<p dir="ltr">Cancer biological mechanisms are a vastly researched area in the field, yet they are not well understood in the various contexts in which cancer is found. Cancerous tumors often exist in harsh, stressful environments for normal cells, but cancer cells can thrive in these conditions. The tumor microenvironment (TME) typically has low oxygen levels (hypoxia), high acidity, and low nutrition. Exposure to the TME leads to many metabolic changes in the cells, enabling cancer to continue proliferating and migrating. However, these metabolic changes are not well understood, especially at the single-cell level. The ability to monitor cells in real time to determine the physical characteristics they undergo is critical to understanding the impact of these metabolic changes. Conventional methods focus on determining the genomic and proteomic changes in large numbers of cells, which may be overlooked if the changes are homogeneous across samples. In this work, we demonstrate the power of using multiple imaging techniques in combination with biochemical methods to visualize metabolic changes and determine the causes in various cancer cells under extreme hypoxia conditions.</p><p dir="ltr">The changes in the microtubule network that occur under hypoxia at the single-cell level are not widely researched. The use of confocal fluorescence microscopy can determine microtubule polymerization in conjunction with eGFP-transfected EB3, a protein that assists in microtubule polymerization. We have determined that hypoxic HeLa cells produce finger-like protrusions when exposed to hypoxia that help with cell migration and, ultimately, cancer cell metastasis. The formation of these protrusions is facilitated by localized mitochondria activities in the protrusions.</p><p dir="ltr">The metabolic changes in lipid droplets (LDs) under hypoxia at the single-cell level remain an elusive topic. The use of stimulated Raman spectroscopy (SRS) and coherent anti-Stokes Raman scattering (CARS) can determine the quantity and spatial-temporal distribution of LDs in cancer cells. We have found that LDs redistribute to the endoplasmic reticulum (ER) and increase in intensity in hypoxic MIA PaCa-2 and A549 cells. Time-lapse CARS microscopy revealed a release-accumulate process of these LDs on ER in hypoxia. We also studied the impact of carbon sources on LD formation and found that MIA PaCa2 cells prefer direct lipid uptake while glucose is also essential to reduce lipotoxicity. The use of hyperspectral stimulated Raman scattering (hSRS) also reveals that the content of the LDs changes to include less cholesteryl ester and a decrease in lipid saturation level.</p><p dir="ltr">Collectively, these findings shed new light on the understanding of cytoskeleton dynamics and lipid metabolism in hypoxic conditions. The discoveries made within this research would lead to better treatment strategies for effective treatment of hypoxia-resistant cancer cells.</p>

<p>UVOD: Karcinom kolona (KK) je velik javnozdravstveni problem usled visoke incidence i stope mortaliteta. Kod KK je stadijum bolesti najvažniji pojedinačni nezavisni faktor prognoze. U prisustvu nepovoljnih prognostičkih parametara, u koje spadaju visok histolo&scaron;ki gradus, ileus, limfo-vaskularna i perineuralna invazija, nakon potencijalno kurativne operacije se kod pacijenata u stadijumu II indikuje primena adjuvantne hemioterapije koja ima pozitivan uticaj na ukupno preživljavanje i na produženje perioda bez bolesti. Međutim, relapsi bolesti nastaju kod nekih bolesnika bez negativnih prognostičkih