cells, which were decreased when the expressions were downregulated. HER2-HER3-dependent CSCs also mediated the ALK therapy resistance. In conclusion, this study suggests that patients with a favorable immunological tumor profile (high number of cytotoxic T cells and M1-like macrophages in the CT) could be treated in a less-intensive manner, that trastuzumab discontinuation could be feasible for these patients, and that targeting of HER2 and HER3 receptors can lead to more effective killing of cancer stem-like cells and should be further studied. / Tiivistelmä Syöpähoidot ovat kehittyneet huomattavasti, kun kohdennetut hoidot ja immunologiset hoidot ovat tulleet perinteisten hoitojen rinnalle. Usein näiden hoitojen hyötyä kuitenkin rajoittaa jo olemassa oleva lääkeresistenssi tai sen kehittyminen, mikä tulisi ottaa huomioon hoitoja suunniteltaessa. Tässä työssä tutkittiin immunologisia merkkiaineita, joilla voitaisiin ennustaa trastutsumabi-hoidon vastetta sekä potilaiden ennustetta levinneessä HER2-positiivisessa rintasyövässä. Tällä hetkellä trastutsumabi-hoitopäätös tehdään pelkän HER2-geenimonistuman mukaan ilman varmuutta siitä, hyötyykö potilas oikeasti hoidosta. Lisäksi tutkimme HER2- ja HER3-reseptorien merkitystä syövän kantasoluille ALK-translokoituneessa ei-pienisoluisessa keuhkosyövässä (NSCLC), sillä syövän kantasolut ovat yksi merkittävimmistä tekijöistä lääkeresistenssin kehittymisessä ja syövän uusiutumisessa. Työssä havaittiin, että kasvaimen keskellä oleva suuri määrä sytotoksisia T-soluja sekä M1-tyypin makrofageja on yhteydessä potilaiden parempaan ennusteeseen ja että kyseiset merkkiaineet ovat toisistaan riippumattomia. Merkkiaineet pystyivät ennustamaan myös taudin etenemistä sekä trastutsumabi-hoitokeskeytyksen pituutta. HER2- ja HER3-proteiinien tuotto lisäsi ALK-translokoituneiden NSCLC-solujen kantasolumaisia ominaisuuksia, jotka puolestaan vähenivät, kun proteiinien tuotto estettiin. Lisäksi HER2-HER3 -riippuvaiset syövän kantasolut säätelivät lääkeresistenssiä kyseisessä taudissa. Työn tulokset viittaavat siihen, että potilaita, joilla on suotuisa kasvaimen immunoprofiili (suuri määrä sytotoksisia T-soluja ja M1-tyypin makrofageja kasvaimen keskellä) pystyttäisiin hoitamaan keveimmillä hoidoilla ja HER2-hoitokeskeytys voisi olla mahdollinen näillä potilailla. Lisäksi työ korostaa HER2- ja HER3-reseptorien kohdentamista syövän kantasolumaisten solujen tehokkaamman tuhoamisen saavuttamiseksi. / Huomautus/Notice Painetussa virheelliset ISBN -tunnukset: ISBN (print) 978-952-42-2343-8 pitäisi olla 978-952-62-2343-8. ISBN (PDF) 978-952-42-2344-5 pitäisi olla 978-952-62-2344-5. Printed version has incorrect ISBNs: ISBN (print) 978-952-42-2343-8 it should be 978-952-62-2343-8. ISBN (PDF) 978-952-42-2344-5 it should be 978-952-62-2344-5.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome cansignificantly vary among patients within the same stage. Data collected from largecohorts of human cancers has demonstrated the impact of immune-classification, which has a prognostic value that may add largely to the significance of the AJCC/UICC TNM-classification. In our study we examined the immune cells that infiltrated the tumor tissues of colorectal and ovarian cancer patients. In a cohort of newly diagnosed colorectal cancer patients we examined the correlations between the KRAS mutational status, patterns of tumor-infiltrating immune cells and the presence of tumor recurrence. Our data suggest that colorectal cancer patients with low levels of tumor-infiltrating lymphocytes, a high CD1a/DC-LAMP tumor-infiltrating dendritic cells ratio, and a KRAS mutation in codon 13 are at a high risk of disease recurrence. In ovarian cancer patients we focused on the dynamics of the tumor-infiltrating...

Relevance of tumor infiltrating lymphocytes as a prognostic factors in patients with portal vein embolisation (PVE) and patients with PVE and administration of autologous stem cells Background: low future liver remnant volume (FLRV) is the cause of why 75% of patients with colorectal liver metastases (CLM) are primarily inoperable. Portal vein embolisation (PVE) helps to increase FLRV and so increase the operability. But PVE fails in almost 40 % of patients. Usage of stem cells (SCs) could be the way how to support the effect of PVE. Currently, there are studies of interactions of the immune system and malignancies. We do not know about papers focused on relations of the immune system and CLM in patients treated by PVE. There were not described interactions of ABC transporters and CLM at patients after PVE was performed too. Aims: the aim of this dissertation was to verify the effect of PVE and intraportal administration of SCs on the growth of FLRV and progression of the CLM. Other aims were to evaluate the tumor infiltrating lymphocytes, ABCC10 and ABCC11 transportes in patients treated by surgery for CLM after PVE and their clinical relevances. Methods: intraportal administration of SCs after PVE and their effect was explored in a group of 63 patients (43 patients with PVE alone, 20 in the group PVE with...