faktora, &scaron;to ukazuje na moguće postojanje drugih tkivnih faktora lo&scaron;e prognoze. U novije vreme se sve veća pažnja posvećuje fenomenu tumorskog pupljenja koje predstavlja pojavu tumorskih pupoljaka (TP), odnosno oligocelularnih grupa tumorskih ćelija koje se na invazivnom frontu tumora odvajaju od glavne tumorske mase. Ove tumorske ćelije poprimaju fenotip mezenhimnih ćelija i stiču sposobnost ameboidnog kretanja kroz ekstracelularni matriks, uz pomoć citoplazmatskih podija koje se na dvodimenzionalnim histolo&scaron;kim rezovima vizualizuju kao citoplazmatski pseudofragmenti (CPF). Značaj gustine TP i CPF je jo&scaron; uvek nedovoljno ispitan, ali postoje indicije da se radi o moćnom prediktoru biolo&scaron;kog pona&scaron;anja tumora. CILJ: Cilj je bio da se ispita zavisnost dužine perioda bez relapsa, veličine primarnog tumora, gustine peritumorske limfocitne infiltracije i konfiguracije tumorske margine od gustine TP i CPF kod bolesnika sa KK u stadijumu II. METODOLOGIJA: Istraživanjem je obuhvaćeno 114 bolesnika operisanih od KK u stadijumu II na Institutu za onkologiju Vojvodine, bez nepovoljnih prognostičkih faktora i bez indikacija za primenu adjuvantne hemioterapije. Mikroskopskom analizom rutinskih histolo&scaron;kih i imunohistohemijskih preparata utvrđivana je gustina TP i CPF, koja je zatim korelirana sa vremenom pojave relapsa, veličinom primarnog tumora, gustinom peritumorske limfocitne infiltracije i konfiguracijom tumorske margine. REZULTATI: Velika gustina TP i/ili CPF nađena je kod 45 tumora (39,5%). U ovoj grupi se relaps dogodio kod 26 bolesnika (57,8%). U grupi bolesnika sa malom gustinom TP/CPF relaps je registrovan u 4 slučaja (5,8%). Poređenje krivih preživljavanja pokazalo je da je verovatnoća relapsa značajno veća ako se u tumoru nalazi velika gustina TP/CPF (p&lt;0,0001). Tumori sa velikom gustinom TP/CPF su imali najveći prečnik koji je varirao u rasponu od 25 do 100 mm, dok su tumori sa malom gustinom TP/CPF bili najvećeg prečnika od 20 do 110 mm (p=0,6744). Intenzitet peritumorskog limfoidnog odgovora je bio velik kod 13 tumora sa velikom gustinom TP/CPF (28,9%) i kod 17 tumora sa malom gustinom TP/CPF (24,6%), p=0,7747. Konfiguracija tumorske margine je bila infiltrativna u svim tumorima sa velikom gustinom TP/CPF, kao i kod 42 tumora sa malom gustinom TP/CPF (60,9%). ZAKLJUČAK: Velika gustina TP/CPF je nezavisan tkivni indikator lo&scaron;e prognoze kod bolesnika sa KK u stadijumu II, koji je ne korelira ni sa veličinom primarnog tumora ni sa intenzitetom peritumorskog limfoidnog odgovora. Velika gustina TP/CPF nije kompatibilna sa ekspanzivnom konfiguracijom tumorske margine, ali infiltrativna konfiguracija tumorske margine nije prediktor velike gustine TP/CPF.</p> / <p>INTRODUCTION: Colonic carcinoma (CC) is a serious public health problem due to its high incidence and mortality rate. Stage is the single most important independent prognosticator in patients with CC. In the presence of indicators of poor prognosis, including high histologic grade, ileus, lympho-vascular invasion and perineural invasion, there is a need for adjuvant chemotherapy after a potentially curative operation in patients with stage II CC, because the therapy improves both overall survival and disease-free survival. However, some patients with no documented poor prognostic factors suffer recurrences, which indicates that there may be some other tissue features that confer poor prognosis. In the recent publications there is an increasing interest in the phenomenon of tumor budding, a term assigned to the presence of small groups of discohesive tumor cells at the invasive front of the tumor &ndash; tumor buds (TB&#39;s). These cells acquire mesenchymal phenotype and gain the ability to migrate through the extracellular matrix by means of cytoplasmic extrusions which are visible on the two-dimensional immunohistologic sections and are called cytoplasmic pseudofragments (CPF&#39;s). Significance of density of TB&#39;s and CPF&#39;s is still to be evaluated, but the pool of evidence suggests that this is a powerful predictor of biologic behaviour of CC. AIM: The aim of this study was to determine the influence of density of TB&#39;s and CPF&#39;s on the risk of recurrence in patients with stage II CC. This research also attempted to establish whether there is a correlation between the density of TB&#39;s and CPF&#39;s and several other morphologic features such as tumor diameter, peritumoral lymphocytic response and the configuration of the tumor margin. METHODS: 114 patients with stage II CC were enrolled in the study. All the patients received surgery at the Institute of Oncology in Sremska Kamenica and no patient had indication for adjuvant chemotherapy. Microscopic analysis of routine histologic and immunohistochemical slides was performed to establish the density of TB&#39;s and CPF&#39;s, to estimate the intensity of the peritumoral lymphocytic response and to determine the configuration of the tumor margin. RESULTS: High density of TB&#39;s and/or CPF&#39;s was found in 45 tumors (39.5%). In this group recurrence occured in 26 patients (57.8%). In the group of patients with low density of TB/CPF in the tumor tissue 4 patients relapsed (5.8%). Comparison of survival curves showed that the probability of recurrence was significantly greater if the density of TB/CPF&#39;s was high (p&lt;0.0001). Tumors with high density of TB/CPF&#39;s ranged from 25 to 100 mm in greatest diameter, while those with low density measured from 20 to 110 mm (p=0.6744). Intensity of peritumoral lymphocytic response was high in 13 tumors with high density of TB/CPF&#39;s (28.9%) and in 17 tumors with low density of TB/CPF&#39;s (24.6%), p=0.7747. All tumors with high density of TB/CPF&#39;s and 42 tumors with low density of TB/CPF&#39;s (60.9%) had infiltrative configuration of tumor margin. CONCLUSION: High density of TB/CPF&#39;s is an independent indicator of poor prognosis in patients with stage II CC and it correlates neither with tumor diameter nor with intensity of peritumoral lymphocytic response. High density of TB/CPF&#39;s is not compatible with the expansive configuration of tumor margin, but the infiltrative configuration of tumor margin is not a predictor of high density of TB/CPF&#39;s.</p>

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy. / Dissertation

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.

Nierenzellkarzinome (NZKs) gelten als stark immunogene Tumore. Dies ist insbesondere auf die Infiltration durch verschiedene Immunzellpopulationen, wie T-Lymphozyten und Natürliche Killer (NK)-Zellen, sowie das klinische Ansprechen auf immuntherapeutische Strategien zurückzuführen. Bisher existieren jedoch nur sehr wenige Studien zur Rolle von humanen nativen dendritischen Zellen (DCs) in NZK-Geweben und über die Tumor-vermittelte Modulation dieser DCs. DCs nehmen als professionelle Antigen-präsentierende Zellen eine zentrale Schlüsselrolle bei der Induktion und Aufrechterhaltung der angeborenen sowie adaptiven Immunantwort ein. Daher wurde im Rahmen dieser Arbeit erstmals der Effekt von klarzelligen NZKs auf den Phänotyp sowie die immunmodulatorischen