Το καρκίνωμα εκ πλακώδους επιθηλίου ή πλακώδες καρκίνωμα (ΠΚ) του δέρματος είναι ο δεύτερος πιο συχνός καρκίνος του δέρματος και εμφανίζεται συνήθως σε έδαφος ακτινικής υπερκεράτωσης (ΑΚ). Τα νεοπλασματικά κύτταρα εκφράζουν μια ποικιλία αντιγόνων προσελκύοντας με αυτό τον τρόπο λεμφοκύτταρα, τα διηθούντα τον όγκο λεμφοκύτταρα (Tumor Ιnfiltrating Lymphocytes-TILs) στο μικροπεριβάλλον του όγκου. Με βάση το ανοσοφαινοτυπικό τους προφίλ η πλειοψηφία των TILs εκφράζει το μόριο CD3 και αφορά σε Τ-λεμφοκύτταρα. Αυτά με την σειρά τους διαχωρίζονται σε CD8+ Τ-κυτταροτοξικά λεμφοκύτταρα και CD4+ Τ-λεμφοκύτταρα. Η έκφραση του δείκτη CD25+ επιτρέπει τον διαχωρισμό των CD4+ λεμφοκυττάρων σε δύο επιπλέον υποομάδες, τα T-επικουρικά (CD4+/CD25-) και τα Τ-ρυθμιστικά (CD4+/CD25+) κύτταρα. Ο πιο αξιόπιστος δείκτης για την ανίχνευση των Τ-ρυθμιστικών κυττάρων (Tregs) θεωρείται ο Forkhead box P3 (Foxp3). Ο αυξημένος αριθμός των TILs έχει συσχετισθεί με την πρόγνωση και τη θεραπευτική αντιμετώπιση σε μια ποικιλία νεοπλασμάτων, περιλαμβανομένων και καρκίνων του δέρματος όπως το μελάνωμα. Η μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης, ωστόσο, δεν έχει ακόμα αποσαφηνισθεί και ποικίλλει στα διάφορα είδη νεοπλασμάτων. Στα πλαίσια της παρούσας διπλωματικής εργασίας η ανίχνευση των Τ-λεμφοκυττάρων έγινε με ανοσοϊστοχημική μέθοδο με τη χρήση των κατάλληλων αντισωμάτων (CD3, CD4, CD8, Foxp3). Η παρουσία των κυττάρων αξιολογήθηκε με τη χρήση φωτονικού μικροσκοπίου με δυο διαφορετικά ημιποσοτικά συστήματα βαθμολόγησης για όλους τους μελετηθέντες Τ-κυτταρικούς υποπληθυσμούς καθώς και με ένα τρίτο σύστημα ποσοτικής καταμέτρησης για τα Τregs. Σκοπός ήταν να γίνει σύγκριση των μεθόδων ώστε να βρεθεί η καταλληλότερη μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης στο πλακώδες καρκίνωμα του δέρματος, να υπολογιστεί ο αριθμός των Tregs στις μελετούμενες οντότητες χρησιμοποιώντας το βέλτιστο σύστημα βαθμολόγησης και να αναζητηθούν διαφορές στην πυκνότητα των λεμφοκυτταρικών υποπληθυσμών μεταξύ των οντοτήτων, οι οποίες μπορεί να έχουν παθογενετική ή θεραπευτική κλινική σημασία. / Squamous cell carcinoma (SCC) of the skin is the second most common skin cancer. It usually develops in a background of actinic keratosis (AK). Neoplastic cells express a variety of antigens, attracting lymphocytes in the tumor microenvironment (Tumor Ιnfiltrating Lymphocytes-TILs). Based on the immunophenotypic profile, the majority of TILs expresses the CD3 molecule, so they are T-lymphocytes. These in turn are divided into CD8+ cytotoxic T-lymphocytes and CD4+ T-lymphocytes. The expression of CD25 enables the separation of CD4+ lymphocytes in two further subgroups, the T-helper (CD4+/CD25-) and T-regulatory (CD4+/CD25+) cells. The most reliable marker for the detection of T-regulatory cells (Tregs) is considered to be the Forkhead box P3 (Foxp3). The increased number of TILs is associated with prognosis and treatment in a variety of tumors, including skin cancers, such as melanoma. The evaluation methology of lymphocytic infiltration, however, is not yet clarified and varies in different types of tumors. In the context of this thesis, the detection of T-lymphocytes was performed by immunohistochemistry using the appropriate antibodies (CD3, CD4, CD8, Foxp3). The presence of the cells was evaluated using light microscopy with two different semiquantitative scoring systems, for all the T-lymphocyte subpopulations, as well as with a third system of quantification of Tregs. The purpose was to compare the methods in order to find the most suitable methodology for the evaluation of the lymphocytic infiltration in squamous cell carcinoma of the skin, to evaluate the number of Tregs in the studied entities using the best method and examine for differences in the density of lymphocyte subpopulations.