Fähigkeiten von 6-sulfo LacNAc+ (slan)DCs evaluiert. SlanDCs, welche eine große Subpopulation humaner Blut-DCs darstellen, sind neben der Sekretion großer Mengen proinflammatorischer Zytokine dazu befähigt, Tumorzellen direkt zu lysieren. Des Weiteren sind slanDCs in der Lage, die antitumoralen Effekte von NK-Zellen zu fördern und CD4+ T-Helfer-Zellen sowie Tumor-reaktive CD8+ T-Lymphozyten effizient zu stimulieren. Angesichts dieser proinflammatorischen Eigenschaften können slanDCs wesentlich an einer Tumor-gerichteten Immunantwort beteiligt sein. Auf dieser Grundlage erfolgte im Rahmen der vorliegenden Arbeit der immunhistochemische Nachweis von slanDCs in klarzelligen NZK-Geweben. Im Vergleich zu Tumor-freiem Nierengewebe trat in den primären Tumorgeweben eine erhöhte Zahl infiltrierender slanDCs auf. Zudem wurde die Präsenz von slanDCs in Lymphknoten- sowie Fernmetastasen von NZK-Patienten beobachtet. Weiterführende Untersuchungen an frischen klarzelligen NZK-Geweben demonstrierten, dass NZK-infiltrierende slanDCs einen unreifen Phänotyp ausprägen und Interleukin-10 produzieren. Ausgehend von diesen Erkenntnissen erfolgten funktionelle Analysen, bei denen der Einfluss der kommerziell erhältlichen klarzelligen NZK-Linien ACHN und Caki-1 sowie der primären klarzelligen NZK-Linien MZ1257RC und MZ2877RC auf bedeutende immunmodulatorische Fähigkeiten von slanDCs untersucht wurde. In diesem Zusammenhang zeigte sich, dass NZK-Zellen effektiv in der Lage sind, sowohl die slanDC-vermittelte Proliferation von CD4+ und CD8+ T-Lymphozyten, als auch die slanDC-induzierte Differenzierung naïver CD4+ T-Lymphozyten in proinflammatorische T-Helfer 1-Zellen zu inhibieren. Darüber hinaus wurde demonstriert, dass NZK-Zellen das Potenzial von slanDCs zur Aktivierung von NK-Zellen hemmen. Untersuchungen der zugrunde liegenden Mechanismen zeigten, dass die funktionelle Inhibition von slanDCs durch klarzellige NZK-Zellen über membranständige Moleküle vermittelt wird. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse weisen darauf hin, dass NZKs die Ausreifung sowie wesentliche funktionelle Eigenschaften von DCs inhibieren. Dies deutet auf einen neuen Immunescape-Mechanismus klarzelliger NZKs hin, welcher auf einer Tumorzell-vermittelten Generierung von tolerogenen slanDCs basiert und eine unzureichende Aktivierung der angeborenen sowie adaptiven Tumor-gerichteten Immunantwort zur Folge hat. Diese neuen Erkenntnisse können einen Beitrag zu einem besseren Verständnis der Interaktion von NZKs mit nativen humanen DCs leisten und die Konzeption neuer therapeutischer Strategien ermöglichen, welche auf einer Verstärkung der antitumoralen Eigenschaften von DCs beruhen.

Abstract Desmoglein 3 (Dsg3) is an adhesion receptor in desmosomes, but relatively little is known about its role in cancer. In this study, the function of Dsg3 was investigated in oral squamous cell carcinoma (SCC) cell lines in vitro using locally established human leiomyoma tumor microenvironment (TME) matrices. Since Dsg3 has been identified as a key regulator in cell adhesion, we hypothesized that it may play a role in oral SCC cells adhesion and motility. Thus, one aim of the study was to explore this hypothesis by both gain and loss of function methods in four human buccal mucosa SCC SqCC/Y1 cell lines: transduction of vector control (Ct), full-length (FL) or two different C-terminally truncated Dsg3 mutants (&#916;238 and &#916;560). Live cell imaging was performed for 2D migration and 3D sandwich, alongside other assays. In 3D sandwich, we tested the effects of the monoclonal antibody, AK23, targeting the extracellular domain of Dsg3 in SqCC/Y1 cells. Our results showed that loss of Dsg3 disrupted cell adhesion and protein expression. In 2D assays, FL and Dsg3 mutants migrated faster with higher accumulated distances than Ct. In contrast with 2D, mutants showed accelerated invasion over the Ct in 3D models. The AK23 antibody inhibited only the invasion of FL cells. The TME in vivo consists of cellular and matrix elements playing a leading role in carcinoma progression. To study carcinoma cells invasion in vitro, mouse Matrigel® and rat type 1 collagen are the most commonly used matrices in 3D models. Since they are non-human in origin, they do not perfectly mimic human TME. To address this, we have developed a solid organotypic myoma disc model derived from human uterus leiomyoma tumor. Here, we introduce a novel Myogel, prepared from leiomyoma similar to Matrigel®. We validated Myogel for cell-TME interactions in 3D models, using SqCC/Y1 and HSC-3 cell lines. Compared with Matrigel® and type I collagen, oral SCC cell lines invaded more efficiently in Myogel containing matrices. This study describes promising 3D models using human TME mimicking Myogel which is suitable to analyze oral SCC cells both in carcinoma monocultures and in co-cultures, such as with TME fibroblasts. We also introduce a possible novel therapeutic target against Dsg3 to suppress cancer cell invasion. / Tiivistelmä Desmogleiini 3 (Dsg3) on desmosomien adheesioreseptori, jonka merkityksestä syövässä tiedetään vähän. Koska Dsg3 on tärkeä epiteelisolujen välisissä liitoksissa, oletimme sillä olevan vaikutusta myös suun karsinoomasolujen tarttumisessa ja niiden liikkuvuudessa. Testasimme hypoteesiamme muuttamalla Dsg3:n toimintaa ihmisen posken karsinoomasolulinjassa SqCC/Y1, josta oli aiemmin valmistettu neljä erilaista muunnosta: tyhjän vektorin sisältävä kontrollisolulinja (Ct), kokopitkää Dsg3 tuottava solulinja (FL), sekä kaksi Dsg3 C-päästä lyhennettyä mutanttisolulinjaa (&#916;238 ja &#916;560). Immunofluoresenssi-menetelmää käyttäen analysoimme solulinjoissamme solujen välisiä liitoksia. Lisäksi mittasimme solujen liikkeitä 2D-migraatio- ja 3D-sandwich-kokeissa. Testasimme myös Dsg3:n solunulkoista osaa tunnistavan monoklonaalisen vasta-aineen (AK23) vaikutusta solujen invaasioon. Osoitimme, että Dsg3:n rakenteen muuttaminen ja toiminnan estyminen häiritsi solujen tarttumista. 2D-kokeissa sekä FL että mutanttilinjat (&#916;238 ja &#916;560) migroivat kontrollisoluja nopeammin ja pidemmälle, mutta 3D-kokeissa vain mutanttilinjat invasoituivat kontrollisoluja tehokkaammin. AK23-vasta-aine esti vain FL-solujen invaasiota. Syöpäsolujen 3D-invaasiota mittaavissa kokeissa käytetään yleensä hiiren kasvaimesta valmistettua kaupallista Matrigeeliä® tai rotan kudoksista eristettyä tyypin I kollageenia. Tutkimusryhmämme on jo aiemmin kehittänyt organotyyppisen myoomamallin, jossa valmistamme myoomakudosnapit ihmisen kohdun leiomyoomakasvaimista. Tässä työssä valmistimme leiomyoomasta Myogeelia, vertasimme sitä Matrigeeliin®, sekä tutkimme tarkemmin Myogeeli-valmisteen soveltuvuutta 3D-tutkimuksiin. Totesimme, että kielen (HSC-3) ja posken (SqCC/Y1) karsinoomasolut invasoituivat tehokkaimmin Myogeeli-pitoisissa matrikseissa kuin Matrigeeliä® tai kollageeniä sisältävissä kasvatusalustoissa. Tutkimustulostemme perusteella Myogeeli-pohjaiset 3D-mallit soveltuvat hyvin sekä syöpäsolulinjojen invaasiotutkimuksiin että yhteisviljelmiin, joissa syöpäsoluja viljellään yhdessä syöpäkasvaimen ympärillä olevien solujen, kuten fibroblastien, kanssa.

Pancreatic cancer is characterized by its desmoplastic tumor microenvironment and the infiltration of immunosuppressive cells. It is a devastating disease where most patients are diagnosed at a late stage and the treatment options are few. The development of new treatments is surly needed. One treatment option explored is the use of immunotherapy with the intent to activate the immune system and change the balance from pro-tumor to anti-tumor. This thesis presents the idea of using oncolytic adenoviruses called LOAd-viruses that are armed with immunostimulatory- and microenvironment-modulating transgenes. For effective treatment of pancreatic cancer, the virus needs to be able to be given in addition to standard therapy, the chemotherapy gemcitabine. In paper I, the immunomodulatory effect of gemcitabine was evaluated in blood from pancreatic cancer patients receiving their first 28-day cycle of treatment with infusions day 1, 8 and 15 followed by a resting period. Gemcitabine reduced the level of immunosup-pressive cells and molecules but the effect did not last throughout the resting period. On the other hand, gemcitabine did not affect the level or proliferative function of effector T cells indicating that gemcitabine could be combined with immunotherapy. The LOAd700 virus expresses a novel membrane-bound trimerized form of CD40L (TMZ-CD40L). In paper II, LOAd700 showed to be oncolytic in pancreatic cancer cell lines as well as being immunostimulatory as shown by its capacity to activate dendritic cells (DCs), myeloid cells, endothelium, and to promote expansion of antigen-specific T cells. In paper III, LOAd703 armed with both 4-1BBL and TMZ-CD40L was evaluated. LOAd703 gave a more profound effect than LOAd700 on activation of DCs and the virus was also capable of reducing factors in stellate cells connected to the desmo-plastic and immunosuppressive microenvironment. In paper IV, LOAd713 armed with TMZ-CD40L in combination with a single-chain variable fragment against IL-6R was evaluated. The virus could kill pancreatic cancer cells lines through oncolysis and could also reduce factors involved in desmoplasia in stellate cells. Most interestingly, LOAd713 could reduce the up-regulation of PD-1/PD-L1 in DCs after CD40 activation. Taken together, LOAd703 and LOAd713 seem to have interesting features with their combination of immunostimulation and microenvironment modulation. At present, LOAd703 is evaluated in a clinical trial for pancreatic cancer (NCT02705196).

<div>Cancer is responsible for the second highest cause of death in the United States, and lung cancer accounts for 13% of new cancer diagnoses, with the highest rate of cancer death at 24%. Almost 85% of these cases represent non-small cell lung cancer (NSCLC), which includes lung adenocarcinoma, the most common NSCLC subtype. Traditional cancer treatments often only temporarily stop the spread of the disease, but immunotherapies, which are becoming a standard of care, are much more promising. Natural killer (NK) cells are powerful effectors of innate immunity, and genetically engineered NK cells as immunotherapies have had encouraging clinical responses in the treatment of various cancers. However, more progress is needed for solid tumor treatment, especially for lung adenocarcinoma. The activation of cancer-associated ectoenzymes, CD39 and CD73 catalyze the phosphorylation of ATP to AMP to produce extracellular adenosine (ADO), which is a highly immunosuppressive mechanism contributing to the pathogenesis of solid tumors. Understanding adenosine effects on NK cells will help develop more robust immunotherapeutic treatments to improve cytotoxicity against solid tumors. Here, we established that tumor microenvironment ADO results in impaired metabolic and anti-tumor functions of cytokine-primed NK cells. Specifically, peripheral blood-derived NK cells stimulated with IL-2, IL-15, or a combination of IL-12 and IL-15 showed suppressed anti-tumor immunity due to ADO. This was observed by the downregulation of activation receptor expression, cytotoxicity inhibition, impairment of metabolic activity, and alterations in gene expression. To target ADO-producing CD73 on cancer cells, we redirected NK cells by fusing CD73 ScFv with intracellular and transmembrane regions of NK cell specific signaling components derived from FCyRIIIa (CD16). Engineered NK cells were shown to be cytotoxic against lung adenocarcinoma <i>in vitro</i> and impede tumor growth in a lung adenocarcinoma mouse model <i>in vivo</i>. Engineered cells also had higher levels of degranulation and cytokine release, as well as more infiltration into tumors and longer survival time in mice. In summary, the microenvironment of solid tumors is highly immunosupressive, and redirecting NK cell function using a NK-specific anti-CD73 targeting construct will help to promote anti-tumor immunity and</div><div>inhibit cancer growth for a potentially powerful new immunotherapy against solid tumors.</div>

Nierenzellkarzinome (NZKs) gelten als stark immunogene Tumore. Dies ist insbesondere auf die Infiltration durch verschiedene Immunzellpopulationen, wie T-Lymphozyten und Natürliche Killer (NK)-Zellen, sowie das klinische Ansprechen auf immuntherapeutische Strategien zurückzuführen. Bisher existieren jedoch nur sehr wenige Studien zur Rolle von humanen nativen dendritischen Zellen (DCs) in NZK-Geweben und über die Tumor-vermittelte Modulation dieser DCs. DCs nehmen als professionelle Antigen-präsentierende Zellen eine zentrale Schlüsselrolle bei der Induktion und Aufrechterhaltung der angeborenen sowie adaptiven Immunantwort ein. Daher wurde im Rahmen dieser Arbeit erstmals der Effekt von klarzelligen NZKs auf den Phänotyp sowie die immunmodulatorischen Fähigkeiten von 6-sulfo LacNAc+ (slan)DCs evaluiert. SlanDCs, welche eine große Subpopulation humaner Blut-DCs darstellen, sind neben der Sekretion großer Mengen proinflammatorischer Zytokine dazu befähigt, Tumorzellen direkt zu lysieren. Des Weiteren sind slanDCs in der Lage, die antitumoralen Effekte von NK-Zellen zu fördern und CD4+ T-Helfer-Zellen sowie Tumor-reaktive CD8+ T-Lymphozyten effizient zu stimulieren. Angesichts dieser proinflammatorischen Eigenschaften können slanDCs wesentlich an einer Tumor-gerichteten Immunantwort beteiligt sein. Auf dieser Grundlage erfolgte im Rahmen der vorliegenden Arbeit der immunhistochemische Nachweis von slanDCs in klarzelligen NZK-Geweben. Im Vergleich zu Tumor-freiem Nierengewebe trat in den primären Tumorgeweben eine erhöhte Zahl infiltrierender slanDCs auf. Zudem wurde die Präsenz von slanDCs in Lymphknoten- sowie Fernmetastasen von NZK-Patienten beobachtet. Weiterführende Untersuchungen an frischen klarzelligen NZK-Geweben demonstrierten, dass NZK-infiltrierende slanDCs einen unreifen Phänotyp ausprägen und Interleukin-10 produzieren. Ausgehend von diesen Erkenntnissen erfolgten funktionelle Analysen, bei denen der Einfluss der kommerziell erhältlichen klarzelligen NZK-Linien ACHN und Caki-1 sowie der primären klarzelligen NZK-Linien MZ1257RC und MZ2877RC auf bedeutende immunmodulatorische Fähigkeiten von slanDCs untersucht wurde. In diesem Zusammenhang zeigte sich, dass NZK-Zellen effektiv in der Lage sind, sowohl die slanDC-vermittelte Proliferation von CD4+ und CD8+ T-Lymphozyten, als auch die slanDC-induzierte Differenzierung naïver CD4+ T-Lymphozyten in proinflammatorische T-Helfer 1-Zellen zu inhibieren. Darüber hinaus wurde demonstriert, dass NZK-Zellen das Potenzial von slanDCs zur Aktivierung von NK-Zellen hemmen. Untersuchungen der zugrunde liegenden Mechanismen zeigten, dass die funktionelle Inhibition von slanDCs durch klarzellige NZK-Zellen über membranständige Moleküle vermittelt wird. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse weisen darauf hin, dass NZKs die Ausreifung sowie wesentliche funktionelle Eigenschaften von DCs inhibieren. Dies deutet auf einen neuen Immunescape-Mechanismus klarzelliger NZKs hin, welcher auf einer Tumorzell-vermittelten Generierung von tolerogenen slanDCs basiert und eine unzureichende Aktivierung der angeborenen sowie adaptiven Tumor-gerichteten Immunantwort zur Folge hat. Diese neuen Erkenntnisse können einen Beitrag zu einem besseren Verständnis der Interaktion von NZKs mit nativen humanen DCs leisten und die Konzeption neuer therapeutischer Strategien ermöglichen, welche auf einer Verstärkung der antitumoralen Eigenschaften von DCs beruhen.

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>